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WO2020078378A1 - Procédés et systèmes de profilage de microbes - Google Patents

Procédés et systèmes de profilage de microbes Download PDF

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Publication number
WO2020078378A1
WO2020078378A1 PCT/CN2019/111447 CN2019111447W WO2020078378A1 WO 2020078378 A1 WO2020078378 A1 WO 2020078378A1 CN 2019111447 W CN2019111447 W CN 2019111447W WO 2020078378 A1 WO2020078378 A1 WO 2020078378A1
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subject
microbe
microbes
biological sample
susceptibility
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English (en)
Inventor
Xiang Li
Xiaodong Liu
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Coyote Diagnostics Lab (beijing) Co Ltd
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Coyote Diagnostics Lab (beijing) Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C12Q1/06—Quantitative determination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • Microbes including bacteria, fungi, protozoa and viruses, reside on the human body, including but not limited to the surface and in deep layers of skin (including in mammary glands) , in the saliva and oral mucosa, in the conjunctiva, and in the gastrointestinal tracts. Microbes can outnumber human cells 10 to 1. Many microbes associated with humans appear to be not harmful at all, but rather assist in maintaining processes conducive to health. However, some microbes cause deleterious conditions and disease.
  • Negative health effects are also associated with microbial imbalance, sometimes referred to as "dysbiosis, " typically characterized by increased levels of harmful microbes and/or reduced levels of beneficial microbes.
  • Microbial imbalance can arise from a variety of causes, such as repeated and inappropriate antibiotic exposure, alcohol misuse, or inappropriate diet. Microbial imbalance is reported to be associated with diseases such as chronic fatigue syndrome, obesity, and cancer.
  • An aspect of the disclosure provides a method of assessing a susceptibility of a subject to a health condition associated with a plurality of microbes.
  • the method includes: (a) obtaining at least one biological sample of the subject; (b) assaying the at least one biological sample of the subject to assess a quantity or concentration of each of a plurality of microbes in the at least one biological sample, where the plurality of microbes comprises a first microbe and a second microbe, which first microbe has been identified to be beneficial with respect to a health of the subject and the second microbe has been identified to be harmful with respect to the health of the subject; (c) determining a quantity or relative concentration of each of the plurality of microbes relative to a reference (s) , to identify a dearth or abundance of each of the plurality of microbes; and (d) outputting a report indicative of the susceptibility of the subject to the health condition associated with the plurality of microbes, which susceptibility is determined based at least in part on
  • the plurality of types of microbes comprises at least two types of first microbes, at least three types of first microbes, at least four types of first microbes or at least five types of first microbes. In some embodiments, the plurality of types of microbes comprises at least two types of second microbes, least three types of second microbes, at least four types of second microbes, at least five types of second microbes.
  • the plurality of types of microbes include at least one microbe selected from the group consisting of: bacteria, viruses and fungi.
  • bacteria include Escherichia, Paracolon, Enterobacter, Erwinia, Streptococcous, Faecalibacterium, Enterobacter aerogenes, Proteus, Pseudomonas aeruginosa, Pneumobacillus, Bacillus, Bacteroides, Bifidobacterium, Clostridium, Collinsella, Faecalibacterium, Lactobacillus, Ruminococcus, Enterococcus, Dorea, Listeria, Streptococcus, Staphyloccocus, Corynebacterium, Propionibacterium, Clostridium butyricum, Campylobacter, Aeromonas, Plesiomonas shigelloides, Campylobacter, Clostridium difficile, Escherichia coli O157, Enteroaggregative E.
  • EAEC Enteropathogenic E. coli
  • EPEC Enteropathogenic E. coli
  • EHEC Enterohemorrhagic E. coli
  • ETEC Enterotoxigenic E. coli
  • EIEC Shiga-like Toxin producing E.
  • Non-limiting examples of viruses include Adenovirus 40/41, Norovirus GI/GII, Rotavirus, Rotavirus A, Astrovirus, Sapovirus, herpes simplex virus, hepatovirus, and CMV.
  • Non-limiting examples of fungi include Candida albicans, Hirsutellia, and Saccharomyces.
  • the plurality of types of microbes include at least one microbe selected from the group consisting of Lactobacillus, Bacteroides, Bifidobacterium, Escherichia, Faecalibacterium, Ruminococcus, and Lactobacillus rhamnosus GG strain.
  • the subject is up to 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 year (s) or 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 month (s) of age at a time at which the biological sample is obtained.
  • the subject is aged from six months to twelve years old. In some embodiments, the subject is aged from six months to six years old. In some embodiments, the subject is aged from six months to two years old. In some embodiments, the subject is aged from six months to one year old.
  • the reference comprises normal levels of the plurality of microbes, where a normal level of a first microbe comprises a first threshold and optionally, a second threshold, and a normal level of a second microbe comprises a second threshold and optionally, a first threshold, and where (c) comprises determining whether the quantity or concentration is above the first threshold and/or below the second threshold.
  • the first threshold is at most 10 15 , 10 14 , 10 13 , 10 13 , 10 12 , 10 11 , 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , or less of a given microbe of the plurality of types of microbes per gram of the biological sample.
  • the second threshold is at least 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or more copies of a given microbe of the plurality of types of microbes per gram of the biological sample.
  • the first threshold is 10 15 , 10 14 , 10 13 , 10 13 , 10 12 , 10 11 , 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , or less colony-forming units (CFU) of a given microbe of the plurality of types of microbes per gram of the biological sample.
  • CFU colony-forming units
  • the second threshold is 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or more CFU of a given microbe of the plurality of types of microbes per gram of the biological sample.
  • the method further comprises assessing susceptibility to a health condition of the subject if the quantity or concentration of at least one first microbe is lower than the first threshold and/or the quantity or concentration of the at least one second microbe is higher than the second threshold.
  • the condition is selected from the group consisting of a gut condition, diarrhea, jaundice, inflammatory bowel disease, Crohn's Disease, irritable bowel syndrome, a stomach ulcer, colitis, neonatal necrotizing enterocolitis, gastroesophageal reflux disease, constipation, functional bloating, gastritis, lactose intolerance, visceral hyperalgesia, colic, pouchitis, diverticulitis, diabetes, cholera, obesity, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, colorectal cancer, pathogen infection, purulent or suppurative inflammation, toxinosis, allergic diseases, typhoid fever, acute enteritis, sepsis,
  • the reference comprises normal levels of the plurality of microbes in a population to which the subject belongs. In some embodiments, the reference comprises normal levels of the plurality of microbes of the subject in a healthy state.
  • the assaying in (b) comprises conducting nucleic acid amplification reaction on nucleic acid molecules derived from the at least one biological sample.
  • the nucleic acid amplification reaction is selected from the group consisting of polymerase chain reaction (PCR) , real-time PCR, isothermal amplification, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, solid phase amplification, nucleic acid sequence-based amplification (NASBA) , linear amplification, and digital PCR.
  • PCR polymerase chain reaction
  • real-time PCR isothermal amplification
  • strand displacement amplification strand displacement amplification
  • rolling circle amplification rolling circle amplification
  • ligase chain reaction transcription-mediated amplification
  • solid phase amplification nucleic acid sequence-based amplification
  • digital PCR nucleic acid sequence-based amplification
  • the nucleic acid amplification is PCR.
  • the at least one biological sample is subjected to the PCR without purification. In some embodiments, the at least one biological sample is subjected to the PCR without deoxynucleic acid (DNA) or ribonucleic acid (RNA) enrichment. In some embodiments, the at least one biological sample is subjected to the PCR without pre-culturing. In some embodiments, the at least one biological sample is subjected to the PCR without non-selective enrichment. In some embodiments, the at least one biological sample is subjected to the PCR without selective enrichment. In some embodiments, the at least one biological sample is subjected to the PCR without plating on differential medium. In some embodiments, the at least one biological sample is subjected to the PCR without presumptive biomedical identification.
  • DNA deoxynucleic acid
  • RNA ribonucleic acid
  • the assaying in (b) comprises conducting sequencing on nucleic acid molecules derived from the at least one biological sample.
  • the quantity or concentration is a relative quantity or concentration of each of the plurality of types of microbes in the at least one biological sample.
  • the method further comprises determining a change in the quantity or concentration of at least a subset of the plurality of types of microbes in the at least one biological sample at multiple geographic locations.
  • the method further comprises determining a change in the quantity or concentration of at least a subset of the plurality of types of microbes in the at least one biological sample over a time period of at least 1 minute (min) .
  • the period of time is at least 10 min, 30 min, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 1 year.
  • the biological sample is a stool sample.
  • the subject is aged from zero (0) to one (1) months old, zero (0) to two (2) months old, zero (0) to three (3) months old, zero (0) to four (4) months old, zero (0) to five (5) months old, zero (0) to six (6) months old, zero (0) to seven (7) months old, zero (0) to eight (8) months old, zero (0) to nine (9) months old, zero (0) to ten (10) months old, zero (0) to eleven (11) months old, zero (0) to twelve (12) months old at the time at which the stool sample is obtained.
  • the reference comprises normal levels of Lactobacillus, Escherichia, Bifidobacterium, Ruminococcus, Bacteroides, Faecalibacterium, and Lactobacillus rhamnosus GG strain corresponding to an amount of copies of bacteria in the stool sample of copies/g of stool sample, respectively.
  • subject is aged from six (6) months to twelve (1) year old, six (6) months to two (2) years old, six (6) months to three (3) years old, six (6) months to four (4) years old, six (6) months to five (5) years old, six (6) months to six (6) years old, six (6) months to seven (7) years old, six (6) months to eight (8) years old, six (6) months to nine (9) years old, six (6) months to ten (10) years old, six (6) months to eleven (11) years old, or six (6) months to twelve (12) years old at the time at which the stool sample is obtained.
  • the reference comprises normal levels of Lactobacillus, Escherichia, Bifidobacterium, Ruminococcus, Bacteroides, Faecalibacterium, and Lactobacillus rhamnosus GG strain corresponding to an amount of copies of bacteria in the stool sample of copies/g of stool sample, respectively.
  • (d) comprises outputting a report indicative of the susceptibility of the subject to allergy and/or eczema based at least in part on an abundance of E. coli and a dearth of Bacteroides. In some embodiments, (d) comprises outputting a report indicative of the susceptibility of the subject to enteritis and/or diarrhea based at least in part on an abundance of E. coli and a dearth of Faecalibacterium.
  • the method further comprises providing the subject with a questionnaire to access additional information before (d) , which additional information, when combined with an assessment of a dearth of the first microbe and/or abundance of the second microbe in the subject, is indicative of the susceptibility.
  • additional information when combined with an assessment of a dearth of the first microbe and/or abundance of the second microbe in the subject, is indicative of the susceptibility.
  • susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject, in combination with the additional information.
  • the additional information is selected from the group consisting of information regarding the subject, information regarding the mother of the subject, and information regarding living environment of the subject.
  • the information regarding the subject is selected from the group consisting of age, sex, ethnicity, height, weight, head circumference, mode of birth, gestational age at birth, geological location of birth, feeding status, prebiotic intake, probiotic intake, medical history, congenital disease, bowel movement, and stool appearance of the subject.
  • the feeding status includes breastfeeding, formula feeding, solid food feeding, and combinations thereof.
  • the mode of birth includes vaginal birth or caesarean section, presence or absence of labor induction, and combinations thereof.
  • the medical history includes prior or concurrent medication, prior or concurrent conditions, prior or concurrent symptoms, and combinations thereof.
  • the medication includes antibiotics.
  • the conditions include allergy, congenital diseases, gastrointestinal diseases, infectious disease, and combinations thereof.
  • the allergy is allergy to one or more allergens selected from the group consisting of milk, soy, wheat, lactose, and yogurt.
  • the stool appearance includes color, form, shape, consistency, fluidity, and combinations thereof.
  • the symptoms include constipation, diarrhea, jaundice, colic, itching, eczema and bloating.
  • the information regarding the mother of the subject is selected from age at birth, height, weight, medical history, allergy,
  • the medical history comprises the presence or absence of one or more diseases selected from the group consisting of urticarial, asthma, and gestational diabetes.
  • the allergy is allergy to one or more allergens selected from the group consisting of milk, soy, wheat, lactose, and yogurt.
  • the information regarding the living environment of the subject is selected from access to pets, sanitization of clothing and/or personal items, and sanitization of living environment.
  • the susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject at an accuracy of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%or higher.
  • An additional aspect of the disclosure provides a system for assessing a susceptibility of a subject to a health condition associated with a plurality of microbes.
  • the system includes: an input module that receives a user request to assess a susceptibility of a subject to a health condition associated with a plurality of microbes; an assay module that assays at least one biological sample of the subject to assess a quantity or concentration of each of a plurality of microbes in the at least one biological sample, where the plurality of microbes comprises a first microbe and a second microbe, which first microbe has been identified to be beneficial with respect to a health of the subject and the second microbe has been identified to be harmful with respect to the health of the subject; and one or more computer processor operatively coupled to the assay module.
  • the one or more computer processors can be individually or collectively programmed to: (a) determine a quantity or relative concentration of each of the plurality of microbes relative to a reference (s) , to identify a dearth or abundance of each of the plurality of microbes; and (b) output a report indicative of the susceptibility of the subject to the health condition associated with the plurality of microbes, which susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject at an accuracy of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%or higher.
  • the system further comprises a user interface for providing the subject with a questionnaire to access additional information, which additional information, when combined with an assessment of a dearth of the first microbe and/or abundance of the second microbe in the subject, is indicative of the susceptibility.
  • additional information when combined with an assessment of a dearth of the first microbe and/or abundance of the second microbe in the subject, is indicative of the susceptibility.
  • susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject, in combination with the additional information.
  • the additional information is selected from the group consisting of information regarding the subject, information regarding the mother of the subject, and information regarding living environment of the subject.
  • the information regarding the subject is selected from the group consisting of age, sex, ethnicity, height, weight, head circumference, mode of birth, gestational age at birth, geological location of birth, feeding status, prebiotic intake, probiotic intake, medical history, congenital disease, bowel movement, and stool appearance of the subject.
  • the feeding status includes breastfeeding, formula feeding, solid food feeding, and combinations thereof.
  • the mode of birth includes vaginal birth or caesarean section, presence or absence of labor induction, and combinations thereof.
  • the medical history includes prior or concurrent medication, prior or concurrent conditions, prior or concurrent symptoms, and combinations thereof.
  • the medication includes antibiotics.
  • the conditions include allergy, congenital diseases, gastrointestinal diseases, infectious disease, and combinations thereof.
  • the allergy is allergy to one or more allergens selected from the group consisting of milk, soy, wheat, lactose, and yogurt.
  • the stool appearance includes color, form, shape, consistency, fluidity, and combinations thereof.
  • the symptoms include constipation, diarrhea, jaundice, colic, itching, eczema and bloating.
  • the information regarding the mother of the subject is selected from age at birth, height, weight, medical history or an allergy.
  • the medical history comprises the presence or absence of one or more diseases selected from the group consisting of urticarial, asthma, and gestational diabetes.
  • the allergy is allergy to one or more allergens selected from the group consisting of milk, soy, wheat, lactose, and yogurt.
  • the information regarding the living environment of the subject is selected from access to pets, sanitization of clothing and/or personal items, and sanitization of living environment.
  • An additional aspect of the disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, upon execution by one or more computer processors, implements a method of assessing a susceptibility of a subject to a health condition associated with a plurality of microbes.
  • Such a method can include: (a) obtaining at least one biological sample of the subject; (b) assaying the at least one biological sample of the subject to assess a quantity or concentration of each of a plurality of microbes in the at least one biological sample, where the plurality of microbes comprises a first microbe and a second microbe, which first microbe has been identified to be beneficial with respect to a health of the subject and the second microbe has been identified to be harmful with respect to the health of the subject; (c) determining a quantity or relative concentration of each of the plurality of microbes relative to a reference (s) , to identify a dearth or abundance of each of the plurality of microbes; and (d) outputting a report indicative of the susceptibility of the subject to the health condition associated with the plurality of microbes, which susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject at an accuracy of at least about 50%, 55%, 60%, 65%
  • An additional aspect of the disclosure provides a method for determining and/or treating a health condition of a subject.
  • the method comprises: (a) determining at least one symptom from the subject; (b) performing an intestinal microflora detection and optionally a pathogen detection on the subject, where the intestinal microflora detection is conducted according to the method of the disclosure to assess the susceptibility of a subject to the health condition; and (c) upon determining that the at least one symptom of the subject matches the health condition of (b) , prescribing a drug and/or probiotic intervention to the subject.
  • the at least one symptom from the subject is determined by recording complaints from the subject or taking oral inquiry from the subject.
  • the method further comprises performing a second intestinal microflora detection on the subject, where the second intestinal microflora detection is conducted to assess the susceptibility of the subject to the health condition.
  • the method further comprises outputting a report indicative of recovery of the subject from the health condition if the susceptibility of the subject to the health condition has not been determined.
  • the method further comprises repeating (c) and (d) if the susceptibility of the subject to the health condition has been determined. In some embodiments, if the at least one symptom does not match the health condition of (b) , prior to (c) (i) taking a second inquiry from the subject based on the results of (b) ; (ii) optionally performing other detections.
  • the intestinal microflora detection and the second intestinal microflora detection are performed at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months apart or at a greater interval.
  • An additional aspect of the disclosure provides a method for determining and/or treating a health condition of a subject suspected of dysbiosis of intestinal microflora.
  • the method comprises: (a) determining whether one or more symptoms of the dysbiosis are severe or mild; (b) if the one or more symptoms of the dysbiosis are mild, performing an intestinal microflora detection, where the intestinal microflora detection is conducted according to the method of the disclosure to assess a susceptibility of the subject to the health condition; if the one or more symptoms of the subject match the health condition of (b) , prescribing a probiotic intervention to the subject.
  • a mild of the dysbiosis is selected from the group consisting of loss of appetite, weakness, obesity, wasting, light diarrhea and constipation.
  • a severe symptom of the dysbiosis includes severe diarrhea and/or infectious diarrhea.
  • whether the one or more symptoms of the dysbiosis are severe or mild is determined by recording complaints from the subject or taking oral inquiry from the subject.
  • the method further comprises performing an additional intestinal microflora detection on the subject, where the additional intestinal microflora detection is conducted according to the method of the disclosure to assess the susceptibility of the subject to the health condition.
  • the method further comprises outputting a report indicative of recovery of the subject from the health condition if the susceptibility of the subject to the health condition is not determined. In some embodiments, the method further comprises repeating (c) if the susceptibility of the subject to the health condition is determined. In some embodiments, if the one or more symptoms of the dysbiosis are severe, the method further comprises performing a pathogen detection on the subject. In some embodiments, if the pathogen detection reveals an infectious disease, the method further comprises, performing a drug intervention on the subject. In some embodiments, the drug intervention comprises providing or recommending an antibiotic. In some embodiments, the method comprises proceeding to (b) after the drug intervention.
  • the method further comprises (i) taking a second inquiry from the; (ii) optionally performing one or more other detections; and (iii) proceeding to (c) .
  • Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.
  • Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto.
  • the computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.
  • FIG. 1 shows an illustration of an example system programmed or configured to implement various methods of the present disclosure
  • FIG. 2 shows a flow diagram of an example method for sample processing
  • FIG. 3 shows example characterization of the baseline of microbe quantities in stool samples from healthy populations in different age groups (0-6 months and 6 months above) .
  • FIGs. 4.1-4.9 shows examples of the microbe quantities as indicators for health conditions or disorders in subjects, wherein the panels (a) illustrate the original microbe quantities in the samples and the panels (b) illustrate the microbe quantities after interventions such as probiotics prescribed by physicians.
  • FIG. 5 shows an example computer system that is programmed or configured to implement the present disclosure.
  • FIG. 6 shows a principal coordinate analysis between eczema and healthy groups in conjunction with Example 6.
  • FIG. 7 shows Beta diversity of eczema and healthy samples in different age groups in conjunction with Example 6.
  • FIG. 8 shows different bacterial compositions in eczema and health samples among different age groups in conjunction with Example 6.
  • FIG. 9 shows Alpha diversity of eczema and healthy samples in different age groups in conjunction with Example 6.
  • a cell includes a plurality of cells, including mixtures thereof.
  • the term "about” refers to a range that is no more than 10%greater than or less than a stated numerical value unless the context dictates otherwise.
  • the range may be 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5%greater than or less than a stated numerical value.
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure and may perform any function.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • loci locus defined from linkage analysis, exons, introns, messenger RNA (mRNA) , transfer RNA, ribosomal RNA, short interfering RNA (siRNA) , short-hairpin RNA (shRNA) , micro-RNA (miRNA) , ribozymes, cDNA,
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • target polynucleotide refers to a nucleic acid molecule or polynucleotide in a starting population of nucleic acid molecules having a target sequence whose presence, amount, and/or nucleotide sequence, or changes in one or more of these, are to be determined.
  • target sequence refers to a nucleic acid sequence on a single strand of nucleic acid.
  • the target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA, miRNA, rRNA, or others.
  • the target sequence may be a target sequence from a sample or a secondary target such as a product of an amplification reaction.
  • a “nucleotide probe, " “probe” or “tag oligonucleotide” refers to a polynucleotide used for detecting or identifying its corresponding target polynucleotide in a hybridization reaction by hybridization with a corresponding target sequence.
  • a nucleotide probe is hybridizable to one or more target polynucleotides.
  • Tag oligonucleotides can be perfectly complementary to one or more target polynucleotides in a sample or contain one or more nucleotides that are not complemented by a corresponding nucleotide in the one or more target polynucleotides in a sample.
  • Hybridization generally refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a component in a more extensive process, such as the initiation of PCR, or the enzymatic cleavage of a polynucleotide by an endonuclease.
  • a sequence capable of hybridizing with (e.g., "hybridizable" to) a given sequence is referred to as the "complement" of the given sequence.
  • “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond (s) with another nucleic acid sequence by either traditional Watson-Crick or other non-traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10, being 50, 60, 70, 80, 90, and 100 complementary) .
  • Perfectly complementary refers to a scenario where all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • Substantially complementary refers to a degree of complementarity that is at least 60 , 65 , 70 , 75 , 80 , 85 , 90 , 95 , 97 , 98 , 99 , or 100 over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions. Sequence identity, such as for the purpose of assessing percent complementarity, may be measured by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see e.g., the EMBOSS Needle aligner available at www. ebi. ac.
  • uk/Tools/psa/emboss_needle/nucleotide. html optionally with default settings
  • the BLAST algorithm see e.g., the BLAST alignment tool available at blast. ncbi. nlm. nih. gov/Blast. cgi, optionally with default settings
  • the Smith-Waterman algorithm see e.g., the EMBOSS Water aligner available at www. ebi. ac. uk/Tools/psa emboss water/nucleotide. html, optionally with default settings
  • Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.
  • stringent conditions for hybridization generally refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with a target sequence, and substantially does not hybridize to non-target sequences.
  • Stringent conditions are generally sequence-dependent and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.
  • Non-limiting examples of stringent conditions are described in detail in Tijssen (1993) , Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter “Overview of principles of hybridization and the strategy of nucleic acid probe assay” , Elsevier, N.Y.
  • subject “ “individual, “ and “patient” are used interchangeably herein to refer to a vertebrate, such as a mammal, such as a human.
  • Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.
  • the subject may be of various ages.
  • the age of the subject is defined as the age when a biological sample is obtained from the subject.
  • a subject may be of a relatively young age.
  • a subject may be at most about 15 years old, at most about 14 years old, at most about 13 years old, at most about 12 years old, at most about 11 years old, at most about 10 years old, at most about 9 years old, at most about 8 years old, at most about 7 years old, at most about 6 years old, at most about 5 years old, at most about 4 years old, at most about 3 years old, at most about 2 years old, at most about 1 year old, at most about 11 months old, at most about 10 months old, at most about 9 months old, at most about 8 months old, at most about 7 months old, at most about 6 months old, at most about 5 months old, at most about 4 months old, at most about 3 months old, at most about 2 months old, at most about 1 month old, at most about two weeks old or at most about one week old.
  • a subject may be from about 0 years old to about 15 years old, from about 0 years old to about 10 years old, from about 0 years old to about 8 years old, from about 0 years old to about 6 years old, from about 0 years old to about 4 years old, from about 0 years old to about 2 years old, between 0 years old and 1 year old, or between 0 years old and 6 months old.
  • a subject may be from about 6 months old to about 15 years old, from about 6 months old to about 16 months old, from about 6 months old to about 8 years old, from about 6 months old to about 6 years old, from about 6 months old to about 4 years old, from about 6 months old to about 2 years old, or between 6 months old and 1 year old.
  • a subject may be of a relatively old age.
  • a subject may be at least about 15 years old, at least about 20 years old, about 25 years old, about 30 years old, at least about 35 years old, at least about 40 years old, at least about 45 years old, at least about 50 years old, at least about 55 years old, at least about 60 years old, at least about 65 years old, at least about 70 years old, at least about 75 years old, at least about 80 years old, at least about 85 years old, at least about 90 years old, at least about 95 years old or at least about 100 years old.
  • a subject may be from about 15 years old to about 100 years old, from about 20 years old to about 100 years old, from about 30 years old to about 100 years old, from about 40 years old to about 100 years old, from about 50 years old to about 100 years old, from about 60 years old to about 100 years old, from about 70 years old to about 100 years old, or from about 80 years old to about 100 years old. In some cases, a subject may be between any of the above ages.
  • a subject may be from about 1 years old to about 12 years old, from about 2 years old to about 15 years old, from about 3 years old to about 20 years old, from about 4 years old to about 25 years old, from about 5 years old to about 30 years old, from about 6 years old to about 35 years old, from about 7 years old to about 40 years old, from about 8 years old to about 45 years old, from about 9 years old to about 50 years old, from about 10 years old to about 55 years old, from about 11 years old to about 60 years old, or from about 12 years old to about 70 years old, or any combination thereof.
  • the terms “amplifying” and “amplification” are used interchangeably and generally refer to generating one or more copies or “amplified product” of a nucleic acid.
  • the term “DNA amplification” generally refers to generating one or more copies of a DNA molecule or “amplified DNA product” .
  • the term “reverse transcription amplification” generally refers to the generation of deoxyribonucleic acid (DNA) from a ribonucleic acid (RNA) template via the action of a reverse transcriptase.
  • cycle threshold generally refers to the cycle during thermocycling in which an increase in a detectable signal due to amplified product reaches a statistically significant level above background signal.
  • incubating and “incubation” are used interchangeably and generally refer to keeping a sample, a mixture or a solution at certain temperature for a certain period of time, with or without shaking or stirring.
  • An “incubation temperature” generally refers to a temperature at which incubation is permitted to occur.
  • An “incubation time period” generally refers to an amount of time allotted for incubation to occur.
  • Denaturing and “denaturation” are used interchangeably and generally refer to the full or partial unwinding of the helical structure of a double-stranded nucleic acid, and in some cases the unwinding of the secondary structure of a single stranded nucleic acid.
  • Denaturation may include the inactivation of the cell wall (s) of a pathogen or the shell of a virus, and the inactivation of the protein (s) of inhibitors.
  • Conditions at which denaturation may occur include a "denaturation temperature” that generally refers to a temperature at which denaturation is permitted to occur and a “denaturation duration” that generally refers to an amount of time allotted for denaturation to occur.
  • elongation generally refers to the incorporation of nucleotides to a nucleic acid in a template directed fashion. Elongation may occur via the aid of an enzyme, such as, for example, a polymerase or reverse transcriptase. Conditions at which elongation may occur include an "elongation temperature” that generally refers to a temperature at which elongation is permitted to occur and an “elongation duration” that generally refers to an amount of time allotted for elongation to occur.
  • primer extension reaction generally refers to the denaturing of a double-stranded nucleic acid, binding of a primer to one or both strands of the denatured nucleic acid, followed by elongation of the primer (s) .
  • a template nucleic acid may be single –stranded (e.g., partially single-stranded) without denaturation, and a primer may bind to the single-stranded nucleic acid, followed by elongation of the primer (s) .
  • reaction mixture generally refers to a composition comprising reagents necessary to complete nucleic acid amplification (e.g., DNA amplification, RNA amplification) , with non-limiting examples of such reagents that include primer sets having specificity for target RNA or target DNA, DNA produced from reverse transcription of RNA, a DNA polymerase, a reverse transcriptase (e.g., for reverse transcription of RNA) , suitable buffers (including zwitterionic buffers) , co-factors (e.g., divalent and monovalent cations) , dNTPs, and other enzymes (e.g., uracil-DNA glycosylase (UNG) ) , etc. ) .
  • reaction mixtures can also comprise one or more reporter agents.
  • a “reporter agent” generally refers to a composition that yields a detectable signal, the presence or absence of which can be used to detect the presence of amplified product.
  • biological sample generally refers to any specimen taken by sampling from a living body or an environment.
  • the biological sample can be used for disease diagnosis and research.
  • Examples of biological sample can comprise feces, blood, urine, saliva, nasal mucus, plasma of a living body, as well as food, dust and soil of an environment.
  • microbe refers to a microscopic organism which may be single celled, multicellular, or non-cellular.
  • Non-limiting examples of microbes include bacteria, fungi, protozoa, and viruses.
  • bacteria and “bacteria” as used herein refer to prokaryotic organisms, including those that may not be traditionally considered as bacteria such as blue algae (cyanobacteria) , as well as those that may no longer been classified as eubacteria such as archaea (archaebacteria) .
  • cyanobacteria blue algae
  • eubacteria archaea
  • archaebacteria archaebacteria
  • major subdivisions within bacteria include Firmicutes, Proteobacteria, Actinobacteria, and non-Proteobacteria Gram-negative bacteria.
  • Non-limiting examples of bacterium include those of genera Aeromonas, Bacillus, Bacteroides, Bifidobacterium, Bordetella, Borrelia, Brucella, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Collinsella, Coprococcus, Corynebacterium, Enterobacter, Erwinia, Enterococcus, Escherichia, Faecalibacterium, Francisella, Haemophilus, Helicobacter, Lactobacillus, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Paracolon, Propionibacterium, Plesiomonas, Prevotella, Proteus, Pneumobacillus, Pseudomonas, Rickettsia, Roseburia, Ruminococcus, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Veillon
  • fungus and “fungi” generally refer to heterotrophic eukaryotes with a cell wall, including those organisms generally recognized as fungi.
  • major subdivisions within fungi include Ascomycota, Basidiomycota, Zygomycota, and Microsporidia.
  • fungus/fungi also encompasses Mesomycetozoa and Oomycota.
  • Non-limiting examples of fungus include those of genera Candida, Aspergillus, Cryptococcus, Hirsutellia, Histoplasma, Pneumocystis, Saccharomyces, and Stachybotrys.
  • protozoa generally refer to unicellular, heterotrophic eukaryotes that typically lack a cell wall, including those organisms generally recognized as protozoa.
  • protozoa include those of genera Trichomonas.
  • viruses generally refer to non-cellular infectious agents that replicate in living cells, including those infectious agents generally recognized as viruses.
  • Viruses include DNA viruses, RNA viruses, and reverse transcribing viruses.
  • Non-limiting examples of viruses include those of family Adenoviridae, Herpesviridae, Papillomaviridae, Polyomaviridae, Poxviridae, Hapadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronaviridae, Flaviviridae, Togaviridae, Hepeviridae, Retroviridae, Orthomyxoviridae, Arenaviridae, Bunyaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae, and Reoviridae, as well as Bacteriophages.
  • the viruses include adenovirus, norovirus, rotavirus,
  • pathogenic microbe refers to microbes whose presence or increase in quantity is associated with a disease, condition, disorder, or a symptom thereof, or other negative impacts to the health of a subject. Pathogenic microbes may be identified by taxonomic classification, and/or by the presence of genes the expression of which is associated with a disease, condition, disorder, or a symptom thereof, or other negative impacts on the health of a subject (also referred to as "pathogenic genes" ) . Due to an ability of microbes to transfer genetic information, a microbe having a beneficial taxonomic classification may be designated as pathogenic based on harboring a pathogenic gene.
  • beneficial microbe refers to microbes whose absence or decrease in quantity is associated with a disease, condition, disorder, or a symptom thereof, or other negative impacts to the health of a subject.
  • the beneficial microbes help maintain the normal functions of a subject.
  • the beneficial microbes help protect a subject from the implantation of or invasion by one or more pathogenic microbes.
  • the beneficial microbes check the growth of overgrowing pathogenic microbes in a subject.
  • Beneficial microbes include a variety of microbe types, genera, and species.
  • Beneficial microbes can include, without limitation, Escherichia coli, Paracolon, Enterobacter aerogenes, Proteus, Pseudomonas aeruginosa, pneumobacillus, Bacillus, Saccharomyces, Bacteroides, Bifidobacterium, Clostridium, Collinsella, Lactobacillus, Ruminococcus, Enterococcus, Hirsutellia, Lactobacillus, Dorea, Listeria, Streptococcus, Staphyloccocus, Corynebacterium, Propionibacterium, Clostridium butyricum, Streptococcus, Staphylococcus, Candida albicans, Fungus, Campylobacter, and Bacteriophages.
  • Pathogenic microbes include a variety of microbe types, genera, and species. Pathogenic microbes include, without limitation: bacteria, such as Aeromonas, Plesiomonas shigelloides, Campylobacter, Clostridium difficile, Toxin A/B, Escherichia coli O157, Enteroaggregative E. coli (EAEC) , Enteropathogenic E. coli (EPEC) , Enterotoxigenic E. coli (ETEC) LT/ST, Enteroinvasive E. coli (EIEC) , Shiga-like Toxin producing E.
  • bacteria such as Aeromonas, Plesiomonas shigelloides, Campylobacter, Clostridium difficile, Toxin A/B, Escherichia coli O157, Enteroaggregative E. coli (EAEC) , Enteropathogenic E. coli (EPEC) , Enterotoxigenic E. coli (ETEC) LT/ST, Enteroinvasive
  • coli stx1/stx2, Salmonella, Shigella, Vibrio cholera 5, and Yersinia enterocolitica
  • viruses such as Adenovirus 40/41, Norovirus GI/GII, Rotavirus A, Astrovirus, Sapovirus, herpes simplex virus, hepatovirus, HIV, and CMV
  • protozoa such as Cryptosporidium, Entamoeba histolytica, Giardia, and Cyclospora cayetanensis
  • fungi fungi.
  • the present disclosure provides methods of profiling a plurality of microbes.
  • the method comprises conducting a nucleic acid amplification assay to assess the relative or absolute quantity or concentration of at least one beneficial microbe and at least one pathogenic microbe in the plurality of microbes, thereby profiling the plurality of microbes in at least one biological sample.
  • the method further comprises determining the relative quantity or relative concentration of the at least one pathogenic microbe and the at least one beneficial microbe, thereby assessing susceptibility of a subject to an infectious condition associated with the at least one pathogenic microbe.
  • the method is used for assessing susceptibility of a subject to an infectious condition inflicted by a pathogenic microbe.
  • detecting a microbe or type of microbe comprises detecting a marker for the presence or absence of the microbe or type of microbe.
  • Markers include any detectable feature associated with the microbe to be detected, including proteins, antigens, and nucleic acid sequences.
  • Nucleic acid sequences useful as markers include sequences useful for taxonomic classifications (e.g., ribosomal RNA sequences) , and genes associated with particular microbes or microbes having a particular activity (e.g., so-called "pathogenic genes” , including genes associated with antibiotic resistance, and genes encoding microbial toxins) .
  • the present disclosure provides a method for processing a plurality of types of microbes in at least one biological sample of a subject.
  • the method may comprise (a) obtaining the at least one biological sample of the subject; (b) assaying the at least one biological sample obtained in (a) to assess a quantity or concentration of each of a plurality of types microbes in the at least one biological sample; and (c) determining whether the quantity or concentration for each of the plurality of types of microbes is above or below at least one threshold as compared to a reference (s) .
  • the method may further comprise (d) outputting a report that is indicative of whether the quantity or concentration for each of the plurality of types of microbes is above or below the at least one threshold as compared to the reference.
  • methods of the present disclosure comprise conducting a nucleic acid amplification reaction.
  • a nucleic acid amplification reaction can involve any of a variety of methods for nucleic acid amplification.
  • "amplification” refers to any process by which the copy number of a target sequence, or a complement thereof, is increased.
  • a variety of suitable nucleic acid amplification reactions are available.
  • Non-limiting examples of nuclei acid amplification reactions include polymerase chain reaction (PCR) , real-time polymerase chain reaction, isothermal amplification, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, solid phase amplification, nucleic acid sequence-based amplification (NASBA) , linear amplification, and digital PCR reaction.
  • PCR polymerase chain reaction
  • NASBA nucleic acid sequence-based amplification
  • digital PCR reaction digital PCR reaction
  • PCR polymerase chain reaction
  • RT-PCR reverse transcriptase (RT) is used to make a complementary DNA (cDNA) from RNA, and the cDNA is then amplified by PCR to produce multiple copies of DNA (see e.g., U.S. Pat. Nos. 5,322,770 and 5,310,652) .
  • the nucleic acid amplification reaction is a PCR reaction.
  • Conditions favorable to the amplification of target sequences by PCR can be determined, can be optimized at a variety of stages in the process, and depend on characteristics of elements in the reaction, such as target type, target concentration, sequence length to be amplified, sequence of the target and/or one or more primers, primer length, primer concentration, polymerase used, reaction volume, ratio of one or more elements to one or more other elements, and others, some or all of which can be altered.
  • PCR involves denaturation of the target to be amplified (if double stranded) , hybridization of one or more primers to the target, and extension of the primers by a DNA polymerase, and can be repeated (or "cycled” ) in order to amplify the target sequence.
  • Stages in this process can be optimized for various outcomes, such as to enhance yield, decrease the formation of spurious products, and/or increase or decrease specificity of primer annealing. Methods of optimization may include adjustments to the type or amount of elements in the amplification reaction and/or to the conditions of a given stage in the process, such as temperature at a particular stage, duration of a particular stage, and/or number of cycles.
  • an amplification reaction comprises at least 5, 10, 15, 20, 25, 30, 35, 50, or more cycles. In some embodiments, an amplification reaction comprises no more than 5, 10, 15, 20, 25, 35, 50, or more cycles.
  • Cycles can contain any number of stages, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more stages, and cycled stages may be preceded and/or followed by one or more stages not included in those stages that are cycled (e.g., an initial melting stage or an additional incubation) . Stages can comprise any temperature or gradient of temperatures, suitable for achieving the purpose of the given stage, including but not limited to, primer annealing, primer extension, and strand denaturation.
  • a stage can have a duration of greater than or equal to about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 180, 240, 300, 360, 420, 480, 540, 600 seconds, or more.
  • a stage can have a duration of less than or equal to about 600, 540, 480, 420, 360, 300, 240, 180, 120, 100, 90, 80, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 5, 1 second or less.
  • a stage may be continuous until interrupted (e.g., manually or automatically interrupted) . Cycles of any number comprising different stages can be combined in any order. In some embodiments, different cycles comprising different stages are combined such that the total number of cycles in the combination is about, less that about, or more than about 5, 10, 15, 20, 25, 30, 35, 50, or more cycles.
  • the nucleic acid amplification reaction comprises 3'-end extension of one or more primers, e.g., about, more than about, or less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primers.
  • primer extension in the nucleic acid amplification reaction involves only one pair of primers. In other embodiments, primer extension in the nucleic acid amplification reaction involves multiple pairs of primers, such as 2, 3, 4, 5, or more primer pairs.
  • a pair of primers comprises a first primer and a second primer, wherein the first primer comprises a sequence that is hybridizable to at least a portion of one or more target polynucleotides, and further wherein the second primer comprises a sequence that is hybridizable to at least a portion of the complement of a first primer extension product.
  • the sequence of the second primer that is hybridizable to at least a portion of the complement of a first primer extension product may also be hybridizable to at least a portion of the complementary strand of the target polynucleotide.
  • an amplification primer can be of any suitable length.
  • a length of the amplification primer can be less than or equal to about 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 or 10.
  • a length of the amplification primer can be greater than or equal to about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or 100.
  • any portion or all of the amplification primer may be complementary to the corresponding target sequence (e.g., about, less than about, or more than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides) .
  • a primer comprises a complementary portion and a non-complementary portion, the portion that is complementary to a target sequence is located at the 3'-end of the primer.
  • Primer pairs can be designed to amplify a target sequence of any length.
  • amplicon refers to the target sequence that is amplified from the target polynucleotide in the nucleic acid amplification reaction, in single-or double-stranded form.
  • the amplicon When an amplicon is amplified by a pair of primers, the amplicon is generally flanked by the pair of primers, such that one primer hybridizes at the 5' end of the target sequence and the other primer hybridizes to the complement of the 3' end of the target sequence.
  • the amplicon can be less than or equal to about 1000, 900, 800, 700, 600, 500, 400, 300, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30 or 25 nucleotides in length.
  • the amplicon can be more than or equal to about 50, 100, 200, 300, 400, 500, 750 or 1000 nucleotides in length.
  • amplicon length is between any two of these endpoints, such as 25-1000, 30-500, 50-400, 50-250, 50-150, or 100-200 nucleotides in length.
  • Primers may be selected based on conformance to any of a variety of design considerations, which may be used alone or in combination with any other design consideration disclosed herein.
  • primers include: avoiding runs of the same nucleotide (e.g., 3, 4, 5, or more of the same nucleotide in a row) ; proximity to, without overlapping, probe hybridization site (e.g., less than or equal to about 100, 75, 50, 40, 30, 25, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides between the 3' end of a primer and the 5' end of a probe along the same strand) ; G-C content within about 20%-80%; melting temperature (T m ) within a selected range (e.g., about 55-65°C, 58-62°C, or 58-60°C) ; having no more than two G and/or C bases within the last five nucleotides at the 3' end; primers in a pair having similar T m (e.g., the same T m , or T m 's within about 1-2°C of each other) ; minimal secondary structure (
  • primers specifically amplify amplicons that are at least about 25, 50, 75, 100, 125, 150, or 175 nucleotides in length.
  • Primer extension in a nucleic acid amplification reaction can be carried out by any suitable polymerase, such as a DNA polymerase, many of which are commercially available.
  • DNA polymerases can comprise DNA-dependent DNA polymerase activity, RNA-dependent DNA polymerase activity, or DNA-dependent and RNA-dependent DNA polymerase activity.
  • DNA polymerases can be thermostable or non-thermostable.
  • DNA polymerases include, but are not limited to, Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerases, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Klenow fragment, and variants, modified products and derivatives thereof.
  • enzymes produced using bacteria are highly purified
  • nucleic acid amplification products are detected during and/or at the completion of the amplification process.
  • Amplification product detection can be conducted in real time in an amplification assay.
  • the amplified products can be directly visualized with fluorescent DNA-binding agents including but not limited to DNA intercalators and DNA groove binders. Because the amount of the intercalators incorporated into the double-stranded DNA molecules is typically proportional to the amount of the amplified DNA products, one can conveniently determine the amount of the amplified products by quantifying the fluorescence of the intercalated dye using conventional optical systems.
  • Non-limiting examples of DNA-binding dyes include green, SYBR blue, DAPI, propidium iodine, Hoechst, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, and the like.
  • sequence specific oligonucleotide probes are employed in the nucleic acid amplification reaction to facilitate the detection and/or quantification of the amplified products.
  • Probe-based quantitative amplification relies on the sequence-specific detection of an amplified product, such as by specific hybridization between a probe and a target sequence within an amplification product. Examples of target-specific probes include, without limitation, probes and molecular beacons. Generic methods for performing probe-based quantitative amplification may be used (see e.g., U.S. Pat. No. 5,210,015) . Hybridization can be performed under various stringencies.
  • Suitable hybridization conditions are generally such that the recognition interaction between the probe and target polynucleotide is both sufficiently specific and sufficiently stable as to provide preferential hybridization between an oligonucleotide probe and/or primer and the intended target sequence.
  • Conditions that increase the stringency of a hybridization reaction may be used and may include optimization of annealing temperature and/or salt concentration.
  • An oligonucleotide probe can be of any suitable length, such as greater than or equal to about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90 or 100 nucleotides.
  • the oligonucleotide prove can have a length that is less than or equal to about 100, 90, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 10 nucleotides, or less.
  • a portion or all of the oligonucleotide probe may be complementary to the corresponding target sequence (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides, less than about 50, 45, 40, 35, 30, 25, 20, 15, 10 or 5 nucleotides, or more than about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides) .
  • a plurality of probes is used in a single nucleic acid amplification reaction.
  • a number of the probes can be greater than or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25.
  • the number of probes can be less than or equal to about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3 or 2.
  • a single nucleic acid amplification reaction contains only one probe, such as a probe that specifically hybridizes to a sequence that is identical among a plurality of different microbes (e.g., more than or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40 or 50 microbes) and/or identical among microbes from a plurality of different genera (e.g., more than or equal to about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40 or 50 genera) . Probes may be selected based on conformance to any of a variety of design considerations, which may be used alone or in combination with any other design consideration disclosed herein.
  • a probe specifically hybridizes to amplicons that are at least about 25, 50, 75, 100, 125, 150, or 175 nucleotides in length.
  • nucleotide probes can be conjugated to a detectable label.
  • Suitable detectable labels can include any composition detectable by photochemical, biochemical, spectroscopic, immunochemical, electrical, optical, or chemical approaches.
  • a wide variety of appropriate detectable labels may be used, which include fluorescent labels, chemiluminescent labels, radioactive isotope labels, enzymatic labels, and ligands.
  • the detection methods used to detect or quantify the hybridization intensity will typically depend upon the label selected above. For example, radiolabels may be detected using photographic film or a phosphoimager. Fluorescent markers may be detected and quantified using a photodetector to detect emitted light.
  • each of a plurality of probes in a single reaction is conjugated to a different detectable label (e.g., fluorescent dyes with different emission spectra) , such that signal corresponding to amplification of different targets can be differentiated.
  • detectable label e.g., fluorescent dyes with different emission spectra
  • Enzymatic labels can be detected by providing the enzyme with a substrate and measuring the reaction product produced by the action of the enzyme on the substrate; and colorimetric labels can be detected by simply visualizing the colored label.
  • hybridization of a bound probe is detected using a TaqMan assay (PE Biosystems, Foster City, Calif.; See e.g., U.S. Pat. Nos. 5,962,233 and 5,538,848, each of which is herein incorporated by reference) .
  • the assay is performed during a PCR reaction.
  • the TaqMan assay exploits the 5'-3' exonuclease activity of DNA polymerases such as AMPLITAQ DNA polymerase.
  • a sequence-specific probe is included in the PCR reaction.
  • a typical TaqMan probe is an oligonucleotide with a 5'-reporter dye (e.g., a fluorescent dye) and a 3'-quencher dye.
  • the 5'-3' nucleolytic activity of the AMPLITAQ polymerase cleaves the probe between the reporter and the quencher dye.
  • the separation of the reporter dye from the quencher dye results in an increase of fluorescence.
  • the signal accumulates with each cycle of PCR and can be monitored with a fluorimeter.
  • a variety of reporter-quencher pairs may be used. Some pairs interact through fluorescence resonance energy transfer (FRET) .
  • Molecules commonly used in FRET as reporters or quenchers include, but are not limited to, fluorescein dyes (e.g., FAM, JOE, and HEX) , rhodamine dyes (e.g., R6G, TAMRA, ROX) , cyanine dyes (e.g., Cy3, Cy3.5, Cy5, Cy5.5, and Cy7) , DABCYL, and EDANS. Whether a fluorescent dye acts as a reporter or a quencher is defined by its excitation and emission spectra, and by the fluorescent dye with which it is paired.
  • fluorescein dyes e.g., FAM, JOE, and HEX
  • rhodamine dyes e.g., R6G, TAMRA, ROX
  • cyanine dyes e.g., Cy3, Cy3.5, Cy5, Cy5.5, and Cy7
  • DABCYL DABCYL
  • EDANS Whether a fluorescent dye acts as a
  • FAM is most efficiently excited by light with a wavelength of 488 nm and emits light with a spectrum of 500 to 650 nm, and an emission maximum of 525 nm.
  • FAM is a suitable reporter label for use with, e.g., TAMRA as a quencher, which has its excitation maximum at 514 nm.
  • TAMRA fluorescent indicator
  • non-fluorescent or dark quenchers that dissipate energy absorbed from a fluorescent dye include the Black Hole Quenchers TM marketed by Biosearch Technologies, Inc, (Novato, Calif., USA) .
  • the Black Hole Quenchers TM are structures comprising at least three radicals selected from substituted or unsubstituted aryl or heteroaryl compounds, or combinations thereof, wherein at least two of the residues are linked via an exocyclic diazo bond (see, e.g., International Publication No. WO2001086001) .
  • Other dark quenchers include Iowa Black quenchers (e.g., Iowa Black FQ TM and Iowa Black RQ TM ) , Dark Quenchers (Epoch Biosciences, Inc, Bothell, Wash. ) , and Zen TM quenchers (Integrated DNA Technologies, Inc.; Coralville, IA) . Additional non-limiting examples of quenchers are also provided in U.S. Pat. No. 6,465,175.
  • hybridization of a bound probe is detected using a molecular beacon oligonucleotide probe, such as described in U.S. Pat. No. 5,925,517, PCT Application No. WO1995013399, and U.S. Pat. No. 6,150,097.
  • a central target-recognition sequence is flanked by arms that hybridize to one another when the probe is not hybridized to a target strand, forming a hairpin structure, in which the target-recognition sequence is in the single-stranded loop of the hairpin structure, and the arm sequences form a double-stranded stem hybrid.
  • a FRET pair such as the fluorophore EDANS and the quencher DABCYL (or other pairs described herein) , may be attached to the arms by alkyl spacers.
  • the molecular beacon is not hybridized to a target strand, the fluorophore's emission is quenched.
  • the FRET pair is separated, and the fluorophore's emission is not quenched. Emitted fluorescence signals the presence of target strands. Signal can be detected during a nucleic acid amplification reaction, such as with a fluorimeter at the end of each cycle in a PCR reaction. Signal intensity increases with an increasing amount of target sequence.
  • a Scorpion Primer carries a 5' extension comprising a probe element, a pair of self-complimentary stem sequences, and a fluorophore/quencher pair. The extension is "protected” from being copied by the inclusion of a blocking hexethylene glycol (HEG) monomer.
  • HOG hexethylene glycol
  • primers and probes are selected to maximize sensitivity of target polynucleotide detection.
  • sensitivity is measured in terms of cycle threshold (C T ) value.
  • C T cycle threshold
  • a fixed fluorescence threshold can be set above the baseline.
  • the parameter C T represents the cycle number at which the fluorescence passes the fixed threshold, typically an intensity that is statistically significant above the baseline or background and in the log-linear phase of amplification.
  • RNA for calculating the threshold level of fluorescence in a given reaction or set of reactions are typically included in real-time PCR analysis software packages.
  • One common method for setting the threshold is determining the baseline (background) average signal and setting a threshold 10-fold higher than the baseline average signal.
  • a threshold value may be set at about 10 times the standard deviation of baseline emission.
  • a plot of the log of initial target copy number for a set of standards versus C T is typically a straight line. Quantification of the amount of target in unknown samples is accomplished by measuring C T and using the standard curve to determine starting copy number.
  • detection has a linear range of detection over about or more than about 3, 4, 5, 6, 7, 8, or more logs.
  • amplification of about or less than about 10pg, 5pg, 4pg, 3pg, 2pg, 1pg, 0.5pg, 0.1pg, or range between any of these (e.g., 0.5-4pg, 1pg-5pg, 1pg-3pg, etc. ) of genomic DNA from any one of the bacterial species detectable by a probe in the amplification reaction has a C T of less than 30.
  • amplification of about or less than about 15000, 10000, 5000, 2500, 1500, 1000, 500, 200, 100, 50, or fewer starting copies of a target sequence detectable by a probe in the amplification reaction has a C T of less than 30.
  • amplification of about 1pg of genomic DNA from any one of the bacterial species detectable by a probe in the amplification reaction has a C T of less than or equal to about 30, 29, 28, 27, 26, 25, or lower.
  • the C T for a negative control sample is at least 2 cycles (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles) higher than the C T for a sample containing about 100pg, 10pg, 5pg, 4pg, 3pg, 2pg, 1pg, 0.5pg, 0.1pg, or range between any of these (e.g., 0.5-4pg, 1pg-5pg, 1pg-3pg, 5pg-10pg, etc.
  • a negative control is an amplification reaction that has all reaction reagents, but no template is added (e.g., add water instead of template, or polynucleotides known to lack a target amplicon, such as human genomic DNA in the case of a bacteria-specific amplicon) .
  • primer pairs are immobilized on a solid support.
  • solid supports include, but are not limited to, inorganic materials such as silica based substrates (e.g., glass, quartz, fused silica, silicon, or the like) , other semiconductor materials, and organic materials such as polymer materials (e.g., polymethyl methacrylate, polyethylene, polypropylene, polystyrene, cellulose, agarose, or any of a variety of organic substrate materials conventionally used as supports for reactive media) .
  • inorganic materials such as silica based substrates (e.g., glass, quartz, fused silica, silicon, or the like)
  • organic materials such as polymer materials (e.g., polymethyl methacrylate, polyethylene, polypropylene, polystyrene, cellulose, agarose, or any of a variety of organic substrate materials conventionally used as supports for reactive media) .
  • solid support structures may be in any of a variety of physical configurations, including but not limited to microparticles, beads, nanoparticles, nanocrystals, fibers, microfibers, nanofibers, nanowires, nanotubes, mats, planar sheets, planar wafers or slides, multiwell plates, optical slides including additional structures, capillaries, microfluidic channels, and the like.
  • amplification on a solid support comprises bridge amplification. General methods of bridge amplification may be used. See for example WO1998/044151 and WO2000/018957, which are herein entirely incorporated by reference.
  • a target sequence amplified in the nucleic acid amplification reaction is the sequence of a portion of a conserved bacterial polynucleotide.
  • an amplified portion of a conserved polynucleotide exhibits more than or equal to about 80%, 85%, 90%, 95%or 97.5%homology across different bacterial genera.
  • conserved polynucleotide sequences include, but are not limited to, nucleotide sequences found in the 16S rRNA gene, 23S rRNA gene, 5S rRNA gene, 5.8S rRNA gene, 12S rRNA gene, 18S rRNA gene, 28S rRNA gene, gyrB gene, rpoB gene, fusA gene, recA gene, cox1 gene and nifD gene.
  • the conserved polynucleotide is a portion of a 16S rRNA polynucleotide (e.g., rRNA, rDNA, amplification product, or combination of these) .
  • an amplicon is at least about 25, 50, 75, 100, 125, 150 or 175 nucleotides in length.
  • primer sequences capable of specific hybridization to regions conserved amongst at least 5, 10, 15 or 20 different bacterial genomes.
  • a forward and a reverse primer each hybridizes to a separate region that is conserved amongst at least 5, 10, 15 or 20 different bacterial genomes are selected.
  • These conserved regions include but not limited to from 9 to 28, from 32 to 48, from 522 to 545, from 888 to 903, from 916 to 937, from 939 to 973, from 975 to 994, from 957 to 981, from 1093 to 1125, from 1184 to 1206, from 1231 to 1252, from 1378 to 1396, from 1398 to 1422, or from 1496 to 1516 of 16S rRNA of Staphylococcus aureus (GenBank accession Number NC_007622) or the corresponding regions in any one of the bacterial genomes: Staphylococcus aureus Mu3; Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus pyogenes, Streptococcus pneumonia, Escherichia coli, Citrobacter koseri, Clostridium perfringens, Enterococcus faecalis, Klebsiella pneumonia, Lactobacillus acidophilus
  • the subject primers are capable of specific hybridization to conserved regions of at least 5, 10, 15 or 20 different bacterial genomes, and hence allow specific amplification and detection of any type of the at least 5, 10, 15 or 20 different bacterial genomes.
  • the design of such primers and sets thereof allows for simultaneous determination of bacterial infection across a wide set of bacterial strains.
  • detection occurs in a single amplification reaction with a single pair of primers, and optionally with one or more optional primers to provide additional bacterial coverage.
  • These primers and sets thereof can be used in conjunction with probes disclosed herein or other labeling molecules such as DNA-binding dyes (e.g., Green) and the like.
  • Amplification methods may involve changes in temperature (such as in heat denaturation) or may be isothermal processes that do not include heat denaturation.
  • An example of an isothermal amplification method is strand displacement amplification, commonly referred to as SDA, which uses cycles of annealing pairs of primer sequences to opposite strands of a target sequence, primer extension in the presence of a dNTP to produce a duplex hemiphosphorothioated primer extension product, endonuclease-mediated nicking of a hemi-modified restriction endonuclease recognition site, and polymerase-mediated primer extension from the 3'-end of the nick to displace an existing strand and produce a strand for the next round of primer annealing, nicking, and strand displacement, resulting in geometric amplification of product (see e.g., U.S. Pat. No.
  • Thermophilic SDA uses thermophilic endonucleases and polymerases at higher temperatures in essentially the same method (see e.g., European Pat. No. 0684315) .
  • SDA may be described as follows.
  • a single stranded target nucleic acid is contacted with an SDA primer.
  • An "SDA primer” generally has a length of 25-100 nucleotides, and in some cases has a length of approximately 35 nucleotides.
  • An SDA primer is substantially complementary to a region at the 3' end of the target sequence, and the primer has a sequence at its 5' end (outside of the region that is complementary to the target) that is a recognition sequence for a restriction endonuclease, sometimes referred to as a "nicking enzyme” or a "nicking endonuclease.
  • the SDA primer then hybridizes to the target sequence.
  • the SDA reaction mixture also contains a polymerase (an "SDA polymerase” ) and a mixture of all four deoxynucleoside-triphosphates (also called deoxynucleotides or dNTPs, i.e. dATP, dTTP, dCTP and dGTP, commonly used in primer extension reactions) , at least one species of which is a substituted or modified dNTP; thus, the SDA primer is extended, to form a modified primer, sometimes referred to as a "newly synthesized strand. " The substituted dNTP is modified such that it will inhibit cleavage in the strand containing the substituted dNTP but will not inhibit cleavage on the other strand.
  • Suitable substituted dNTPs include, but are not limited, 2' deoxyadenosine 5'-O- (1-thiotriphosphate) , 5-methyldeoxycytidine 5'-triphosphate, 2'-deoxyuridine 5'-triphosphate, and 7-deaza-2'-deoxyguanosine 5'-triphosphate.
  • the substitution of the dNTP may occur after incorporation into a newly synthesized strand; for example, a methylase may be used to add methyl groups to the synthesized strand.
  • the polymerase may have 5' ⁇ 3' exonuclease activity.
  • the polymerase may lack 5' ⁇ 3' exonuclease activity.
  • the recognition site/endonuclease pair can be any of a wide variety of combinations.
  • the endonuclease is chosen to cleave a strand either at the recognition site, or either 3' or 5' to it, without cleaving the complementary sequence, either because the enzyme only cleaves one strand or because of the incorporation of the substituted nucleotides.
  • Suitable recognition site/endonuclease pairs may be used, including but not limited to HincII, HindII, AvaI, Fnu4HI, TthIIII, NcII, BstXI, BamHI, etc.
  • a chart depicting suitable enzymes, and their corresponding recognition sites and the modified dNTP to use is found in U.S. Pat. No. 5,455,166, hereby incorporated by reference.
  • the polymerase chosen can initiate 5' ⁇ 3' polymerization at a nick site, can also displace the polymerized strand downstream from the nick, and can lack 5' ⁇ 3' exonuclease activity (this may be additionally accomplished by the addition of a blocking agent) .
  • Suitable polymerases in SDA include, but are not limited to, the Klenow fragment of DNA polymerase I, SEQUENASE 1.0 and SEQUENASE 2.0 (U.S. Biochemical) , T5 DNA polymerase and Phi29 DNA polymerase.
  • SDA does not require thermocycling. The temperature of the reaction is generally set to be high enough to prevent non-specific hybridization but low enough to allow specific hybridization; this is typically from about 37°C.
  • a second primer extension reaction can be done using the complementary target sequence, resulting in a substantial increase in amplification during a set period of time. That is, a second primer nucleic acid is hybridized to a second target sequence, that is substantially complementary to the first target sequence, to form a second hybridization complex. The addition of the enzyme, followed by disassociation of the second hybridization complex, results in the generation of a number of newly synthesized second strands. Accordingly, amplification may be linear or non-linear (e.g., exponential) .
  • the method includes hybridizing chimeric RNA/DNA amplification primers to the probes or target.
  • the DNA portion of the probe can be 3' to the RNA.
  • the method includes hybridizing a polynucleotide comprising a termination polynucleotide sequence to a region of the template that is 5' with respect to hybridization of the composite primer to the template. Following hybridization of the primer to the template, the primer is extended with DNA polymerase.
  • RNA is cleaved from the composite primer with an enzyme that cleaves RNA from an RNA/DNA hybrid.
  • an additional RNA/DNA chimeric primer is hybridized to the template such that the first extended primer is displaced from the target probe.
  • the extension reaction is repeated, whereby multiple copies of the probe sequence are generated.
  • the amplification reaction can proceed linearly.
  • a reverse SPIA primer with complementarity to the first primer extension product is also used, the amplification reaction is non-linear.
  • amplification methods include rolling circle amplification (RCA) (e.g., Lizardi, “Rolling Circle Replication Reporter Systems, " U.S. Pat. No. 5,854,033) ; helicase dependent amplification (HDA) (e.g., U.S. Pat. Appl. US 20040058378) , and loop-mediated isothermal amplification (LAMP) (e.g., Notomi et al., "Process for Synthesizing Nucleic Acid, " U.S. Pat. No. 6,410,278) .
  • RCA rolling circle amplification
  • HDA helicase dependent amplification
  • LAMP loop-mediated isothermal amplification
  • nucleic acid amplification reactions include transcription-based amplification methods such as nucleic acid sequence-based amplification, also referred to as NASBA (e.g., Malek et al., U.S. Pat. No. 5,130,238) ; methods which rely on the use of an RNA replicase to amplify the probe molecule itself, commonly referred to as replicase (e.g., Lizardi, P.et al. (1988) BioTechnol. 6, 1197-1202) ; and self-sustained sequence replication (e.g., Guatelli, J. et al. (1990) Proc. Natl. Acad. Sci.
  • NASBA nucleic acid sequence-based amplification
  • TMA transcription- mediated amplification
  • nucleic acid amplification methods include ligase chain reaction (see e.g., U.S. Pat. Nos. 5,494,810 and 5,830,711) , and solid-phase amplification methods (e.g., bridge amplification with primers attached to a solid surface, such as a slide or a bead; see e.g., U.S. Pat. Nos. 5,641,658 and 7,985,565) .
  • NASBA is generally described in U.S. Pat. No. 5,409,818; Sooknanan et al., Nucleic Acid Sequence-Based Amplification, Ch. 12 (pp. 261-285) of Molecular Methods for Virus Detection, Academic Press, 1995; and "Profiting from Gene-based Diagnostics” , CTB International Publishing Inc., N.J., 1996, all of which are incorporated by reference.
  • NASBA is very similar to both TMA and QBR.
  • Transcription mediated amplification (TMA) is generally described in U.S. Pat. Nos. 5,399,491, 5,888,779, 5,705,365, 5,710,029, all of which are incorporated by reference.
  • NASBA utilizes the addition of RNAse H to effect RNA degradation
  • TMA relies on inherent RNAse H activity of a reverse transcriptase.
  • these techniques may be described as follows.
  • a single stranded target nucleic acid usually an RNA target sequence (sometimes referred to as “the first target sequence” or “the first template” )
  • a first primer generally referred to herein as a "NASBA primer” (although "TMA primer” is also suitable) .
  • NASBA primer a first primer
  • TMA primer is also suitable
  • the first primer can be a DNA primer that has at its 3' end a sequence that is substantially complementary to the 3' end of the first template.
  • the first primer also has an RNA polymerase promoter at its 5' end (or its complement (antisense) , depending on the configuration of the system) .
  • the first primer is then hybridized to the first template to form a first hybridization complex.
  • the reaction mixture also includes a reverse transcriptase enzyme (a "NASBA reverse transcriptase" ) and a mixture of the four dNTPs, such that the first NASBA primer is extended, to form a modified first primer, comprising a hybridization complex of RNA (the first template) and DNA (the newly synthesized strand) .
  • NASBA reverse transcriptase a reverse transcriptase enzyme
  • RNA-directed DNA polymerase an enzyme capable of synthesizing DNA from a DNA primer and an RNA template.
  • Suitable RNA-directed DNA polymerases include, but are not limited to, avian myeloblastosis virus reverse transcriptase ( "AMV RT” ) and the Moloney murine leukemia virus RT.
  • AMV RT avian myeloblastosis virus reverse transcriptase
  • the reverse transcriptase enzyme can further comprise RNA degrading activity.
  • the NASBA reaction also includes an RNA degrading enzyme, also sometimes referred to herein as a ribonuclease, that will hydrolyze RNA of a RNA: DNA hybrid without hydrolyzing single-or double-stranded RNA or DNA.
  • RNA degrading enzyme also sometimes referred to herein as a ribonuclease
  • Suitable ribonucleases include, but are not limited to, RNase H from E. coli and calf thymus.
  • the ribonuclease activity degrades the first RNA template in the hybridization complex, resulting in a disassociation of the hybridization complex leaving a first single stranded newly synthesized DNA strand, sometimes referred to as "the second template.
  • the NASBA reaction also includes a second NASBA primer, generally comprising DNA (although as for all the probes and primers herein, nucleic acid analogs may also be used) .
  • This second NASBA primer has a sequence at its 3' end that is substantially complementary to the 3' end of the second template, and also contains an antisense sequence for a functional promoter and the antisense sequence of a transcription initiation site.
  • this primer sequence when used as a template for synthesis of the third DNA template, contains sufficient information to allow specific and efficient binding of an RNA polymerase and initiation of transcription at the target site.
  • the antisense promoter and transcription initiation site can be that of the T7 RNA polymerase, although other RNA polymerase promoters and initiation sites can be used as well.
  • the second primer hybridizes to the second template, and a DNA polymerase, also termed a "DNA-directed DNA polymerase, " also present in the reaction, synthesizes a third template (a second newly synthesized DNA strand) , resulting in second hybridization complex comprising two newly synthesized DNA strands.
  • RNA polymerase ribonucleoside triphosphates
  • NTPs ribonucleotides
  • RNA polymerases include, but are not limited to, T7 RNA polymerase, and other bacteriophage RNA polymerases including those of phage T3, phage Salmonella phage sp6, or Pseudomonas phage gh-1.
  • TMA and NASBA are used with starting DNA target sequences, a first primer comprising the RNA polymerase promoter, and a DNA polymerase enzyme to generate a double stranded DNA hybrid with the newly synthesized strand comprising the promoter sequence. The hybrid can then be denatured, and the second primer can be added.
  • the confronting ends of the probe elements can be joined by ligation, e.g., by treatment with ligase.
  • the ligated probe element is then assayed, evidencing the presence of the target sequence.
  • the ligated probe elements act as a template for a pair of complementary probe elements.
  • the target sequence is amplified linearly, allowing very small amounts of target sequence to be detected and/or amplified. This approach is an example of a ligase detection reaction.
  • ligase chain reaction which achieves exponential amplification of target sequences. See e.g., F. Barany, Proc. Nat'l Acad. Sci. USA, 88: 189-93 (1991) and F. Barany, PCR Methods and Applications, 1: 5-16 (1991) .
  • Jou, et al., Human Mutation 5: 86-93 (1995) relates to the use of a so called "gap ligase chain reaction" process to amplify simultaneously selected regions of multiple exons with the amplified products being read on an immunochromatographic strip having antibodies specific to the different haptens on the probes for each exon.
  • nucleic amplification comprises digital PCR.
  • features or embodiments for a digital PCR can be found in PCT Patent Publication Nos. WO/2010/036352 and WO/2014/186440; U.S. Patent Publication Nos. 2010/0173394, 2011/0053798, 2011/0086780, 2011/0092373, 2011/0092376, 2011/0092392, 2011/0159499, 2011/0177586, 2012/0028311, 2013/0302792 and 2014/0200164; and U.S. Patent Nos.
  • a biological sample may be solid matter (e.g., biological tissue) or may be a fluid (e.g., a biological fluid) .
  • a biological fluid can include any fluid associated with living organisms.
  • Non-limiting examples of a biological sample include blood (or components of blood –e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location (e.g., tissue, circulatory system, bone marrow) of a subject, cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, breath, bone marrow, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, breast, pancreas, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, cavity fluids, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluid, tears, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, skin cells, plasma,
  • the biological sample is a tissue sample selected from the group consisting of thyroid, lymph, skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, prostate, reproductive organ, urogenital, anus, and esophageal tissues.
  • the biological sample is a biological fluid selected from the group consisting of blood, serum, bone marrow aspirate, synovial fluid, cerebrospinal fluid, saliva, sweat, tears, sputum, mucus, breast milk, urine, lymphatic fluid, amniotic fluid, placental fluid, urogenital fluid, anal fluid, and effusions.
  • nucleic acids may be derived from a variety of sample sources. Nucleic acids may optionally, but not necessarily, be isolated and/or purified before further manipulation, such as in a nucleic acid amplification reaction. For example, a biological sample may be subjected to a polymerase chain reaction (PCR) procedure without separate extraction, such that cell-free nucleic acids are amplified from an unpurified sample. As a further example, a sample may be subjected to cell lysis conditions, either immediately before or during a nucleic acid amplification reaction, without purifying the nucleic acids away from other cellular components.
  • PCR polymerase chain reaction
  • nucleic acids e.g., DNA, RNA, or both
  • a biological sample before subjecting the nucleic acid to an amplification reaction.
  • nucleic acid purification may be used and may vary with the type of biological sample.
  • biological samples may include tissue and/or fluid from a subject.
  • a biological fluid includes any treated or untreated fluid associated with living organisms, including, but not limited to, blood, including whole blood, warm or cold blood, and stored or fresh blood; treated blood, such as blood diluted with at least one physiological solution, including but not limited to saline, nutrient, and/or anticoagulant solutions; blood components, such as platelet concentrate (PC) , platelet-rich plasma (PRP) , platelet-poor plasma (PPP) , platelet-free plasma, plasma, fresh frozen plasma (FFP) , components obtained from plasma, packed red cells (PRC) , transition zone material or buffy coat (BC) ; analogous blood products derived from blood or a blood component or derived from bone marrow; red cells separated from plasma and resuspended in physiological fluid or a cryoprotective fluid; and platelets separated from plasma and resuspended in physiological fluid or a cryoprotective fluid.
  • PC platelet concentrate
  • PRP platelet-rich plasma
  • PPP platelet-poor plasma
  • biological samples include skin, heart, lung, kidney, bone marrow, breast, pancreas, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluid, tears, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, skin cells, plasma, nasal swab or nasopharyngeal wash, spinal fluid, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, cord blood, emphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and/or other excretions or body tissues.
  • the sample to be tested is whole blood. In some embodiments, the sample to be tested is buffy coat.
  • the tissue analyzed is a portion of a tissue to be transplanted or surgically grafted, such as an organ (e.g., heart, kidney, liver, lung, etc. ) , skin, bone, nervous tissue, tendons, blood vessels, fat, cornea, blood, or a blood component.
  • the sample is from a subject, such as a mammal, including but not limited to murines, simians, humans, farm animals, sport animals, or pets.
  • the biological sample is a stool sample.
  • the stool sample may be a solid stool sample or a liquid stool sample.
  • the liquid stool sample may be a watery diarrhea stool.
  • the stool sample may be obtained by any suitable approach, such as a swab.
  • Nucleic acids may be extracted from a biological sample using any suitable method, which may not include extraction of nucleic acids from cells. When no extraction is included, typically the nucleic acids amplified will be cell-free nucleic acids. When extraction is included, for example, nucleic acids can be purified by organic extraction with phenol, phenol/chloroform/isoamyl alcohol, or similar formulations, including TRIzol and TriReagent.
  • extraction techniques include: (1) organic extraction followed by ethanol precipitation, e.g., using a phenol/chloroform organic reagent (Ausubel et al., 1993) , with or without the use of an automated nucleic acid extractor, e.g., the Model 341 DNA Extractor available from Applied Biosystems (Foster City, Calif. ) ; (2) stationary phase adsorption methods (U.S. Pat. No. 5,234,809; Walsh et al., 1991) ; and (3) salt-induced nucleic acid precipitation methods (Miller et al., (1988) , such precipitation methods being typically referred to as "salting-out" methods.
  • an automated nucleic acid extractor e.g., the Model 341 DNA Extractor available from Applied Biosystems (Foster City, Calif. )
  • stationary phase adsorption methods U.S. Pat. No. 5,234,809; Walsh et al., 1991
  • nucleic acid isolation and/or purification includes the use of magnetic particles (e.g., beads) to which nucleic acids can specifically or non-specifically bind, followed by isolation of the particles using a magnet, and washing and eluting the nucleic acids from the particles (see e.g., U.S. Pat. No. 5,705,628) .
  • the above isolation methods may be preceded by enzyme digestion to help eliminate unwanted protein from the sample, e.g., digestion with proteinase K, or other like proteases. See, e.g., U.S. Pat. No. 7,001,724. If some cases, RNase inhibitors may be added to the lysis buffer.
  • protein denaturation/digestion can be added to the protocol.
  • Purification methods may be directed to isolate DNA, RNA (including but not limited to mRNA, rRNA, tRNA) , or both. When both DNA and RNA are isolated together during or subsequent to an extraction procedure, further operations may be employed to purify one or both separately from the other. Sub-fractions of extracted nucleic acids can also be generated, for example, purification by size, sequence, or other physical or chemical characteristic. In addition to initial nucleic acid isolation, purification of nucleic acids can be performed after subsequent manipulation, such as to remove excess or unwanted reagents, reactants, or products.
  • Polynucleotides from a sample may be fragmented prior to further processing. Fragmentation may be accomplished by any of a variety of methods, including chemical, enzymatic, and mechanical fragmentation.
  • the fragments have an average or median length from about 10 to about 1,000 nucleotides in length, such as between10-800, 10-500, 50-500, 90-200, or 50-150 nucleotides.
  • the fragments have an average or median length of less than or equal to about 1500, 1000, 800, 600, 500, 300, 200 or 100 nucleotides.
  • the fragments range from about 90-200 nucleotides, and/or have an average length of about 150 nucleotides.
  • the fragmentation is accomplished mechanically comprising subjecting sample polynucleotides to acoustic sonication.
  • the fragmentation comprises treating the sample polynucleotides with one or more enzymes under conditions suitable for the one or more enzymes to generate double-stranded nucleic acid breaks.
  • enzymes useful in the generation of polynucleotide fragments include sequence specific and non-sequence specific nucleases.
  • nucleases include DNase I, Fragmentase, restriction endonucleases, variants thereof, and combinations thereof. For example, digestion with DNase I can induce random double-stranded breaks in DNA in the absence of Mg++ and in the presence of Mn++.
  • fragmentation comprises treating the sample polynucleotides with one or more restriction endonucleases. Fragmentation can produce fragments having 5' overhangs, 3' overhangs, blunt ends, or a combination thereof. In some embodiments, such as when fragmentation comprises the use of one or more restriction endonucleases, cleavage of sample polynucleotides leaves overhangs having a predictable sequence. Fragmented polynucleotides may be subjected to size selecting the fragments via standard methods such as column purification or isolation from an agarose gel.
  • the microbes as described herein may be classified into two categories.
  • the first category of microbes also referred to as “first microbe (s) " herein, is identified to be beneficial with respect to health of a subject.
  • the second category of microbes also referred to as “second microbe (s) " herein, is identified to be harmful with respect to health of a subject.
  • the first microbe may be a probiotic.
  • a probiotic is a microbe that can contribute to any beneficial effect on the subject when introduced into the subject.
  • the first microbe may not be usually identified as a probiotic, but presence or abundance of it may be associated with any beneficial effect on the subject.
  • association it not only includes the circumstances where the presence or abundance of the first microbe causes the beneficial effect on the subject, but also includes the circumstances where the presence or abundance of the first microbe is simply the result of the beneficial effect, or the beneficial effect and the presence or abundance of the first microbe are not directly causal to each other, but are both the results of an underlying cause.
  • the second microbe may be a pathogen as described herein.
  • the first microbe may not be usually identified as a pathogen, but presence or abundance of it may be associated with any harmful effect on the subject.
  • association it not only includes the circumstances where the presence or abundance of the second microbe causes the harmful effect on the subject, but also includes the circumstances where the presence or abundance of the second microbe is simply the result of the harmful effect, or the harmful effect and the presence or abundance of the second microbe are not directly causal to each other, but are both the results of an underlying cause.
  • a method may further comprise determining the health state of an individual, such as diagnosing one or more diseases, conditions, disorders, or symptoms of any of these.
  • the disease, condition, or disorder may be an infection or an infectious disease. Infection typically refers to invasion and multiplication of infectious agents in body tissues of a subject, as well as reaction of host tissues to the infectious agents and toxins, if exists, produced by the infectious agents. An infectious disease is a disease caused by infection. Infectious diseases include, but are not limited to bacterial diseases, viral diseases, fungal infection, parasitic diseases, and the like.
  • the infectious disease may be a bacterial disease.
  • the bacterial disease may be a bacterial disease caused by Firmicutes.
  • Firmicutes are a group of mostly Gram positive bacteria, but some of them may be Gram negative, characterized by low G+C contents.
  • Firmicutes infectious diseases may include bacterial diseases caused by bacteria from the genera Streptococcus, Enterococcus, Staphylococcus, Bacillus, Listeria, Clostridium, Peptostreptoccous, Ureaplasma, Mycoplasma, Erysipelothrix, or the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Streptococcus include pneumococcal infection, infection caused by an optochin resistant (S. viridans, S. mitis, S. mutans, S. oralis, S. sanguinis, S. sobrinus, milleri group, etc. ) , Group A streptococcal infection, streptococcal pharyngitis, scarlet fever, erysipelas, rheumatic fever, Group B streptococcal infection, cutaneous Streptococcus iniae infection, Streoptoccus bovis infection, and the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Enterococcus include urinary tract infection, Enterococcus faecium infection, and the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Staphylococcus include staphylococcal scalded skin syndrome, toxic shock syndrome, Methicillin-resistant Staphylococcus aureus (MRSA) infection, infection caused by novobiocin susceptible and novobiocin resistant Cg-(coagulase negative) Staphyloccoccus, and the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Bacillus include anthrax, food poisoning caused by Bacillus, and the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Listeria include listeriosis and the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Clostridium include pseudomembranous colitis, botulism, tetanus, gas gangrene, clostridial necrotizing enteritis, and the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Peptostreptococcus include infection caused by Peptostreptococcus magnus and the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Ureaplasma include ureaplasma infection and the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Mycoplasma include mycoplasma pneumonia, infection caused by Mycoplasma genitalium, and the like.
  • Non-limiting examples of Firmicutes infectious diseases caused by Erysipelothrix include erysipeloid and the like.
  • the bacterial disease may be a bacterial disease caused by Proteobacteria.
  • Proteobacteria are a major group of Gram-negative bacteria.
  • Proteobacteria can be further divided into Alphaprocteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, and Epsilonproteobacteria.
  • Bacterial diseases caused by Proteobacteria can be caused by any of the aforesaid divisions.
  • Bacterial disease caused by Alphaproteobacteria may include those caused by genera Rickettsia, Orientia, Anaplasma, Ehrlichia, Brucella, Bartonella, or the like.
  • Non-limiting examples of bacterial diseases caused by Rickettsia include murine typhus, epidemic typhus, Brill-Zinsser disease, flying squirrel typhus, Rocky Mountain spotted fever, boutonneuse fever, Japanese spotted fever, North Asian tick typhus, Queensland tick typhus, Flinders Island spotted fever, African tick bite fever, American tick bite fever, Rickettsia aeschlimannii infection, rickettsialpox, flee-borne spotted fever, and the like.
  • Non-limiting examples of bacterial diseases caused by Orientia include scrub typhus and the like.
  • Non-limiting examples of bacterial diseases caused by Anaplasma include human granulocytic anaplasmosis, anaplasmosis, and the like.
  • Non-limiting examples of bacterial diseases caused by Ehrlichia include human monocytotropic ehrlichiosis, Ehrlichiosis ewingii infection, and the like.
  • Non-limiting examples of bacterial diseases caused by Brucella include brucellosis and the like.
  • Non-limiting examples of bacterial diseases caused by Bartonellosis include cat-scratch disease, trench fever, bacillary angiomatosis, Carrion's disease, verruga peruana, and the like.
  • Bacterial disease caused by Betaproteobacteria may include those caused by genera Neisseria, Eikenella, Chromobacerium, Burkholderia, or the like.
  • Non-limiting examples of bacterial diseases caused by Bartonellosis include meningococcal disease, Waterhouse-Friderichsen syndrome, meningococcal septicaemia, gonorrhea, and the like.
  • Non-limiting examples of bacterial diseases caused by Eikenella include HACEK endocarditis and the like.
  • Non-limiting examples of bacterial diseases caused by Chromobacterium include Chromobacteriosis infection and the like.
  • Non-limiting examples of bacterial diseases caused by Burkholderia include melioidosis, glanders, infection caused by Burkholderia cepacia complex, pertussis, and the like.
  • Bacterial disease caused by Gammaproteobacteria may include those caused by genera Klebsiella, Escherichia, Enterobacter, Serratia, Salmonella, Shigella, Proteus, Yersinia, Haemophilus, Pasteurella, Actinobacillus, Aggregatibacter, Legionella, Coxiella, Francisella, Vibrio, Plesiomonas, Pseudomonas, Moraxella, Acinetobacter, Stenotrophomonas, Cardiobacterium, Aeromonas, or the like.
  • Non-limiting examples of bacterial diseases caused by Klebsiella include rhinoscleroma, Klebsiella pneumonia, donovanosis or granuloma inguinale, infection caused by Klebsiella oxytoca, and the like.
  • Non-limiting examples of bacterial diseases caused by Escherichia include infection caused by E. coli (enterotoxigenic, enteroinvasive, enterohemorrhagic, O157: H7, O104: H4) , hemolytic-uremic syndrome, and the like.
  • Non-limiting examples of bacterial diseases caused by Serratia include Serratia infection and the like.
  • Non-limiting examples of bacterial diseases caused by Citrobacter include infection caused by Citrobacter koseri/Citrobacter freundii.
  • Non-limiting examples of bacterial diseases caused by Salmonella include typhoid fever, paratyphoid fever, salmonellosis, and the like.
  • Non-limiting examples of bacterial diseases caused by Shigella include Shigellosis, bacillary dysentery, and the like.
  • Non-limiting examples of bacterial diseases caused by Proteus include infection caused by Proteus mirabillis/Proteus vulgaris and the like.
  • Non-limiting examples of bacterial diseases caused by Yersinia include plague/bubonic plague, yersiniosis, Far East scarlet-like fever, and the like.
  • Non-limiting examples of bacterial diseases caused by Haemophilus include haemophilus meningitis, Brazian purpuric fever, chancroid, HACEK endocarditis, and the like.
  • Non-limiting examples of bacterial diseases caused by Pasteurella include pasteurellosis and the like.
  • Non-limiting examples of bacterial diseases caused by Actinobacillus include actinobacillosis and the like.
  • Non-limiting examples of bacterial diseases caused by Aggregatibacter include HACEK endocarditis and the like.
  • Non-limiting examples of bacterial diseases caused by Legionella include legionnaires' disease and the like.
  • Non-limiting examples of bacterial diseases caused by Coxiella include Q fever and the like.
  • Non-limiting examples of bacterial diseases caused by Francisella include tularemia and the like.
  • Non-limiting examples of bacterial diseases caused by Vibrio include cholera, infection caused by other Vibrio species (V. vulnificus, V. parahaemolyticus, V. alginolyticus, and the like) , and the like.
  • Non-limiting examples of bacterial diseases caused by Plesiomonas include infection caused by Plesiomonas shigelloides and the like.
  • Non-limiting examples of bacterial diseases caused by Pseudomonas include pseudomonas infection and the like.
  • Non-limiting examples of bacterial diseases caused by Moraxella include infection caused by Moraxella catarrhalis and the like.
  • Non-limiting examples of bacterial diseases caused by Acinetobacter include infection caused by Acinetobacter baumannii and the like.
  • Non-limiting examples of bacterial diseases caused by Stenotrophomonas include infection caused by Stenotrophomonas maltophilia and the like.
  • Non-limiting examples of bacterial diseases caused by Cardiobacterium include HACEK endocarditis and the like.
  • Non-limiting examples of bacterial diseases caused by Aeromonas include Aeromonas infection and the like.
  • Bacterial disease caused by Epsilonproteobacteria may include those caused by genera Campylobacter, Helicobacter, or the like.
  • Non-limiting examples of bacterial diseases caused by Campylobacter include campylobacteriosis, Guillan-Barre syndrome, and the like.
  • Non-limiting examples of bacterial diseases caused by Helicobacter include peptic ulcer, MALT lymphoma, gastric cancer, helicobacter cellulitis, and the like.
  • the bacterial disease may be a bacterial disease caused by a non-Proteobacteria Gram-negative bacterium.
  • Bacterial disease caused by non-proteobacteria Gram-negative bacteria may include those caused by genera Treponema, Borrelia, Leptospira, Spirillum, Chlamydophila, Chlamydia, Bacteroides, Tannerella, Capnocytophaga, Porphyromonas, Prevotella, Fusobacterium, Streptobacillus, or the like.
  • Non-limiting examples of bacterial diseases caused by Treponema include syphilis/bejel, yaws, pinta, infection caused by Treponema denticola, and the like.
  • Non-limiting examples of bacterial diseases caused by Borrelia include lyme disease, erythema chronicum migrains, neuboborreliosis, louse borne relapsing fever, tick borne relapsing fever, and the like.
  • Non-limiting examples of bacterial diseases caused by Leptospira include leptosipirosis and the like.
  • Non-limiting examples of bacterial diseases caused by Spirillum include rat-bite fever/sodoku and the like.
  • Non-limiting examples of bacterial diseases caused by Chlamydophila include psittacosis, infection caused by pneumoniae, and the like.
  • Non-limiting examples of bacterial diseases caused by Chlamydia include chlamydia, lymphogranuloma venereum, trachoma, and the like.
  • Non-limiting examples of bacterial diseases caused by Bacteroides include infection caused by Bacteroides fragilis and the like.
  • Non-limiting examples of bacterial diseases caused by Tannerella include infection caused by Tannerella forsythia and the like.
  • Non-limiting examples of bacterial diseases caused by Capnocytophaga include infection caused by Capnocytophaga canimorsus and the like.
  • Non-limiting examples of bacterial diseases caused by Porphyromonas include infection caused by Porphyromonas gingivalis and the like.
  • Non-limiting examples of bacterial diseases caused by Prevotella include infection caused by Prevotella intermedia and the like.
  • Non-limiting examples of bacterial diseases caused by Fusobacterium include Lemierre's syndrome, infection caused by Fusobacterium nucleatum and Fusobacterium polymorphum, and the like.
  • Non-limiting examples of bacterial diseases caused by Streptobacillus include rat-bite fever/haverhill fever and the like.
  • the infectious disease is a viral disease.
  • the viral disease may be a neoplasm caused by an oncovirus.
  • neoplasms caused by oncoviruses include heptaocellular carcinoma, cervical cancer, anal cancer, penile cancer, vulvar cancer, vaginal cancer, oropharyngeal cancer, Kaposi's sarcoma, nasopharynx cancer, Burkitt's lymphoma, Hodgkin's lymphoma, follicular dendritic cell sarcoma, nasal type NK/T-cell lymphoma, Merkel cell carcinoma, heptocellular carcinoma, splenic marginal zone lymphoma, adult T-cell leukemia/lymphoma, and the like.
  • the viral disease may be an immune disorder such as acquired immune deficiency syndrome (AIDS) , and the like.
  • AIDS acquired immune deficiency syndrome
  • the viral disease may be a central nervous system or eye viral disease.
  • central nervous system or eye viral diseases include progressive multifocal leukoencephalopathy, subacute slerosing panencephalitis, lymphocitic choriomeningitis, arbovirus encephalitis, encephalitis lethargica, rabies, infection caused by Chandipura virus, herpesviral meningitis, Ramsay Hunt syndrome type II, poliomyelitis, post-polio syndrome, tropical spastic paraparesis, cytomegalovirus retinitis, herpes of the eye, and the like.
  • the viral disease may be a cardiovascular viral disease.
  • cardiovascular viral diseases include pericarditis, myocarditis, and the like.
  • the viral disease may be a respiratory system viral disease such as an acute viral nasopharyngitis or viral pneumonia.
  • respiratory system viral diseases include EBV infection/infectious mononucleosis, infection caused by Cytomegalovirus, severe acute respiratory syndrome (SARS) , influenza, avian influenza, parainfluenza, infection caused by human respiratory syncytial virus (RSF) , infection caused by human metapneumovirus (hMPV) , and the like.
  • the viral disease may be a digestive system viral disease.
  • digestive system viral diseases include mumps, cytomegalovirus esophagitis, gastroenteritis/diarrhea caused by a DNA virus (adenovirus infection or the like) , gastroenteritis/diarrhea caused by an RNA virus (Rotavirus, Norovirus, Astrovirus, Coronavirus, or the like) , hepatitis caused by a DNA virus (hepatitis B) , hepatitis caused by an RNA virus (hepatitis A, hepatitis C, hepatitis D, hepatitis E, hepatitis G, or the like) , hepatitis or pancreatitis caused by Coxsackie B virus (CBV) , and the like.
  • CBV Coxsackie B virus
  • the viral disease may be a urogenital viral disease.
  • urogenital viral diseases include infection caused by BK virus, mumps, and the like.
  • the infectious disease is a parasitic disease caused by protozoa such as trichomoniasis.
  • the infectious disease is a fungal infection.
  • Fungi include Ascomycota, Basidiomycota, Zygomycota, and Microsporidia.
  • the term "fungus/fungi” also encompasses mesomycetozoa. Therefore, infection caused by mesomycetozoea such as phinosporidiosis is also encompassed in the scope of this disclosure.
  • Non-limiting examples of fungal infections caused by Ascomycota include tinea barbae/tineacapitis (kerion) , tinea corporis (ringworm or dermatophytids) , tinea cruris, tinea manuum, tinea pedis (athlete's foot) , tinea unguium/onychomycosis (white superficial onychomycosis, distal subungual onychomycosis, proximal subungual onychomycosis) , tinea corporis gladiatorum, tinea faciel, tinea imbricata, tinea incognito, favus, tinea nigra, black piedra, coccidioidomycosis, disseminated coccidioidomycosis, primary cutaneous coccidioidomycosis, primary pulmonary coccidioidomycosis, histoplasmosis, primary cutaneous histoplasmosis, primary
  • Non-limiting examples of fungal infections caused by Basidiomycota include tinea versicolor, pityrosporium folliculitis, white piedra, cryptococcosis, trichosporonosis, and the like.
  • Non-limiting examples of fungal infections caused by Zygomycota include mucormycosis, entomophthoramycosis such as basidobolomycosis and conidiobolomycosis, and the like.
  • Non-limiting examples of fungal infections caused by Microsporidia include microsporidiosis caused by Enterocytozoon bieneusi/Encephalitozoon intestinalis, and the like.
  • infectious diseases include Acinetobacter infections, Actinomycosis, African sleeping sickness (African trypanosomiasis) , AIDS (Acquired immunodeficiency syndrome) , Amebiasis, Anaplasmosis, Angiostrongyliasis, Anisakiasis, Anthrax, Arcanobacterium haemolyticum infection, Argentine hemorrhagic fever, Ascariasis, Aspergillosis, Astrovirus infection, Babesiosis, Balantidiasis, Bartonellosis Baylisascaris infection, BK virus infection, Black piedra, Blastocystis hominis infection, Blastomycosis, Venezuelan hemorrhagic fever, Botulism (and Infant botulism) , Brazilian hemorrhagic fever, Brucellosis, Bubonic plague, Burkholderia infection, Buruli ulcer, Calicivirus infection (Norovirus and Sapovirus
  • the disease, condition or disorder may be a disease, condition or disorder associated with dysbiosis, or microbial imbalance.
  • these colonies exhibit a decreased ability to check each's growth, which can then lead to overgrowth of one or more of the disturbed colonies which may further damage some of the other smaller beneficial ones.
  • more colonies of the first microbes are damaged, making the imbalance more pronounced, more overgrowth issues occur because the damaged colonies are less able to check the growth of the overgrowing colonies.
  • the disease, condition or disorder associated with microbial imbalance includes, but is not limited to obesity; malnutrition; diabetes (both type-1 and type-2) ; atherosclerosis and heart disease; multiple sclerosis; asthma and eczema; liver disease; numerous diseases of the intestines, including bowel cancer; and autism.
  • the first microbe comprises one or more microbes selected from the group consisting of Lactobacillus, Bifidobacterium, Ruminococcus, Escherichia, Bacteroides, Faecalibacterium, and Lactobacillus rhamnosus GG strain.
  • the second microbe is Escherichia.
  • Human intestinal microflora are a complex system including various microbes such as bacteria, archeae, yeasts and filamentous fungi. For each individual, about 1,000 types of bacteria may colonize his/her intestinal tract.
  • Common intestinal microbes in human include, but are not limited to Acnietobacter, Akkermansia, Alistipes, Anaerotruncus, Bacteroides, Bifidobacterium, Bilophila, Blautia, Brachyspira, Butyrivibrio, Cardiobacterium, Clostridium, Coprococcus, Collinsella, Dialiser, Dorea, Enterococcus, Enterobacter, Erwinia, Escherichia, Eubacterium, Faecalibacterium, Fusobacterium, Haemophilus, Holdemania, Klebsiella, Lactobacillus, Mogibacterium, Neisseria, Parabacteroides, Peptostreptococcus, Porphyromonas, Prevotella, Pseu
  • Colonized bacteria in human intestinal tract form symbiotic relationship with their host and contribute to the hemostasis of their host.
  • the host provides shelter and nutrients to the bacteria, while the bacteria serve various physiological functions for the host, including, but not limited to carbohydrate metabolism, synthesis of co-factors and vitamins, transportation of carbohydrates, purine and pyrimidine metabolism, ATP synthesis, transportation of carbonate and amino acids, nucleosomal function, and aromatic amino acid metabolism.
  • the bacteria also interact with the host to regulate expression of host genes, which play various roles, such as metabolism, development and maturation of the immune system, development of tissues or organs, etc.
  • human intestinal microflora (including, but not limited to bacteria and archaea colonized in the human intestinal tract) helps host carbohydrate metabolism.
  • the human intestinal microflora can degrade and process carbohydrates that are difficult to be directly digested and utilized by the human intestinal tract.
  • the metabolites of these carbohydrates usually smaller carbohydrates such as monosaccharides, can be readily absorbed by the human intestinal tract.
  • intestinal microflora helps amino acid metabolism and absorption by the human intestinal tract.
  • the intestinal microflora may directly process or modify many drugs or prodrugs, or indirectly modify the response of the host to these drugs or prodrugs through the interaction between the host and the microflora, such that the efficacy and toxicity of the drugs or prodrugs are modified.
  • the human intestinal microflora also plays roles in regulation of immune response and host defense.
  • various diseases or conditions are associated with disruption or imbalance of normal human intestinal microflora.
  • the diseases or conditions that may be associated with the disruption or imbalance include, but are not limited to obesity, diabetes, Crohn’s disease, inflammatory bowel disease, irritable bowel syndrome, celiac disease, gastric cancer, anorexia, allergies, autism, colorectal cancer, infectious diseases, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, non-alcoholic fatty liver disease, and neural and behavior diseases.
  • the subject methods can involve assessing relative or absolute quantity or concentration of at least one type of microbe in at least one biological sample.
  • the quantity of a microbe in the biological sample may be represented as the mass, the amount of substance, or the number of the microbe.
  • the concentration of a microbe in the biological sample may be a mass concentration, a molar concentration, or a number concentration. If the biological sample is a fluid, the mass concentration, molar concentration or number concentration can be represented as the mass/amount of substance/number of the microbe divided by the volume of the fluid.
  • the volume of the lysate, suspension, extract, or homogenate thus produced may be used in place of the volume of the sample.
  • the microbe is a bacterium or a fungus
  • the quantity of the microbe may be measured in colony-forming units (CFU) . Accordingly, the concentration of the microbe may be represented in CFU divided by the volume of the sample as defined herein, for example, in CFU/mL.
  • the quantity or the concentration of the microbe may be normalized such that the quantity or the concentration of the microbe may be comparable across different biological samples.
  • the different biological samples may be biological samples taken from different sources.
  • the different biological samples may be biological samples taken from the same source at different times.
  • the different biological samples may be different aliquots of biological samples taken from the same source.
  • the quantity or the concentration may be normalized by an internal standard.
  • the internal standard may represent the quantity or the concentration of a relatively constant component of the biological sample.
  • a component may be considered relatively constant if the quantity or the concentration of the component does not fluctuate across different biological samples under the conditions where the biological sample is taken (e.g., within 10%, 5%, 1%, or less of each other) .
  • the internal standard may be the quantity or concentration of another microbe.
  • the relative quantity of a microbe in the biological sample may be represented as a ratio between the quantity of the microbe and an internal quantity.
  • the internal quantity is the quantity of one or more subgroups of or all microbes in the biological sample.
  • the internal quantity is the quantity of one or more subgroups of or all first microbes.
  • the internal quantity is the quantity of one or more subgroups of or all second microbes.
  • the internal quantity is the quantity of a specific microbe.
  • the internal quantity of a microbe is the quantity of another specific microbe (either a first or second microbe) .
  • the relative concentration of a microbe in the biological sample may be represented as a ratio between the concentration of the microbe and another internal concentration.
  • the internal concentration is the concentration of one or more subgroups of or all microbes in the biological sample.
  • the internal concentration is the concentration of one or more subgroups of or all first microbes.
  • the internal concentration is the concentration of one or more subgroups or all second microbes.
  • the internal concentration is the concentration of a specific microbe.
  • the relative tissue distribution of a microbe may be represented as the ratio between the quantity or concentration of the microbe in one tissue and the quantity or concentration of the microbes in all tissues or in another tissue.
  • the microbes as described herein comprise two categories of microbes, namely, first microbes and second microbes.
  • the first microbes and the second microbes are any first microbes and second microbes as described elsewhere herein.
  • the biological sample of the present disclosure comprises at least one type of first microbes. In some embodiments, the biological sample of the present disclosure comprises at least two type of first microbes.
  • the at least one type of first microbe and the at least one type of second microbe assessed according to the present disclosure are from the same classification, wherein the classifications are: viruses, bacteria, fungi, and protozoa.
  • the at least one type of first microbe and the at least one type of second microbe are viruses.
  • the at least one type of first microbe and the at least one type of second microbe are fungi.
  • the at least one type of first microbe and the at least one type of second microbe are bacteria.
  • the at least one type of first microbe and the at least one type of second microbe are protozoa.
  • the at least one type of first microbe are from any one of the classifications viruses, bacteria, fungi, and protozoa, while the at least one type of second microbe are from a different classification of the classifications viruses, bacteria, fungi, and protozoa.
  • the at least one type of first microbe assessed according to the present disclosure are from at least two different classifications (e.g., 2, 3, or 4) of the classifications of viruses, bacteria, fungi, and protozoa.
  • the at least one type of second microbe assessed according to the present disclosure are from at least two (e.g., 2, 3, or 4) different classifications of the classifications of viruses, bacteria, fungi, and protozoa.
  • the presence, absence, quantity, or concentration of 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 500, 1000, 5000 or more types of first microbes and 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 500, 1000, 5000 or more types of second microbes are assessed.
  • the presence, absence, quantity, or concentration of 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 500, 1000, 5000 or more types of first microbes and 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 500, 1000, 5000 or more types of second microbes are detected in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100 or 200 biological samples to determine the relative tissue distribution.
  • the presence, absence, relative quantity, or relative concentration of 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 500, 1000, 5000 or more types of first microbes and 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 500, 1000, 5000 or more types of second microbes are detected in at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 100 or 200 biological samples to determine the relative tissue distribution.
  • the presence, absence, relative quantity, or relative concentration of 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 500, 1000, 5000 or more types of first microbes and 1, 2, 3, 4, 5, 10, 15, 25, 50, 100, 500, 1000, 5000 or more types of second microbes are detected over a certain period of time to determine the change of relative quantity or concentration of the first microbes and the second microbes in the plurality of microbes, and/or the change of tissue distribution of the first microbes and the second microbes over the period of time.
  • the period of time is about or at least 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 1 year.
  • the quantity or concentration, relative quantity or concentration, and relative tissue distribution of the at least one type of first microbe and the at least one type of second microbe may be weighted.
  • each type of first and second microbe may be assigned a coefficient which represents the relevant significance of the impact the microbe in question on the benefits or harm of the subject.
  • the method of the present disclosure may comprise manipulating the quantity and concentration, relative quantity and concentration, and relative tissue distribution so that the quantity or concentration, relative quantity or concentration, and relative tissue distribution of the at least one type of first microbe and the at least one type of second microbe are weighted.
  • the coefficients may be used to calculate weighted sums of the quantity and concentration, relative quantity and concentration, and relative tissue distribution of the first and second microbes as described herein. Such weighted sums may indicate whether the benefits outweigh the harm when the assessed microbes are taken as a whole, or vice versa.
  • the coefficient may be positive or negative.
  • the coefficients for a type of first microbe and a type of second microbe may be of the same or opposite signs. In some embodiments, the coefficients for the first microbes and the second microbes are of the same signs. In such embodiments, the weighted sums or averages for the first microbes and the second microbes are calculated separately and then compared. In some embodiments, weighted sums or averages for the first microbes that are higher than the weighted sums or averages for the second microbes indicate that the benefits outweigh the harm when the assessed microbes are taken as a whole. In some embodiments, weighted sums or averages for the first microbes that are lower than the weighted sums or averages for the second microbes indicate that the benefits are outweighed by the harm when the assessed microbes are taken as a whole.
  • the coefficients for a type of first microbe and a type of second microbe may be of the opposite signs. In some embodiments, the coefficients for the first microbes are positive and the coefficients for the second microbes are negative, or vice versa. In some embodiments, where the coefficients for the first microbes are positive and the coefficients for the second microbes are negative, a weighted sum or mean for the first and the second microbes that as a whole is positive indicates that the benefits outweigh the harm; otherwise, a weighted sum or mean for the first and the second microbes that as a whole are negative indicates that benefits are outweighed by the harm.
  • the coefficients for the first microbes are negative and the coefficients for the second microbes are positive.
  • a negation of the weighted sums or averages for the first and the second microbes that as a whole are positive indicates that the benefits outweigh the harm; otherwise, a negation of the weighted sums or averages for the first and the second microbes that as a whole are negative indicates that the benefits are outweighed by the harm.
  • the results of the profiling performed on the biological sample of the subject may be compared to reference.
  • the reference may be a result from a biological sample that is known or appears to be normal.
  • a biological sample is normal if it is obtained from a subject that is known or appears to be normal.
  • a subject is normal or appears normal if it is free of any disease, condition, or disorder, or any symptom associated with the disease, condition, or disorder.
  • the disease, condition, disorder, or symptom may be any disease, condition, disorder, or symptom described herein.
  • the normal sample may be from a different subject from the subject being tested, or from the same subject.
  • the normal sample is a stool sample of a subject such as the subject being tested for example. The normal sample may be assayed at the same time, or at a different time from the test sample.
  • the reference may comprise one or more thresholds. In some embodiments, the reference comprises one threshold. In some embodiments, the reference comprises two thresholds. In some embodiments, the reference comprises three or more thresholds.
  • the threshold may be a quantity or threshold as defined herein.
  • the results of an assay on the biological sample may be compared to the results of the same assay on a normal sample.
  • the results of the assay on the normal sample are from a database, or a reference value.
  • the results of the assay on the normal sample can be a known or generally accepted value.
  • the comparison is qualitative. In other cases, the comparison is quantitative.
  • qualitative or quantitative comparisons may involve but are not limited to one or more of the following: comparing colony numbers, colony forming units, specifically stained cell numbers, cell counts, cytometric results, fluorescence values, spot intensities, absorbance values, chemiluminescent signals, histograms, critical threshold values, statistical significance values, gene product expression levels, gene product expression level changes, alternative exon usage, changes in alternative exon usage, protein levels, DNA polymorphisms, copy number variations, indications of the presence or absence of one or more DNA markers or regions, nucleic acid concentration or quantity, or nucleic acid sequences.
  • the assay is conducted to measure the quantity or concentration of at least one microbe as described herein.
  • the assay is conducted to measure the quantity or concentration of at least one first microbe and at least one second microbe as described herein.
  • the profiling results are evaluated using methods for correlating the quantity or concentration of the at least one microbe to the health state, such as presence/absence, susceptibility, onset, progression, amelioration, or type of any disease, condition, disorder, or symptom as described herein, or healthiness (e.g., free of any disease, condition, disorder, or symptom as described herein) of the subject.
  • a specified statistical confidence level may be determined in order to provide a diagnostic confidence level. For example, it may be determined that a confidence level of greater than 90%may be a useful predictor of the health state of the subject. In other embodiments, more or less stringent confidence levels may be chosen.
  • a confidence level of approximately 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, 99.5%, or 99.9% may be chosen as a useful predictor of the health state of the subject.
  • the confidence level provided may in some cases be related to the quality of the sample, the quality of the data, the quality of the analysis, the specific methods used, and the number of microbes profiled.
  • the specified confidence level for providing a diagnosis may be chosen on the basis of the expected number of false positives or false negatives and/or cost.
  • Methods for choosing parameters for achieving a specified confidence level or for identifying markers with diagnostic power include but are not limited to Receiver Operator Curve analysis (ROC) , binormal ROC, principal component analysis, partial least squares analysis, singular value decomposition, least absolute shrinkage and selection operator analysis, least angle regression, and the threshold gradient directed regularization method.
  • ROC Receiver Operator Curve analysis
  • binormal ROC principal component analysis
  • partial least squares analysis singular value decomposition
  • least absolute shrinkage and selection operator analysis least angle regression
  • threshold gradient directed regularization method include but are not limited to Receiver Operator Curve analysis (ROC) , binormal ROC, principal component analysis, partial least squares analysis, singular value decomposition, least absolute shrinkage and selection operator analysis, least angle regression, and the threshold gradient directed regularization method.
  • Raw quantity or concentration data may in some cases be improved through the application of algorithms configured to normalize and or improve the reliability of the data.
  • the data analysis requires a computer or other device, machine or apparatus for application of the various algorithms described herein due to the large number of subject data points that are processed.
  • a “machine learning algorithm” refers to a computational-based prediction methodology, or “classifier” , employed for characterizing a gene expression profile.
  • the signals corresponding to certain quantity or concentration of microbes, which are obtained by, e.g., nucleic acid amplification or sequencing assays, are typically subjected to the algorithm in order to classify the microbial profile.
  • Supervised learning generally involves "training" a classifier to recognize the distinctions among classes and then “testing" the accuracy of the classifier on an independent test set. For new, unknown samples the classifier can be used to predict the class in which the samples belong.
  • Methods of data analysis of the quantity or concentration of microbes may further include the use of a classifier algorithm as provided herein.
  • a support vector machine (SVM) algorithm, a random forest algorithm, or a combination thereof is provided for classification of microarray data.
  • identified markers that distinguish samples e.g., presence/absence of any disease, condition, disorder, or symptom
  • FDR Benjamini Hochberg correction for false discovery rate
  • the classifier algorithm may be supplemented with a meta-analysis approach such as that described by Fishel and Kaufman et al. 2007 Bioinformatics 23 (13) : 1599-606.
  • the classifier algorithm may be supplemented with a meta-analysis approach such as a repeatability analysis.
  • the repeatability analysis selects markers that appear in at least one predictive microbe set.
  • the results of feature selection and classification may be ranked using a Bayesian post-analysis method.
  • profiling data may be extracted, normalized, and summarized using any method such as the methods provided herein.
  • the data may then be subjected to a feature selection operation.
  • classification such as any of the classification methods such as the use of any of the algorithms or methods provided herein including but not limited to the use of SVM or random forest algorithms.
  • the results of the classifier algorithm may then be ranked by according to a posterior probability function.
  • the posterior probability function may be derived from examining known profiling results, such as results obtained in the present or previous study, to derive prior probabilities from type I and type II error rates of assigning a marker to a category (e.g., health state of the subject as described herein) .
  • error rates may be calculated based on reported sample size for each study using an estimated fold change value (e.g., 1.1, 1.2., 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.2, 2.4, 2.5, 3, 4, 5, 6, 7, 8, 9, 10 or more) .
  • These prior probabilities may then be combined with a profiling dataset of the present disclosure to estimate the posterior probability of differential microbial profiling.
  • the posterior probability estimates may be combined with a second dataset of the present disclosure to formulate one or more additional posterior probabilities of differential profiling.
  • the posterior probabilities may be used to rank the markers provided by the classifier algorithm.
  • markers may be ranked according to their posterior probabilities and those that pass a chosen threshold may be chosen as markers whose differential expression is indicative of or diagnostic for samples corresponding to various health state of the subject.
  • Illustrative threshold values include prior probabilities of 0.7, 0.75, 0.8, 0.85, 0.9, 0.925, 0.95, 0.975, 0.98, 0.985, 0.99, 0.995 or higher.
  • a statistical evaluation of the results of the microbial profiling may provide a quantitative value or values indicative of one or more of the following: the likelihood of diagnostic accuracy, the likelihood of disorder, disease, condition, or symptom, the likelihood of a particular disorder, disease, condition, or symptom, the likelihood of the success of a particular therapeutic or nutritional intervention.
  • a physician who may not be trained in genetics or molecular biology, need not understand the raw data. Rather, the data is presented directly to the physician in its most useful form to guide patient care.
  • the results of the microbial profiling can be statistically evaluated using a number of methods including, but not limited to: the students T test, the two sided T test, pearson rank sum analysis, hidden markov model analysis, analysis of q-q plots, principal component analysis, one way ANOVA, two way ANOVA, and the like.
  • the use of profiling alone or in combination with other analysis may provide a diagnosis that is between about 85%accurate and about 99%or about 100%accurate.
  • the institute conducting the profiling may through the use of the profiling and/or other analysis provide a diagnosis of the health state of the subject that is about 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, 99.75%, 99.8%, 99.85%, or 99.9%accurate.
  • accuracy may be determined by tracking the subject over time to determine the accuracy of the original diagnosis. In other cases, accuracy may be established in a deterministic manner or using statistical methods. For example, receiver operator characteristic (ROC) analysis may be used to determine the optimal assay parameters to achieve a specific level of accuracy, specificity, positive predictive value, negative predictive value, and/or false discovery rate. Methods for using ROC analysis in diagnosis of health state of the subject may be available and have been described for example in US Patent Application No. 2006/019615 herein incorporated by reference in its entirety.
  • ROC receiver operator characteristic
  • microbe (s) which is determined to exhibit the greatest difference in quantity or concentration between presence/absence of any disease, condition, disorder, or symptom may be chosen for use as target for profiling in the present disclosure.
  • Such gene expression products may be particularly useful by providing a wider dynamic range, greater signal to noise, improved diagnostic power, lower likelihood of false positives or false negative, or a greater statistical confidence level than other methods.
  • the use of profiling alone or in combination with other analysis may reduce the number of samples deemed as non-diagnostic by about 100%, 99%, 95%, 90%, 80%, 75%, 70%, 65%or 60%when compared to the use of other methods.
  • the methods of the present invention may reduce the number of samples deemed as indeterminate by about 100%, 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%or 60%when compared to other methods.
  • the results of the profiling may be classified into one of the following: normal (free of a disorder, disease, or condition) , abnormal (positive diagnosis for a cancer, disease, or condition) , or non-diagnostic (providing inadequate information concerning the presence or absence of a disorder, disease, or condition) .
  • a diagnostic result may further classify the type of disorder, disease or condition.
  • a diagnostic result may indicate a certain molecular or metabolic pathway involved in the disorder, disease or condition, or a certain progression or severity of a particular disorder, disease or condition.
  • a diagnostic result may inform an appropriate therapeutic intervention, such as a specific drug regimen, or a specific dietary regimen such as dietary supplement of beneficial microbes.
  • results are classified using a trained algorithm.
  • Trained algorithms of the present invention include algorithms that have been developed using a reference set of known normal, abnormal, and reference samples including but not limited to the samples associated with Examples and Figures of the present disclosure.
  • Algorithms suitable for categorization of samples include but are not limited to k-nearest neighbor algorithms, concept vector algorithms, naive bayesian algorithms, neural network algorithms, hidden markov model algorithms, genetic algorithms, and mutual information feature selection algorithms or any combination thereof.
  • trained algorithms of the present invention may incorporate data other than microbial data such as but not limited to DNA polymorphism data, gene expression data, gene transcription data, sequencing data, diagnosis by physicians, information provided by the pre-classifier algorithm of the present invention, or information about the medical history of the subject of the present invention.
  • a subject may be monitored using methods and compositions of the present invention.
  • a subject may be diagnosed as in an abnormal state.
  • a subject is abnormal or appears abnormal if it is diagnosed with any disease, condition, or disorder, or exhibits any symptom associated with the disease, condition, or disorder.
  • This initial diagnosis may or may not involve the use of microbial profiling.
  • the subject may be prescribed a therapeutic intervention.
  • the results of the therapeutic intervention may be monitored on an ongoing basis by microbial profiling to detect the efficacy of the therapeutic intervention.
  • the subject may be prescribed a dietary supplement that may contain beneficial microbes.
  • the results of the dietary supplement may be monitored on an ongoing basis by microbial profiling to detect the efficacy of the dietary supplement.
  • a subject may be diagnosed with a disease, condition, or disorder, and the disease, condition, or disorder may be monitored on an ongoing basis by microbial profiling to detect any changes in the state of the disease, condition, or disorder.
  • microbial profiling may be used as a research tool to explore the correlation between microbial profile and various states (normal, abnormal, and the like) of the subject; to monitor the effect of therapeutic intervention or dietary supplement on subject diagnosed with a disorder, disease or condition; or to uncover new role of a certain microbe in intestinal microflora.
  • the change of quantity or concentration, or relative quantity or concentration of a microbe may be represented as a first or second derivative of the relative quantity or concentration of the microbe with time.
  • the change of relative quantity or concentration of a microbe may be a first or second derivative of the relative quantity or concentration of the microbe with time.
  • the change of relative quantity or concentration of the microbe reaching or exceeding a threshold may provide information regarding whether the population of the microbe is expanding, shrinking, idling, growing or declining in an accelerated or decelerated manner, and the like.
  • a method as described herein may comprise optional operations which will or will not be performed depending on such information.
  • the threshold may be zero.
  • the threshold may be a positive threshold, that is, a positive real number.
  • the threshold may be a negative threshold, that is, a negative real number.
  • the first derivative of the relative quantity or concentration of a microbe in the biological sample exceeding a threshold of zero may indicate that the microbe is expanding, that is, accounting for a larger and larger portion among total microbes.
  • the first derivative of the relative quantity or concentration of a microbe in the biological sample reaching or exceeding zero or a positive threshold while the second derivative of the relative quantity or concentration of a microbe exceeding zero may indicate that the growth of the microbe is accelerating.
  • the negation of the first derivative of the relative quantity or concentration of a microbe in the biological sample reaching or exceeding a negative threshold while the negation of the second derivative of the relative quantity or concentration of a microbe exceeding zero may indicate that the growth of the microbe is decelerating.
  • the negation of the first derivative of the relative quantity or concentration of a microbe in the biological sample exceeding a threshold of zero may indicate that the microbe is shrinking, that is, accounting for a smaller and smaller portion among total microbes.
  • the negation of the first derivative of the relative quantity or concentration of a microbe in the biological sample reaching or exceeding zero or a positive threshold while the negation of the second derivative of the relative quantity or concentration of a microbe exceeding zero may indicate that the decline of the microbe is accelerating.
  • the first derivative of the relative quantity or concentration of a microbe in the biological sample reaching or exceeding a negative threshold while the negation of the second derivative of the relative quantity or concentration of a microbe exceeding zero may indicate that the decline of the microbe is decelerating.
  • the optional operation may be the administration of a first microbe to the subject from which the biological sample is taken. In some embodiments, the optional operation of administration of a first microbe to the subject is performed when the first microbe is shrinking or declining, no matter in an accelerated or a decelerated manner. In some embodiments, the optional operation of administration of a first microbe to the subject is only performed when the first microbe is shrinking or declining in an accelerated manner but is not performed when the first microbe is declining in a decelerated manner. In some embodiments, the optional operation of administration of a first microbe to the subject is not performed when the first microbe is expanding or growing, no matter in an accelerated or a decelerated manner. In some embodiments, the optional operation of administration of a first microbe to the subject is not performed when the first microbe is shrinking or declining in an accelerated manner but is performed when the first microbe is growing in a decelerated manner.
  • the optional operation may be the administration of an anti-microbial agent specific for a second microbe to the subject from which the biological sample is taken. In some embodiments, the optional operation of administration of an anti-microbial agent specific for a second microbe to the subject is not performed when the second microbe is shrinking or declining, no matter in an accelerated or a decelerated manner. In some embodiments, the optional operation of administration of an anti-microbial agent specific for a second microbe to the subject is not performed when the second microbe is shrinking or declining in an accelerated manner but is performed when the second microbe is declining in a decelerated manner.
  • the optional operation of administration of an anti-microbial agent specific for a second microbe to the subject is performed when the second microbe is expanding or growing, no matter in an accelerated or a decelerated manner. In some embodiments, the optional operation of administration of an anti-microbial agent specific for a second microbe to the subject is only performed when the second microbe is shrinking or declining in an accelerated manner but is not performed when the second microbe is growing in a decelerated manner.
  • the change of quantity or concentration, or relative quantity or concentration of a microbe at a certain time may be the difference between the quantity or concentration, or relative quantity or concentration of the microbe at that time and a baseline.
  • the present disclosure provides a method of assessing susceptibility of a subject for a condition inflicted by presence of at least one second microbe and/or absence of at least one first microbe.
  • the method comprises: conducting a nucleic acid amplification assay to assess the quantity or concentration of the second microbe and at least one type of first microbe present in at least one biological sample from the subject; and determining the relative quantity or relative concentration of the pathogen and the at least one type of first microbe in the at least one biological sample, thereby assessing susceptibility of the condition inflicted by the presence of at least one second microbe and/or absence of at least one first microbe in the subject.
  • Methods of amplification, sample sources, and microbes can be any of those described above, with regard to any of the various aspects of the disclosure.
  • Microbes may be detected in various ways for the purpose of the present disclosure.
  • a target nucleic acid molecule in a biological sample can be detected, for example, by a method comprising: (a) mixing the biological sample with a first buffer to obtain a mixture; (b) incubating the mixture at a temperature for example at equal to or higher than 15°C for a period of time for example no more than about 15 minutes; (c) adding to the mixture from (b) a lysis buffer to obtain a lysate; (d) adding the lysate from (c) to a reaction vessel comprising reagents necessary for conducting nucleic acid amplification, the reagents comprising (i) a deoxyribonucleic acid (DNA) polymerase and optionally, a reverse transcriptase, and (ii) a primer set for the target nucleic acid molecule, to obtain a reaction mixture; and (e) subjecting the reaction mixture in the reaction vessel to multiple cycles of a primer extension reaction to generate amplified product (s) that is indicative of a presence of
  • Each cycle can comprise (i) incubating the reaction mixture at a denaturing temperature for a denaturing duration, followed by (ii) incubating the reaction mixture at an elongation temperature for an elongation duration, thereby amplifying the target nucleic acid molecule.
  • the present disclosure provides a method for detecting a microbe in a biological sample.
  • the method may comprise: (a) mixing the biological sample with a first buffer to obtain a mixture (b) incubating the mixture at a temperature for example at equal to or higher than 15°C for a period of time for example no more than about 15 minutes; (c) adding to the mixture from (b) a lysis buffer to obtain a lysate; (d) adding the lysate from (c) to a reaction vessel comprising reagents necessary for conducting nucleic acid amplification, the reagents comprising (i) a deoxyribonucleic acid (DNA) polymerase and in some cases a reverse transcriptase, and (ii) one or more primer sets, each capable of specifically binding to a target nucleic acid sequence from a microbial genome or transcriptome, or a variant thereof, to obtain a reaction mixture; and (d) subjecting the reaction mixture in the reaction vessel to a plurality of series of primer extension reactions
  • the biological sample may be a stool sample.
  • the amplified product may be DNA product.
  • the microbe may be any microbe as described elsewhere herein. In some embodiments, the microbe may be selected from the group consisting of Bacteroides, Bifidobacterium, Escherichia, Faecalibacterium, Lactobacillus, Ruminococcus, and Lactobacillus rhamnosus.
  • the "incubation temperature” may be greater than or equal to 15°C,
  • the incubation temperature may be greater than or equal to about 20°C, greater than or equal to about 25°C, greater than or equal to about 30°C, greater than or equal to about 35°C, greater than or equal to about 40°C, greater than or equal to about 45°C, greater than or equal to about 50°C, greater than or equal to about 55°C, greater than or equal to about 60°C, greater than or equal to about 65°C, greater than or equal to about 70°C, greater than or equal to about 75°C, greater than or equal to about 80°C, greater than or equal to about 85°C, greater than or equal to about 90°C, greater than or equal to about 91°C, greater than or equal to about 92°C, greater than or equal to about 93°C, greater than or equal to about 94°C, greater than or equal to about 95°C, greater than or equal to about 96°C, greater than or equal to about 97°C, greater
  • the incubation temperature can be a temperature that is from about 15°C to 95°C.
  • the incubation temperature can be from about 20°C to 100°C, from about 30°C to 99°C, from about 40°C to 98°C, from about 50°C to 97°C, from about 60°C to 96°C, from about 70°C to 95°C, from about 80°C to 100°C, from about 85°C to 99°C, from about 87°C to 98°C, from about 90°C to 97°C, from about 92°C to 97°C, from about 92°C to 96°C, from about 93°C to 95°C, from about 20°Cto 90°C, from about 25°C to 85°C, from about 30°C to 80°C, from about 40°C to 70°C, from about 40°C to 95°C, from about 45°C to 90°C, from about 50°C to 85°C, from about 55°C to 80°C, from about
  • the “incubation time period” may be no more than about 20 minutes.
  • the “incubation time period” may be no more than about 19 minutes, no more than about 18 minutes, no more than about 17 minutes, no more than about 16 minutes, no more than about 15 minutes, no more than about 14 minutes, no more than about 13 minutes, no more than about 12 minutes, no more than about 11 minutes, no more than about 10 minutes, no more than about 9 minutes, no more than about 8 minutes, no more than about 7 minutes, no more than about 6 minutes, no more than about 5 minutes, no more than about 4 minutes, no more than about 3 minutes, no more than about 2 minutes, no more than about 1 minute, no more than about 50 seconds, no more than about 40 seconds, no more than about 30 seconds, no more than about 20 seconds, no more than about 15 seconds, no more than about 10 seconds, no more than about 5 seconds, no more than about 3 seconds, no more than about 2 seconds, or no more than about 1 seconds.
  • the first buffer may be water, saline, or any other suitable buffer.
  • Buffer that can be used in accordance with the present disclosure includes, but is not limited to, sodium chloride solutions of about 0.9%, phosphate buffers, lactated Ringer's solution, acetated Ringer's solution, phosphate buffered saline, citrate buffers, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a histidine buffer, a HEPES buffer, a MOPS buffer, glycine buffers, N-glycylglycine buffers, and combinations thereof.
  • the biological sample prior to being mixed with a first buffer, can be subjected to centrifugation to yield a solution comprising the biological sample and a pellet. In some embodiments, prior to being mixed with a first buffer, the biological sample can be subjected to centrifugation to yield a pellet comprising the biological sample and a supernatant.
  • the biological sample is a stool sample.
  • the stool sample may be preserved in a preservation buffer prior to use.
  • the stool sample may be taken using a scoop.
  • the scoop can be of any size or shape suitable for taking stool samples.
  • one or more (such as two) scoops of stool sample are taken and immediately transferred into a preservation buffer.
  • the preservation buffer may be stored in a sterile stool collection tube before or after transfer of the stool sample into the preservation buffer.
  • the stool sample in the preservation buffer Prior to mixing with the first buffer, the stool sample in the preservation buffer may be spun down to form a pellet.
  • the pellet may be used as the biological sample to be mixed with the first buffer.
  • the preservation buffer, together with the stool sample in it may be used as the biological sample to be mixed with the first buffer.
  • the preservation buffer may be vortexed thoroughly before being spun down or mixed with the first buffer.
  • the stool sample is not mixed with a preservation buffer prior to use.
  • the stool sample may be directly mixed with the first buffer to form a mixture.
  • the mixture after incubating the mixture of the biological sample and the first buffer, the mixture may be subjected to centrifugation to yield a supernatant comprising the biological sample. Then, the supernatant may serve as the mixture in the subsequent operations. For example, the supernatant may then be mixed with the lysis buffer. Alternatively, or additionally, the supernatant may be added to a reaction vessel comprising reagents necessary for conducting nucleic acid amplification.
  • the target nucleic acid molecules may be subjected to one or more denaturing conditions.
  • the one or more denaturing conditions may be selected from a denaturing temperature profile and a denaturing agent.
  • the biological sample before conducting the primer extension reactions, may be pre-heated at a pre-heating temperature from 90°C to 100°C for a pre-heating duration of no more than 10 minutes. In some embodiments, the pre-heating duration is no more than 1 minute.
  • the mixture of the biological sample with the first buffer may be added to the reaction vessel comprising reagents necessary for conducting nucleic acid amplification without undergoing DNA or ribonucleic acid (RNA) extraction.
  • the mixture may be added to the reaction vessel without undergoing purification.
  • the mixture may be added to the reaction vessel without undergoing DNA or RNA concentration.
  • the mixture of the biological sample with the first buffer may be incubated at a temperature that is greater than or equal to about 20°C, greater than or equal to about 25°C, greater than or equal to about 30°C, greater than or equal to about 35°C, greater than or equal to about 40°C, greater than or equal to about 45°C, greater than or equal to about 50°C, greater than or equal to about 55°C, greater than or equal to about 60°C, greater than or equal to about 65°C, greater than or equal to about 70°C, greater than or equal to about 75°C, greater than or equal to about 80°C, greater than or equal to about 85°C, greater than or equal to about 90°C, greater than or equal to about 91°C, greater than or equal to about 92°C, greater than or equal to about 93°C, greater than or equal to about 94°C, greater than or equal to about 95°C, greater than or equal to about 96°C, greater than or equal to about 97
  • the mixture of the biological sample with the first buffer may be incubated for a period of time that is no more than about 20 minutes.
  • the period of time in (b) may be no more than about 19 minutes, no more than about 18 minutes, no more than about 17 minutes, no more than about 16 minutes, no more than about 15 minutes, no more than about 14 minutes, no more than about 13 minutes, no more than about 12 minutes, no more than about 11 minutes, no more than about 10 minutes, no more than about 9 minutes, no more than about 8 minutes, no more than about 7 minutes, no more than about 6 minutes, no more than about 5 minutes, no more than about 4 minutes, no more than about 3 minutes, no more than about 2 minutes, no more than about 1 minute, no more than about 50 seconds, no more than about 40 seconds, no more than about 30 seconds, no more than about 20 seconds, no more than about 15 seconds, or no more than about 10 seconds, no more than about 5 seconds, no more than about 3 seconds, no more than about 2 seconds or no more than about
  • nucleic acid from a biological sample obtained from a subject is amplified.
  • the biological sample may be obtained directly from a source thereof.
  • the biological sample may be obtained directly from a source thereof without pre-culturing, non-selective enrichment, selective enrichment, plating on differential medium, and/or presumptive biomedical identification.
  • Pre-culturing generally refers to a process for expanding one or more target species (e.g., microorganisms) in a sample, or for increasing the number thereof, prior to performing methods of the present disclosure.
  • Non-selective enrichment generally refers to a process of increasing the amount of a majority or all of the species (e.g., microorganisms) in a mixed population non-selectively.
  • Selective enrichment generally refers to a process of increasing the proportion and/or amount of one or more specific species (e.g., microorganisms) in a mixed population while inhibiting other species. Such inhibition may result due to medium constituents such as compounds which are selectively toxic, as well as the end-products of microbial metabolism produced by organisms which utilize the medium constituents.
  • “Differential medium” generally refers to a medium that includes one or more added indicator (s) that allows for the differentiation of particular chemical reactions occurring during growth.
  • Presumptive biomedical identification generally refers to preliminary identification of a microorganism based on observation such as colony characteristics, growth on primary isolation media, gram stain results, etc.
  • the biological sample is cultivated for microbial proliferation.
  • the biological sample prior to being mixed with a first buffer, is subjected to enrichment culturing conditions for a culturing time period.
  • the enrichment culturing conditions may comprise culturing the biological sample in a suitable culture medium (e.g., tryptic soy broth, modified tryptic soy broth, tryptone, nutrient broth, L-broth, gram negative broth, peptone, tryptic soy broth with yeast, or the like) at a suitable temperature (e.g., from 23°C to 40°C, such as 25°C, 30°C, 35°C, or 37°C) with or without shaking.
  • a suitable culture medium e.g., tryptic soy broth, modified tryptic soy broth, tryptone, nutrient broth, L-broth, gram negative broth, peptone, tryptic soy broth with yeast, or the like
  • a suitable temperature e.g., from 23°C
  • the culturing time period may be from about 0.5 hour to 10 hours, e.g., greater than or equal to about 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours.
  • the culturing time period is no more than about 7 hours, e.g., no more than about 6.5 hours, no more than about 6 hours, no more than about 5.5 hours, no more than about 5 hour, no more than about 4.5 hour, no more than about 4 hours, no more than about 3.5 hours, no more than about 3 hours, no more than about 2.5 hours, no more than about 2 hours, no more than about 1.5 hours, no more than about 1 hour, or no more than about 0.5 hour.
  • the biological sample prior to and/or after being subjected to enrichment culturing conditions for a culturing time period, the biological sample is subjected to centrifugation to yield a solution comprising the biological sample and a pellet. In some embodiments, prior to and/or after being subjected to enrichment culturing conditions for a culturing time period, the biological sample is subjected to centrifugation to yield a pellet comprising the biological sample and a supernatant.
  • the biological sample after being subjected to enrichment culturing conditions for a culturing time period, is mixed with the first buffer without selective enrichment, plating on differential medium, and/or presumptive biomedical identification.
  • a first buffer may be added to the mixture.
  • the biological sample is a stool sample.
  • the stool sample is a solid stool sample.
  • the stool sample is a liquid stool sample.
  • the solid stool sample may be suspended in a suitable buffer as a suspended stool sample.
  • the liquid stool sample may be a watery diarrhea sample.
  • the weight of the biological sample may be about 50 mg to about 5 g.
  • the weight of the biological sample may be greater than or equal to about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1.0 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g or 5.0 g.
  • the weight of the biological sample may be any value or range between any two of the aforesaid numeric values.
  • the weight of the stool sample may be greater than or equal to about 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1.0 g, 1.1 g, 1.2 g, 1.3 g, 1.4 g, 1.5 g, 1.6 g, 1.7 g, 1.8 g, 1.9 g, 2.0 g, 2.5 g, 3.0 g, 3.5 g, 4.0 g, 4.5 g, or 5.0 g.
  • the weight of the biological sample may be any value or range between any two of the aforesaid numeric values.
  • the biological sample may be a liquid or a suspension.
  • the volume of the liquid biological sample or the suspended biological sample may be about 50 ⁇ l to about 5 ml.
  • the volume of the biological sample may be greater than or equal to about 50 ⁇ l, 100 ⁇ l, 150 ⁇ l, 200 ⁇ l, 250 ⁇ l, 300 ⁇ l, 350 ⁇ l, 400 ⁇ l, 450 ⁇ l, 500 ⁇ l, 550 ⁇ l, 600 ⁇ l, 650 ⁇ l, 700 ⁇ l, 750 ⁇ l, 800 ⁇ l, 850 ⁇ l, 900 ⁇ l, 950 ⁇ l, 1.0 ml, 1.1 ml, 1.2 ml, 1.3 ml, 1.4 ml, 1.5 ml, 1.6 ml, 1.7 ml, 1.8 ml, 1.9 ml, 2.0 ml, 2.5 ml, 3.0 ml, 3.5 ml, 4.0 ml
  • the volume of the liquid stool sample or the suspended stool sample may be about 50 ⁇ l to about 5 ml.
  • the volume of the biological sample may be greater than or equal to about 50 ⁇ l, 100 ⁇ l, 150 ⁇ l, 200 ⁇ l, 250 ⁇ l, 300 ⁇ l, 350 ⁇ l, 400 ⁇ l, 450 ⁇ l, 500 ⁇ l, 550 ⁇ l, 600 ⁇ l, 650 ⁇ l, 700 ⁇ l, 750 ⁇ l, 800 ⁇ l, 850 ⁇ l, 900 ⁇ l, 950 ⁇ l, 1.0 ml, 1.1 ml, 1.2 ml, 1.3 ml, 1.4 ml, 1.5 ml, 1.6 ml, 1.7 ml, 1.8 ml, 1.9 ml, 2.0 ml, 2.5 ml, 3.0 ml, 3.5 ml, 4.0 ml, 4.5 ml, or 5.0 ml.
  • the volume of the biological sample may
  • a biological sample has not been purified when provided in a reaction vessel.
  • the nucleic acid of a biological sample has not been extracted when the biological sample is provided to a reaction vessel.
  • the RNA or DNA in a biological sample may not be extracted from the biological sample when providing the biological sample to a reaction vessel.
  • a target nucleic acid e.g., a target RNA or target DNA
  • a target nucleic acid present in a biological sample may not be concentrated prior to providing the biological sample to a reaction vessel.
  • the mixture of the biological sample and the first buffer may be added to the reaction vessel without being subject to DNA or RNA extraction. In some cases, the mixture may be added to the reaction vessel without being purified. In some cases, the mixture may be added to the reaction vessel without being subject to DNA or RNA concentration.
  • the reaction vessel may be one that comprises reagents necessary for conducting nucleic acid amplification.
  • the mixture is added to the reaction vessel without being purified.
  • the mixture is added to the reaction vessel without being subject to DNA or RNA extraction.
  • the mixture is added to the reaction vessel without being subject to DNA or RNA concentration.
  • the reaction vessel may be one that comprises reagents necessary for conducting nucleic acid amplification.
  • the supernatant is added to the reaction vessel without being purified.
  • the supernatant is added to the reaction vessel without being subject to DNA or RNA extraction.
  • the supernatant is added to the reaction vessel without being subject to DNA or RNA concentration.
  • the reaction vessel may be one that comprises reagents necessary for conducting nucleic acid amplification.
  • a biological sample may be solid matter (e.g., biological tissue) or may be a fluid (e.g., a biological fluid) .
  • a biological fluid can include any fluid associated with living organisms.
  • Non-limiting examples of a biological sample include blood (or components of blood –e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location (e.g., tissue, circulatory system, bone marrow) of a subject, cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, breath, bone marrow, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, breast, pancreas, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, cavity fluids, sputum, pus, microbiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluid, tears, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, skin cells, plasma,
  • a target nucleic acid is amplified to generate an amplified product.
  • a target nucleic acid may be a target RNA or a target DNA.
  • the target nucleic acid may be from any microbe as described herein.
  • the target nucleic acid molecule may be associated with a disease.
  • the target RNA may be any type of RNA, including types of RNA described elsewhere herein.
  • the target RNA is viral RNA.
  • the viral RNA may be the RNA of any virus as described herein.
  • the viral RNA may be pathogenic to the subject.
  • Non-limiting examples of pathogenic viral RNA include human immunodeficiency virus I (HIV I) , human immunodeficiency virus II (HIV II) , orthomyxoviruses, Ebola virus, Dengue virus, influenza viruses (e.g., H1N1, H3N2, H7N9, or H5N1) , hepesvirus, hepatitis A virus, hepatitis B virus, hepatitis C (e.g., armored RNA-HCV virus) virus, hepatitis D virus, hepatitis E virus, hepatitis G virus, Epstein-Barr virus, mononucleosis virus, cytomegalovirus, SARS virus, West Nile Fever virus, polio virus, measles, enterovirus (such as Coxsackie virus, e.g., Coxsackie virus A16) , and norovirus (e.g., norovirus GI or norovirus GII)
  • the target DNA may be any type of DNA, including types of DNA described elsewhere herein.
  • the target DNA is viral DNA.
  • the viral DNA may be the DNA of any virus as described herein.
  • the viral DNA may be pathogenic to the subject.
  • Non-limiting examples of DNA viruses include herpes simplex virus, smallpox, adenovirus (e.g., Adenovirus Type 55, Adenovirus Type 7) and Varicella virus (e.g., chickenpox) .
  • a target nucleic acid may be a bacterial nucleic acid.
  • the bacterial nucleic acid may be the nucleic acid of any bacteria as described herein.
  • the bacterial nucleic acid may be from a bacterium pathogenic to the subject.
  • the pathogenic bacterium may be a gram-positive or gram-negative pathogenic bacterium.
  • the pathogenic bacterium may be selected from the group consisting of Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, Enterobacter sakazakii, Vibrio Parahemolyticus, Shigella spp., and Mycobacterium tuberculosis (abacterium that can cause tuberculosis) .
  • the pathogenic bacterium is Salmonella.
  • a target DNA may be a DNA from a pathogenic protozoan, such as, for example one or more protozoans of the Plasmodium type that can cause Malaria.
  • the bacterial nucleic acid may be from one or more bacteria selected from Escherichia, Paracolon, Enterobacter aerogenes, Erwinia, Proteus, Pseudomonas aeruginosa, Pneumobacillus, Bacillus, Bacteroides, Bifidobacterium, Clostridium, Collinsella, Faecalibacterium, Lactobacillus, Ruminococcus, Enterococcus, Dorea, Listeria, Streptococcus, Staphyloccocus, Corynebacterium, Propionibacterium, Clostridium butyricum, Campylobacter, Aeromonas, Plesiomonas shigelloides, Campylobacter, Clostridium difficile, Escherichia coli O157, Enteroaggregative E.
  • EAEC Enteroharmful E. coli
  • EPEC Enteroharmful E. coli
  • ETEC Enterotoxigenic E. coli
  • SEIEC Shiga-like Toxin producing E. coli (STEC) stx1/stx2, Salmonella, Shigella, Vibrio cholera 5, Yersinia enterocolitica, Prevotella, Veillonella, Coprococcus, Roseburia, Campylobacter fetus and Lactobacillus rhamnosus GG strain.
  • the bacterial nucleic acid may be from one or more bacteria selected from Bacteroides, Bifidobacterium, Escherichia, Faecalibacterium, Lactobacillus, Ruminococcus, Prevotella, Streptococcus, Veillonella, Coprococcus, Roseburia, and Lactobacillus rhamnosus GG strain.
  • the biological sample may be at a ratio to the suspension buffer of about 5: 1 (wt/vol) to about 1: 500 (wt/vol) , such as about 1: 1 (wt/vol) to about 1: 100 (wt/vol) .
  • the ratio of the biological sample to the suspension buffer may be at least about 5: 1 (wt/vol) , about 4: 1 (wt/vol) , about 3: 1 (wt/vol) , about 2: 1 (wt/vol) , about 1: 1 (wt/vol) , about 1: 2 (wt/vol) , about 1: 3 (wt/vol) , about 1: 4 (wt/vol) , about 1: 5 (wt/vol) , about 1: 6 (wt/vol) , about 1: 7 (wt/vol) , about 1: 8 (wt/vol) , about 1: 9 (wt/vol) , about 1: 10 (wt/vol) , about 1: 20 (wt/vol) , about 1: 30 (wt/vol) , about 1: 40 (wt/vol) , about 1: 50 (wt/vol) , about 1: 60 (wt/vol) , about 1: 70 (wt/vol)
  • the biological sample (such as a stool sample) may be at a ratio to the suspension buffer of about 5: 1 (g/ml) to about 1: 500 (g/ml) , such as about 1: 1 (g/ml) to about 1: 100 (g/ml) .
  • the ratio of the biological sample to the suspension buffer may be at least about 5: 1 (g/ml) , about 4: 1 (g/ml) , about 3: 1 (g/ml) , about 2: 1 (g/ml) , about 1: 1 (g/ml) , about 1: 2 (g/ml) , about 1: 3 (g/ml) , about 1: 4 (g/ml) , about 1: 5 (g/ml) , about 1: 6 (g/ml) , about 1: 7 (g/ml) , about 1: 8 (g/ml) , about 1: 9 (g/ml) , about 1: 10 (g/ml) , about 1: 20 (g/ml) , about 1: 30 (g/ml) , about 1: 40 (g/ml) , about 1: 50 (g/ml) , about 1: 60 (g/ml) , about 1: 70 (g/ml)
  • the first buffer may be at a ratio to the homogenized preparation of about 50: 1 (vol/vol) to about 1: 50 (vol/vol) , such as about 5: 1 (vol/vol) to about 1: 5 (vol/vol) .
  • the ratio of the lysis to the homogenized preparation may be at least about 50: 1 (vol/vol) , 40: 1 (vol/vol) , 30: 1 (vol/vol) , 20: 1 (vol/vol) , 10: 1 (vol/vol) , 9: 1 (vol/vol) , 8: 1 (vol/vol) , 7: 1 (vol/vol) , 6: 1 (vol/vol) , 5: 1 (vol/vol) , about 4: 1 (vol/vol) , about 3: 1 (vol/vol) , about 2: 1 (vol/vol) , about 1: 1 (vol/vol) , about 1: 2 (vol/vol) , about 1: 3 (vol/vol) , about 1: 4 (vol/vol) , about 1: 5 (vol/vol) , about 1: 6 (vol/vol) , about 1: 7 (vol/vol) , about 1: 8 (vol/vol) , about 1: 9 (vol/vol) , about 1: 10
  • a sample is obtained directly from a subject without further processing.
  • a sample is processed prior to a biological or chemical reaction (e.g., nucleic acid amplification) .
  • a reagent may be used for processing the sample.
  • the reagent for processing the sample may be added to the receptacle prior to adding a biological sample and reagents necessary for nucleic acid amplification.
  • the reagent for processing the sample may be directly added to the biological sample. In some embodiments, the reagent for processing the sample may be added to a homogenized preparation as described herein, wherein the homogenized preparation comprises the biological sample. In some embodiments, the reagent for processing the sample may be added to a pellet as described herein, wherein the pellet comprises the biological sample. In some embodiments, the reagent for processing the sample may be added to a supernatant as described herein, wherein the supernatant comprises the biological sample.
  • the reagent for processing the sample may be added to a mixture of the first buffer with the biological sample. In some embodiments, the reagent for processing the sample may be added to a mixture of the first buffer and a homogenized preparation as described herein, wherein the homogenized preparation comprises the biological sample. In some embodiments, the reagent for processing the sample may be added to the mixture of the first buffer and a pellet as described herein, wherein the pellet comprises the biological sample. In some embodiments, the reagent for processing the sample may be added to a mixture of the first buffer and a supernatant as described herein, wherein the supernatant comprises the biological sample.
  • the reagent for processing the sample may comprise a lysis buffer.
  • the lysis buffer may comprise any suitable lysis agent, including commercially available lysis agents.
  • Non-limiting examples of lysis agents include Tris-HCl, EDTA, detergents (e.g., Triton X-100, SDS) , lysozyme, glucolase, proteinase E, viral endolysins, exolysins zymolose, lyticase, proteinase K, endolysins and exolysins from bacteriophages, endolysins from bacteriophage PM2, endolysins from the B.
  • Tris-HCl EDTA
  • detergents e.g., Triton X-100, SDS
  • lysozyme e.g., Triton X-100, SDS
  • lysozyme e.g., Triton X-100, SDS
  • subtilis bacteriophage PBSX endolysins from Lactobacillus prophages Lj928, Lj965, bacteriophage 15 Phiadh, endolysin from the Streptococcus pneumoniae bacteriophage Cp-I, bifunctional peptidoglycan lysin of Streptococcus agalactiae bacteriophage B30, endolysins and exolysins from prophage bacteria, endolysins from Listeria bacteriophages, holin-endolysin, cell 20 lysis genes, holWMY Staphylococcus wameri M phage varphiWMY, Iy5WMY of the Staphylococcus wameri M phage varphiWMY, Tween 20, PEG, KOH, NaCl, and combinations thereof.
  • An example of a lysis buffer is sodium hydroxide (NaOH) .
  • the biological sample is not treated with a
  • the lysis buffer may have a pH from about 7 to 14, such as from about 8 to 13, from about 9 to 12, from about 10 to 11.
  • the lysis buffer may have a pH of greater than or equal to about 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14.
  • the biological sample or the pellet may be at a ratio to the reagent for processing the sample of about 5: 1 (wt/vol) to about 1: 500 (wt/vol) , such as about 1: 1 (wt/vol) to about 1: 100 (wt/vol) .
  • the ratio of the biological sample to the suspension buffer may be at least about 5: 1 (wt/vol) , about 4: 1 (wt/vol) , about 3: 1 (wt/vol) , about 2: 1 (wt/vol) , about 1: 1 (wt/vol) , about 1: 2 (wt/vol) , about 1: 3 (wt/vol) , about 1: 4 (wt/vol) , about 1: 5 (wt/vol) , about 1: 6 (wt/vol) , about 1: 7 (wt/vol) , about 1: 8 (wt/vol) , about 1: 9 (wt/vol) , about 1: 10 (wt/vol) , about 1: 20 (wt/vol) , about 1: 30 (wt/vol) , about 1: 40 (wt/vol) , about 1: 50 (wt/vol) , about 1: 60 (wt/vol) , about 1: 70 (wt/vol)
  • the biological sample such as a stool sample
  • a pellet comprising the biological sample such as a stool sample
  • the biological sample may be at a ratio to the reagent for processing the sample of about 5: 1 (g/ml) to about 1: 500 (g/ml) , such as about 1: 1 (g/ml) to about 1: 100 (g/ml) .
  • the ratio of the biological sample to the suspension buffer may be at least about 5: 1 (g/ml) , about 4: 1 (g/ml) , about 3: 1 (g/ml) , about 2: 1 (g/ml) , about 1: 1 (g/ml) , about 1: 2 (g/ml) , about 1: 3 (g/ml) , about 1: 4 (g/ml) , about 1: 5 (g/ml) , about 1: 6 (g/ml) , about 1: 7 (g/ml) , about 1: 8 (g/ml) , about 1: 9 (g/ml) , about 1: 10 (g/ml) , about 1: 20 (g/ml) , about 1: 30 (g/ml) , about 1: 40 (g/ml) , about 1: 50 (g/ml) , about 1: 60 (g/ml) , about 1: 70 (g/ml)
  • the reagent for processing the sample may be at a ratio to the liquid biological sample, the homogenized preparation, the suspension, or the mixture of about 50: 1 (vol/vol) to about 1: 50 (vol/vol) , such as about 5: 1 (vol/vol) to about 1: 5 (vol/vol) .
  • the ratio of the reagent for processing the sample to the liquid biological sample, the homogenized preparation, the suspension, or the mixture may be great than or equal to about 50: 1 (vol/vol) , 40: 1 (vol/vol) , 30: 1 (vol/vol) , 20: 1 (vol/vol) , 10: 1 (vol/vol) , 9: 1 (vol/vol) , 8: 1 (vol/vol) , 7: 1 (vol/vol) , 6: 1 (vol/vol) , 5: 1 (vol/vol) , 4: 1 (vol/vol) , 3: 1 (vol/vol) , 2: 1 (vol/vol) , 1: 1 (vol/vol) , 1: 2 (vol/vol) , 1: 3 (vol/vol) , 1: 4 (vol/vol) , 1: 5 (vol/vol) , 1: 6 (vol/vol) , 1: 7 (vol/vol) , 1: 8 (vol/vol) , 1: 9 (vol/vol
  • the reagent for processing the sample may comprise a reagent for extracting nucleic acids. DNA or RNA, or both may be extracted.
  • the extracting reagent may comprise ethanol, isopropanol, or phenol-chloroform for DNA extraction, or acid guanidinium thiocynate-phenol-chloroform (such as Trizol) for RNA extraction.
  • the extracting reagent may also be a solid phase such as silica, which binds or adsorbs nucleic acids.
  • the solid phase may be one that used in various spin column-based nucleic acid purification kits.
  • the reagent for processing the sample may comprise enzymes for degrading one or more components of the sample.
  • the enzymes may be DNase, RNase, proteinase, or lipase.
  • the choice of the enzyme may be dependent on the purpose of the sample processing. For example, for DNA extraction, RNase and proteinase may be used to remove RNA and proteins.
  • the reagent for processing the sample may comprise inhibitor of the enzyme.
  • the reagent for processing the sample may comprise an RNase inhibitor.
  • a sample is preserved prior to use, for example, a biological or chemical reaction (e.g., nucleic acid amplification) .
  • a reagent may be used for preserving the sample.
  • the reagent for preserving the sample may be added to the receptacle prior to use, such as adding a biological sample and reagents necessary for nucleic acid amplification.
  • preservation techniques may be selected from the group consisting of freezing, fridging, lyophilization, glassification, sol gel, and other suitable techniques for preserving samples. Accordingly, various reagents may be used for preserving the sample.
  • the reagent for preserving the sample may be a cryoprotectant.
  • a cryoprotectant protects the biological sample from freezing damages.
  • cryoprotectants include glycols such as ethylene glycol, propylene glycol, and glycerol, sugars such as trehalose, sucrose and dextran, dimethyl sulfoxide (DMSO) , etc. Many of these cryoprotectants may also serve as lyoprotectant, which protects the biological sample from damages during freeze drying. Additional cryoprotectant may comprise amino acids such as histidine or asparagine.
  • the reagent for preserving the sample may comprise reagents for facilitating or inhibiting the degradation of one or more components of the sample.
  • the reagent for preserving the sample may comprise a proteinase.
  • the reagent may also be other enzymes as described elsewhere herein.
  • the reagent may be an enzyme inhibitor for inhibiting the degradation of one or more components of the sample.
  • the reagent may comprise one or more DNase inhibitors or RNases inhibitors.
  • the reaction vessel contains reagents necessary for nucleic acid amplification.
  • the reagents include those necessary for reverse transcription amplification (e.g., reverse transcriptase) or DNA amplification (e.g., DNA polymerase) .
  • nucleic acid amplification reaction Any type of nucleic acid amplification reaction may be used to amplify a target nucleic acid and generate an amplified product. Moreover, amplification of a nucleic acid may linear, exponential, or a combination thereof. Amplification may be emulsion based or may be non-emulsion based. Non-limiting examples of nucleic acid amplification methods include reverse transcription, primer extension, polymerase chain reaction, ligase chain reaction, helicase-dependent amplification, asymmetric amplification, rolling circle amplification, and multiple displacement amplification (MDA) . In some embodiments, the amplified product may be DNA.
  • DNA can be obtained by reverse transcription of the RNA and subsequent amplification of the DNA can be used to generate an amplified DNA product.
  • the amplified DNA product may be indicative of the presence of the target RNA in the biological sample.
  • various DNA amplification protocols may be employed.
  • Non-limiting examples of DNA amplification methods include polymerase chain reaction (PCR) , variants of PCR (e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer PCR, multiplex PCR, nested PCR, overlap-extension PCR, thermal asymmetric interlaced PCR, touchdown PCR) , and ligase chain reaction (LCR) .
  • PCR polymerase chain reaction
  • variants of PCR e.g., real-time PCR, allele-specific PCR, assembly PCR, asymmetric PCR, digital PCR, emulsion PCR, dial-out PCR, helicase-dependent PCR, nested PCR, hot start PCR, inverse PCR, methylation-specific PCR, miniprimer
  • nucleic acid amplification reactions described herein may be conducted in parallel.
  • parallel amplification reactions are amplification reactions that occur in the same reaction vessel and at the same time.
  • Parallel nucleic acid amplification reactions may be conducted, for example, by including reagents necessary for each nucleic acid amplification reaction in a reaction vessel to obtain a reaction mixture and subjecting the reaction mixture to conditions necessary for each nucleic amplification reaction.
  • reverse transcription amplification and DNA amplification may be conducted in parallel, by providing reagents necessary for both amplification methods in a reaction vessel to form to obtain a reaction mixture and subjecting the reaction mixture to conditions suitable for conducting both amplification reactions.
  • DNA generated from reverse transcription of the RNA may be amplified in parallel to generate an amplified DNA product. Any suitable number of nucleic acid amplification reactions may be conducted in parallel. In some cases, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleic acid amplification reactions are conducted in parallel.
  • a target nucleic acid e.g., target RNA, target DNA
  • a target nucleic acid may be extracted or released from a biological sample during heating phases of parallel nucleic acid amplification.
  • the biological sample comprising the target RNA can be heated and the target RNA released from the biological sample.
  • the released target RNA can immediately begin reverse transcription (via reverse transcription amplification) to produce complementary DNA.
  • the complementary DNA can then be immediately amplified, often on the order of seconds. Short times between release of a target RNA from a biological sample and reverse transcription of the target RNA to complementary DNA may help minimize the effects of inhibitors in the biological sample that may impede reverse transcription and/or DNA amplification.
  • primer sets directed to a target nucleic acid may be utilized to conduct nucleic acid amplification reaction.
  • the reagents necessary for conducting the nucleic acid amplification reaction may comprise one or more primer sets.
  • Primer sets generally comprise one or more primers.
  • a primer set may comprise about at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primers.
  • a primer set or may comprise primers directed to different amplified products or different nucleic acid amplification reactions.
  • a primer set may comprise a first primer that can be used to generate a first strand of nucleic acid product that is complementary to at least a portion of the target nucleic acid and a second primer complementary to the nucleic acid strand product that can be used to generate a second strand of nucleic acid product that is complementary to at least a portion of the first strand of nucleic acid product.
  • a primer set may be directed to a target RNA.
  • the primer set may comprise a first primer that can be used to generate a first strand of nucleic acid product that is complementary to at least a portion the target RNA.
  • the first strand of nucleic acid product may be DNA.
  • the primer set may also comprise a second primer that can be used to generate a second strand of nucleic acid product that is complementary to at least a portion of the first strand of nucleic acid product.
  • the second strand of nucleic acid product may be a strand of nucleic acid (e.g., DNA) product that is complementary to a strand of DNA generated from an RNA template.
  • a strand of nucleic acid e.g., DNA
  • primer sets Any suitable number of primer sets may be used. For example, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more primer sets may be used. Where multiple primer sets are used, one or more primer sets may each correspond to a particular nucleic acid amplification reaction or amplified product.
  • each primer set of the one or more primer sets is capable of specifically binding to a target nucleic acid sequence from a microbial genome or a microbial transcriptome, or a variant thereof.
  • the microbial genome is a genome of a microbe as described herein.
  • the microbial transcriptome is a transcriptome of a microbe as described herein.
  • the target nucleic acid may be a target RNA or a target DNA.
  • the target nucleic acid may be a target DNA sequence from the genome of a microbe as described herein.
  • the target nucleic acid may be a target RNA sequence from the transcriptome of a microbe as described herein.
  • the target nucleic acid may be other type of target RNA sequences, such as rRNA, tRNA, nRNA, siRNA, snRNA, snoRNA, scaRNA, microRNA, dsRNA, but not limited thereto.
  • the microbe may be a bacterium selected from Bacteroides, Bifidobacterium, Erwinia, Escherichia, Faecalibacterium, Lactobacillus, Ruminococcus, Prevotella, Streptococcus, Veillonella, Coprococcus, Roseburia, and Lactobacillus rhamnosus GG strain.
  • the target nucleic acid is a target RNA
  • it may be any type of RNA, including types of RNA as described elsewhere herein.
  • the target RNA is an mRNA.
  • the target RNA is a mRNA of a microbe as described herein.
  • the target RNA may be selected from an mRNA with a high expression abundance in a microbe as described herein.
  • the target RNA is recA mRNA.
  • the microbe may be a bacterium selected from Bacteroides, Bifidobacterium, Escherichia, Erwinia, Faecalibacterium, Lactobacillus, Ruminococcus, Prevotella, Streptococcus, Veillonella, Coprococcus, Roseburia, and Lactobacillus rhamnosus GG strain.
  • the target nucleic acid is a target DNA
  • it may be any type of DNA, including types of DNA as described elsewhere herein.
  • the target DNA is a genome DNA.
  • the target DNA is a genome DNA of a microbe as described herein.
  • the target DNA may be selected from a gene specific to and/or conservative among the microbes as described herein.
  • the target DNA is the 16SrDNA.
  • the microbe may be a bacterium selected from Bacteroides, Bifidobacterium, Erwinia, Escherichia, Faecalibacterium, Lactobacillus, Ruminococcus, Prevotella, Streptococcus, Veillonella, Coprococcus, Roseburia, and Lactobacillus rhamnosus GG strain.
  • the one or more primer sets may comprise a primer set capable of specifically binding to a target nucleic acid sequence from a bacterial genome or a variant thereof. In some embodiments, the one or more primer sets may comprise a primer set capable of specifically binding to a target nucleic acid sequence from a bacterial transcriptome or a variant thereof. In some embodiments, the one or more primer sets may comprise both a primer set capable of specifically binding to a target nucleic acid sequence from a bacterial genome or a variant thereof and a primer set capable of specifically binding to a target nucleic acid sequence from a bacterial transcriptome or a variant thereof.
  • the reagent may comprise a forward primer.
  • the reagent may comprise any amount of the forward primer suitable for conducting the nucleic acid amplification.
  • the reagent may comprise less than or equal to about 15.0 ⁇ M, 14.0 ⁇ M, 13.0 ⁇ M, 12.0 ⁇ M, 11.0 ⁇ M, 10.0 ⁇ M, 9.0 ⁇ M, 8.0 ⁇ M, 7.0 ⁇ M, 6.0 ⁇ M, 5.0 ⁇ M, 4.5 ⁇ M, 4.0 ⁇ M, 3.5 ⁇ M, 3.0 ⁇ M, 2.5 ⁇ M, 2.0 ⁇ M, 1.9 ⁇ M, 1.8 ⁇ M, 1.7 ⁇ M, 1.6 ⁇ M, 1.5 ⁇ M, 1.4 ⁇ M, 1.3 ⁇ M, 1.2 ⁇ M, 1.1 ⁇ M, 1.0 ⁇ M, 0.9 ⁇ M, 0.8 ⁇ M, 0.7 ⁇ M, 0.6 ⁇ M, 0.5 ⁇ M, 0.4 ⁇ M, 1.3 ⁇ M, 1.2
  • the reagent may comprise greater than or equal to about 0.01 ⁇ M, 0.05 ⁇ M, 0.06 ⁇ M, 0.07 ⁇ M, 0.08 ⁇ M, 0.09 ⁇ M, 0.1 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.5 ⁇ M, 3.0 ⁇ M, 3.5 ⁇ M, 4.0 ⁇ M, 4.5 ⁇ M, 5.0 ⁇ M, 6.0 ⁇ M, 7.0 ⁇ M, 8.0 ⁇ M, 9.0 ⁇ M, 10.0 ⁇ M, 11.0 ⁇ M, 12.0 ⁇ M, 13.0 ⁇ M, 14.0
  • the reagent may comprise a forward primer having any range between the aforesaid numeric values, such as about 0.01 to 10.0 ⁇ M, 0.05 to 5.0 ⁇ M, 0.05 to 4.0 ⁇ M, 0.05 to 3.0 ⁇ M, 0.05 to 2.0 ⁇ M, 0.05 to 1.0 ⁇ M, 0.05 to 0.5 ⁇ M, 0.1 to 5.0 ⁇ M, 0.1 to 4.0 ⁇ M, 0.1 to 3.0 ⁇ M, 0.1 to 2.0 ⁇ M, 0.1 to 1.0 ⁇ M, 0.1 to 0.9 ⁇ M, 0.1 to 0.8 ⁇ M, 0.1 to 0.7 ⁇ M, 0.1 to 0.6 ⁇ M, 0.1 to 0.5 ⁇ M forward primer.
  • the reagent may comprise about 0.1 to 1.0 ⁇ M forward primer.
  • the reagent may comprise a reverse primer.
  • the reagent may comprise any amount of the reverse primer suitable for conducting the nucleic acid amplification.
  • the reagent may comprise less than or equal to about 15.0 ⁇ M, 14.0 ⁇ M, 13.0 ⁇ M, 12.0 ⁇ M, 11.0 ⁇ M, 10.0 ⁇ M, 9.0 ⁇ M, 8.0 ⁇ M, 7.0 ⁇ M, 6.0 ⁇ M, 5.0 ⁇ M, 4.5 ⁇ M, 4.0 ⁇ M, 3.5 ⁇ M, 3.0 ⁇ M, 2.5 ⁇ M, 2.0 ⁇ M, 1.9 ⁇ M, 1.8 ⁇ M, 1.7 ⁇ M, 1.6 ⁇ M, 1.5 ⁇ M, 1.4 ⁇ M, 1.3 ⁇ M, 1.2 ⁇ M, 1.1 ⁇ M, 1.0 ⁇ M, 0.9 ⁇ M, 0.8 ⁇ M, 0.7 ⁇ M, 0.6 ⁇ M, 0.5 ⁇ M, 0.4 ⁇ M, 1.3 ⁇ M, 1.2
  • the reagent may comprise more than or equal to about 0.01 ⁇ M, 0.05 ⁇ M, 0.06 ⁇ M, 0.07 ⁇ M, 0.08 ⁇ M, 0.09 ⁇ M, 0.1 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.5 ⁇ M, 3.0 ⁇ M, 3.5 ⁇ M, 4.0 ⁇ M, 4.5 ⁇ M, 5.0 ⁇ M, 6.0 ⁇ M, 7.0 ⁇ M, 8.0 ⁇ M, 9.0 ⁇ M, 10.0 ⁇ M, 11.0 ⁇ M, 12.0 ⁇ M, 13.0 ⁇ M, 14.0
  • the reagent may comprise a reverse primer having any range between the aforesaid numeric values, such as about 0.01 to 10.0 ⁇ M, 0.05 to 5.0 ⁇ M, 0.05 to 4.0 ⁇ M, 0.05 to 3.0 ⁇ M, 0.05 to 2.0 ⁇ M, 0.05 to 1.0 ⁇ M, 0.05 to 0.5 ⁇ M, 0.1 to 5.0 ⁇ M, 0.1 to 4.0 ⁇ M, 0.1 to 3.0 ⁇ M, 0.1 to 2.0 ⁇ M, 0.1 to 1.0 ⁇ M, 0.1 to 0.9 ⁇ M, 0.1 to 0.8 ⁇ M, 0.1 to 0.7 ⁇ M, 0.1 to 0.6 ⁇ M, 0.1 to 0.5 ⁇ M reverse primer.
  • the reagent may comprise about 0.1 to 1.0 ⁇ M reverse primer.
  • a DNA polymerase is used. Any suitable DNA polymerase may be used, including commercially available DNA polymerases.
  • a DNA polymerase generally refers to an enzyme that is capable of incorporating nucleotides to a strand of DNA in a template bound fashion.
  • Non-limiting examples of DNA polymerases include Taq polymerase, Tth polymerase, Tli polymerase, Pfu polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerases, Sso polymerase, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Klenow fragment, and variants, modified products and derivatives thereof.
  • Hot Start Polymerase denaturation at about 94°C -
  • the reagent necessary for conducting the nucleic acid amplification may comprise a reverse transcriptase.
  • the reagent as described herein may comprise a reverse transcriptase.
  • a reverse transcriptase generally refers to an enzyme that is capable of incorporating nucleotides to a strand of DNA, when bound to an RNA template.
  • Non-limiting examples of reverse transcriptases include HIV-1 reverse transcriptase, M-MLV reverse transcriptase, AMV reverse transcriptase, telomerase reverse transcriptase, and variants, modified products and derivatives thereof.
  • nucleotide analogs include ribonuclotide and deoxyribonucleotide analogs.
  • nucleotide analogs include, adenosine, thymidine, guanosine, cytidine, uracil, and analogs of these bases.
  • the analogs may comprise nucleoside triphosphates, or may include additional phosphate groups, e.g., tetraphosphates, pentaphosphates, hexaphosphates, heptaphosphates, or greater.
  • primer extension reactions are utilized to generate amplified product.
  • Primer extension reactions generally comprise a cycle of incubating a reaction mixture at a denaturation temperature for a denaturation duration and incubating a reaction mixture at an elongation temperature for an elongation duration.
  • reagents necessary for conducting nucleic acid amplification may also include a reporter agent.
  • the reporter agent may yield a detectable signal.
  • the detectable signal may indicate whether the amplified product is present. For example, the presence or absence of the detectable signal may be indicative of the presence of an amplified product.
  • the intensity of the detectable signal may be proportional to the amount of amplified product. In some cases, where amplified product is generated of a different type of nucleic acid than the target nucleic acid initially amplified, the intensity of the detectable signal may be proportional to the amount of target nucleic acid initially amplified.
  • reagents necessary for both reactions may also comprise a reporter agent may yield a detectable signal that is indicative of the presence of the amplified DNA product and/or the target RNA amplified.
  • the intensity of the detectable signal may be proportional to the amount of the amplified DNA product and/or the original target RNA amplified.
  • Reporter agents may be linked with nucleic acids, including amplified products, by covalent or non-covalent interactions.
  • Non-limiting examples of non-covalent interactions include ionic interactions, Van der Waals forces, hydrophobic interactions, hydrogen bonding, and combinations thereof.
  • reporter agents may bind to initial reactants and changes in reporter agent levels may be used to detect amplified product.
  • reporter agents may only be detectable (or non-detectable) as nucleic acid amplification progresses.
  • an optically-active dye e.g., a fluorescent dye
  • Non-limiting examples of dyes include SYBR green, SYBR blue, DAPI, propidium iodine, Hoeste, SYBR gold, ethidium bromide, acridines, proflavine, acridine orange, acriflavine, fluorcoumanin, ellipticine, daunomycin, chloroquine, distamycin D, chromomycin, homidium, mithramycin, ruthenium polypyridyls, anthramycin, phenanthridines and acridines, ethidium bromide, propidium iodide, hexidium iodide, dihydroethidium, ethidium homodimer-1 and -2, ethidium monoazide, and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, acridine orange, 7-AAD, actinomycin D,
  • a reporter agent may be a sequence-specific oligonucleotide probe that is optically active when hybridized with an amplified product. Due to sequence-specific binding of the probe to the amplified product, use of oligonucleotide probes can increase specificity and sensitivity of detection.
  • a probe may be linked to any of the optically-active reporter agents (e.g., dyes) described herein and may also include a quencher capable of blocking the optical activity of an associated dye.
  • Non-limiting examples of probes that may be useful used as reporter agents include TaqMan probes, TaqMan Tamara probes, TaqMan MGB probes, or Lion probes.
  • a reporter agent may be a sequence specific oligonucleotide probe having blocked optical activity upon hybridization with an amplification product.
  • the oligonucleotide probe becomes optically active upon its breakdown.
  • the reporter agent may be an oliognucleotide probe that includes an optically-active dye (e.g., fluorescent dye) and a quencher positioned adjacently on the probe. The close proximity of the dye with the quencher can block the optical activity of the dye.
  • the probe may bind to a target sequence to be amplified.
  • the oligonucleotide probe may be an RNA oligonucleotide probe or a DNA oligonucleotide probe.
  • a reporter agent may be a molecular beacon.
  • a molecular beacon includes, for example, a quencher linked at one end of an oligonucleotide in a hairpin conformation. At the other end of the oligonucleotide is an optically active dye, such as, for example, a fluorescent dye. In the hairpin configuration, the optically-active dye and quencher are brought in close enough proximity such that the quencher is capable of blocking the optical activity of the dye.
  • the oligonucleotide Upon hybridizing with amplified product, however, the oligonucleotide assumes a linear conformation and hybridizes with a target sequence on the amplified product.
  • Linearization of the oligonucleotide results in separation of the optically-active dye and quencher, such that the optical activity is restored and can be detected.
  • sequence specificity of the molecular beacon for a target sequence on the amplified product can improve specificity and sensitivity of detection.
  • an oligonucleotide probe or a molecular beacon as describe herein may be a sequence specific oligonucleotide probe or a sequence specific molecular beacon.
  • the oligonucleotide probe or the molecular beacon as describe herein hybridizes with a region on the target nucleic acid between the primers in the primer set capable of specifically binding to the target nucleic acid.
  • the primer set is used for amplification, an amplified product corresponding to the region on the target nucleic acid between the primers (including the primers) is produced. Therefore, the oligonucleotide probe or the molecular beacon as describe herein may hybridize with the amplified product.
  • the oligonucleotide probe or the molecular beacon as describe herein may hybridize with the amplified product produced from the amplification of the target nucleic acid.
  • the reagent may comprise one or more oligonucleotide probes or molecular beacons.
  • the reagent may comprise any amount of the one or more oligonucleotide probes or molecular beacons suitable for conducting the nucleic acid amplification.
  • the amount of the one or more oligonucleotide probes or molecular beacons may be less than or equal to about 15.0 ⁇ M, 14.0 ⁇ M, 13.0 ⁇ M, 12.0 ⁇ M, 11.0 ⁇ M, 10.0 ⁇ M, 9.0 ⁇ M, 8.0 ⁇ M, 7.0 ⁇ M, 6.0 ⁇ M, 5.0 ⁇ M, 4.5 ⁇ M, 4.0 ⁇ M, 3.5 ⁇ M, 3.0 ⁇ M, 2.5 ⁇ M, 2.0 ⁇ M, 1.9 ⁇ M, 1.8 ⁇ M, 1.7 ⁇ M, 1.6 ⁇ M, 1.5 ⁇ M, 1.4 ⁇ M, 1.3 ⁇ M, 1.2 ⁇ M, 1.1 ⁇ M, 1.0 ⁇ M, 0.9 ⁇ M, 0.8 ⁇ M, 0.7 ⁇ M, 0.6 ⁇ M, 0.5 ⁇ M, 0.4 ⁇ M, 0.3 ⁇ M, 0.2 ⁇ M, 0.1 ⁇ M, 0.09 ⁇ M, 0.08
  • the amount of the one or more oligonucleotide probes or molecular beacons may be greater than or equal to about 0.01 ⁇ M, 0.01 ⁇ M, 0.05 ⁇ M, 0.06 ⁇ M, 0.07 ⁇ M, 0.08 ⁇ M, 0.09 ⁇ M, 0.1 ⁇ M, 0.2 ⁇ M, 0.3 ⁇ M, 0.4 ⁇ M, 0.5 ⁇ M, 0.6 ⁇ M, 0.7 ⁇ M, 0.8 ⁇ M, 0.9 ⁇ M, 1.0 ⁇ M, 1.1 ⁇ M, 1.2 ⁇ M, 1.3 ⁇ M, 1.4 ⁇ M, 1.5 ⁇ M, 1.6 ⁇ M, 1.7 ⁇ M, 1.8 ⁇ M, 1.9 ⁇ M, 2.0 ⁇ M, 2.5 ⁇ M, 3.0 ⁇ M, 3.5 ⁇ M, 4.0 ⁇ M, 4.5 ⁇ M, 5.0 ⁇ M, 6.0 ⁇ M, 7.0 ⁇ M, 8.0 ⁇ M, 9.0 ⁇ M, 1
  • the reagent may comprise one or more oligonucleotide probes or molecular beacons having an amount between the aforesaid numeric values, such as about 0.01 to 10.0 ⁇ M, 0.05 to 5.0 ⁇ M, 0.05 to 4.0 ⁇ M, 0.05 to 3.0 ⁇ M, 0.05 to 2.0 ⁇ M, 0.05 to 1.0 ⁇ M, 0.05 to 0.5 ⁇ M, 0.1 to 5.0 ⁇ M, 0.1 to 4.0 ⁇ M, 0.1 to 3.0 ⁇ M, 0.1 to 2.0 ⁇ M, 0.1 to 1.0 ⁇ M, 0.1 to 0.9 ⁇ M, 0.1 to 0.8 ⁇ M, 0.1 to 0.7 ⁇ M, 0.1 to 0.6 ⁇ M, 0.1 to 0.5 ⁇ M one or more oligonucleotide probes or molecular beacons.
  • the reagent may comprise about 0.1 to 0.5 ⁇ M one or more oligonucleotide probes or molecular beacons.
  • a reporter agent may be a radioactive species.
  • radioactive species include 14 C, 123 I, 124 I, 125 I, 131 I, Tc 99m , 35 S, or 3 H.
  • a reporter agent may be an enzyme that is capable of generating a detectable signal. Detectable signal may be produced by activity of the enzyme with its substrate or a particular substrate in the case the enzyme has multiple substrates.
  • enzymes that may be used as reporter agents include alkaline phosphatase, horseradish peroxidase, I2-galactosidase, alkaline phosphatase, ⁇ -galactosidase, acetylcholinesterase, and luciferase.
  • a plurality of reporter agents may be employed to detect a plurality of amplified products.
  • Each of the plurality of reporter agents may produce a detectable signal, while the plurality of detectable signals produced by the plurality of reporter agents is distinct among one another.
  • Each of the plurality of detectable signals may be indicative of the presence or absence of the corresponding amplified product.
  • the intensity of each of the plurality of detectable signals may be proportional to the amount of the corresponding amplified product.
  • the detectable signal corresponding to one amplified product may be FAM fluorescence
  • the detectable signal corresponding to another amplified product may be ROX fluorescence.
  • Detection of distinct detectable signals in parallel may allow comparison of the presence and/or amount among different amplified products.
  • detection of distinct detectable signals in parallel may allow determination of the amount of the amplified product (s) relative to an internal reference.
  • the reagent may further comprise MgCl 2 .
  • the reagent may comprise any amount of MgCl 2 suitable for conducting the nucleic acid amplification.
  • the reagent may comprise an amount of MgCl 2 less than or equal to about 15.0 mM, 14.0 mM, 13.0 mM, 12.0 mM, 11.0 mM, 10.0 mM, 9.0 mM, 8.0 mM, 7.0 mM, 6.0 mM, 5.0 mM, 4.5 mM, 4.0 mM, 3.5 mM, 3.0 mM, 2.5 mM, 2.0 mM, 1.9 mM, 1.8 mM, 1.7 mM, 1.6 mM, 1.5 mM, 1.4 mM, 1.3 mM, 1.2 mM, 1.1 mM, 1.0 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 1.3 mM,
  • the reagent may comprise an amount of MgCl 2 greater than or equal to about 0.01 mM, 0.01 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM, 10.0 mM, 11.0 mM,
  • the reagent may comprise an amount of MgCl 2 between the aforesaid numeric values, such as about 0.01 to 15.0 mM, 0.05 to 14.0 mM, 0.1 to 13.0 mM, 0.2 to 12.0 mM, 0.3 to 11.0 mM, 0.4 to 10.0 mM, 0.5 to 9.0 mM, 0.6 to 8.0 mM, 0.7 to 7.0 mM, 0.8 to 6.0 mM, 0.9 to 5.0 mM, 1.0 to 4.0 mM, 1.1 to 3.0 mM, 1.2 to 2.5 mM, 1.3 to 2.0 mM, or 1.4 to1.6 mM MgCl 2 .
  • the reagent may comprise about 1.5 mM MgCl 2 .
  • the reagent may further comprise dNTPs.
  • the reagent may comprise any amount of dNTPs suitable for conducting the nucleic acid amplification.
  • the reagent may comprise an amount of dNTPs less than or equal to about 15.0 mM, 14.0 mM, 13.0 mM, 12.0 mM, 11.0 mM, 10.0 mM, 9.0 mM, 8.0 mM, 7.0 mM, 6.0 mM, 5.0 mM, 4.5 mM, 4.0 mM, 3.5 mM, 3.0 mM, 2.5 mM, 2.0 mM, 1.9 mM, 1.8 mM, 1.7 mM, 1.6 mM, 1.5 mM, 1.4 mM, 1.3 mM, 1.2 mM, 1.1 mM, 1.0 mM, 0.9 mM, 0.8 mM, 0.7 mM, 0.6 mM, 1.3 mM, 1.2
  • the reagent may comprise an amount of dNTPs greater than or equal to about 0.01 mM, 0.01 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1.0 mM, 1.1 mM, 1.2 mM, 1.3 mM, 1.4 mM, 1.5 mM, 1.6 mM, 1.7 mM, 1.8 mM, 1.9 mM, 2.0 mM, 2.5 mM, 3.0 mM, 3.5 mM, 4.0 mM, 4.5 mM, 5.0 mM, 6.0 mM, 7.0 mM, 8.0 mM, 9.0 mM, 10.0 mM, 11.0 mM,
  • the reagent may comprise an amount of dNTPs between the aforesaid numeric values, such as about 0.01 to 10.0 mM, 0.05 to 5.0 mM, 0.05 to 4.0 mM, 0.05 to 3.0 mM, 0.05 to 2.0 mM, 0.05 to 1.0 mM, 0.05 to 0.5 mM, 0.1 to 5.0 mM, 0.1 to 4.0 mM, 0.1 to 3.0 mM, 0.1 to 2.0 mM, 0.1 to 1.0 mM, 0.1 to 0.9 mM, 0.1 to 0.8 mM, 0.1 to 0.7 mM, 0.1 to 0.6 mM, 0.1 to 0.5 mM dNTPs.
  • the reagent may comprise about 1.5 mM dNTPs.
  • amplified product e.g., amplified DNA product, amplified RNA product
  • Detection of amplified product, including amplified DNA may be accomplished with any suitable detection method.
  • the particular type of detection method used may depend, for example, on the particular amplified product, the type of reaction vessel used for amplification, other reagents in a reaction mixture, whether or not a reporter agent was included in a reaction mixture, and if a reporter agent was used, the particular type of reporter agent use.
  • detection methods include optical detection, spectroscopic detection, electrostatic detection, electrochemical detection, and the like.
  • Optical detection methods include, but are not limited to, fluorimetry and UV-vis light absorbance.
  • Spectroscopic detection methods include, but are not limited to, mass spectrometry, nuclear magnetic resonance (NMR) spectroscopy, and infrared spectroscopy.
  • Electrostatic detection methods include, but are not limited to, gel based techniques, such as, for example, gel electrophoresis.
  • Electrochemical detection methods include, but are not limited to, electrochemical detection of amplified product after high-performance liquid chromatography separation of the amplified products.
  • the amplified product may be detected in the primer extension reaction. In some embodiments, the amplified product may be detected in a plurality of series of the primer extension reaction. In some embodiments, the amplified product may be detected in each individual series of the plurality of series of the primer extension reaction. In some embodiments, the amplified product may be detected in some individual series, but not other individual series of the plurality of series of the primer extension reaction.
  • the plurality of series of the primer extension reaction may comprise a first series and a second series. The amplified product may be detected not in the first series, but in the second series. Alternatively, the amplified product may be detected not in the second series, but in the first series. Alternatively, the amplified product may be detected in both the first series and the second series, or in neither the first series nor the second series.
  • the detectable signal produced by the reporter agent may be detected in the primer extension reaction. In some embodiments, the detectable signal produced by the reporter agent may be detected in a plurality of series of the primer extension reaction. In some embodiments, the detectable signal produced by the reporter agent may be detected in each individual series of the plurality of series of the primer extension reaction. In some embodiments, the detectable signal produced by the reporter agent may be detected in some individual series, but not other individual series of the plurality of series of the primer extension reaction.
  • the plurality of series of the primer extension reaction may comprise a first series and a second series. The detectable signal produced by the reporter agent may be detected not in the first series, but in the second series. Alternatively, the detectable signal produced by the reporter agent may be detected not in the second series, but in the first series. Alternatively, the detectable signal produced by the reporter agent may be detected in both the first series and the second series, or in neither the first series nor the second series.
  • the time required to complete the elements of a method may vary depending upon the particular components of the method. For example, an amount of time for completing the elements of a method may be from about 5 minutes to about 120 minutes. In other examples, an amount of time for completing the elements of a method may be from about 5 minutes to about 60 minutes. In other examples, an amount of time for completing the elements of a method may be from about 5 minutes to about 30 minutes.
  • an amount of time for completing the elements of a method may be less than or equal to 120 minutes, less than or equal to 90 minutes, less than or equal to 75 minutes, less than or equal to 60 minutes, less than or equal to 45 minutes, less than or equal to 40 minutes, less than or equal to 35 minutes, less than or equal to 30 minutes, less than or equal to 25 minutes, less than or equal to 20 minutes, less than or equal to 15 minutes, less than or equal to 10 minutes, or less than or equal to 5 minutes.
  • the present disclosure provides a computer assisted method of profiling a plurality of microbes in a biological sample.
  • the method comprises: (a) obtaining results of a nucleic acid amplification reaction comprising amplified nucleic acids from at least one beneficial microbe and at least one pathogenic microbe; (b) determining, with a computer system, a quantity or concentration of the at least one beneficial microbe and at least one pathogenic microbe; and (c) generating a report of results from (b) .
  • Methods of amplification, sample sources, and microbes can be any of those described above, with regard to any of the various aspects of the disclosure.
  • a computer for use in the system can comprise one or more processors.
  • Processors may be associated with one or more controllers, calculation units, and/or other units of a computer system, or implanted in firmware.
  • the routines may be stored in any computer readable memory such as in RAM, ROM, flash memory, a magnetic disk, a laser disk, or other suitable storage medium.
  • this software may be delivered to a computing device via any delivery method including, for example, over a communication channel such as a telephone line, the internet, a wireless connection, etc., or via a transportable medium, such as a computer readable disk, flash drive, etc.
  • the various components may be implemented as various blocks, operations, tools, modules and techniques which, in turn, may be implemented in hardware, firmware, software, or any combination of hardware, firmware, and/or software.
  • some or all of the blocks, operations, techniques, etc. may be implemented in, for example, a custom integrated circuit (IC) , an application specific integrated circuit (ASIC) , a field programmable logic array (FPGA) , a programmable logic array (PLA) , etc.
  • IC integrated circuit
  • ASIC application specific integrated circuit
  • FPGA field programmable logic array
  • PDA programmable logic array
  • a client-server, relational database architecture can be used in embodiments of the system.
  • a client-server architecture is a network architecture in which each computer or process on the network is either a client or a server.
  • Server computers are typically powerful computers dedicated to managing disk drives (file servers) , printers (print servers) , or network traffic (network servers) .
  • Client computers include PCs (personal computers) or workstations on which users run applications, as well as any suitable output devices.
  • Client computers rely on server computers for resources, such as files, devices, and even processing power.
  • the server computer handles all of the database functionality.
  • the client computer can have software that handles all the front-end data management and can also receive data input from users.
  • the computer system can regulate various aspects of the methods of the present disclosure, such as, for example, sample preparation, sample amplification, signal detection; signal transformation, and data collection.
  • a results report is generated.
  • a report may be generated in real-time, such as during the nucleic acid amplification reaction, or a result of the nucleic acid amplification reaction being analyzed with periodic updates as the process progresses.
  • a report may be generated at the conclusion of the assessment.
  • the report may be generated automatically.
  • the report is generated in response to instructions from a user.
  • the report may include any number of elements, with non-limiting examples that include information regarding the subject (e.g., sex, age, race, health status, etc.
  • raw data e.g., raw data, processed data (e.g., graphical displays (e.g., figures, charts, data tables, data summaries) , determined quantity or concentration of beneficial and pathogenic microbes, determined relative quantity or concentration of beneficial and pathogenic microbes, determined tissue distribution or relative tissue distribution of beneficial and pathogenic microbes, determined change of relative quantity or concentration of beneficial and pathogenic microbes, determined change of tissue distribution or relative tissue distribution of beneficial and pathogenic microbes, and the like) , and results of the profiling of the beneficial and pathogenic microbes.
  • a report may also contain an analysis based on the above information.
  • the report may include information concerning this association, such as a likelihood that the disease, condition, disorder, or a symptom thereof, or other negative impacts to the health is present, at what level (e.g., diagnosis information, prognosis information, disease information, and any combination thereof) , and optionally a suggestion based on this information (e.g., additional tests, monitoring, or remedial measures) .
  • the report can take any of a variety of forms.
  • Data relating to the present disclosure can be transmitted over such networks or connections (or any other suitable system for transmitting information, including but not limited to mailing a physical report, such as a print-out) for reception and/or for review by a receiver.
  • the receiver can be but is not limited to an individual, or electronic system (e.g., one or more computers, and/or one or more servers) .
  • the present disclosure provides a system for profiling a plurality of microbes in a biological sample.
  • the system comprises: (a) a memory unit configured to store results of a nucleic acid amplification reaction of nucleic acids from at least one beneficial microbe and at least one pathogenic microbe; and (b) an output unit that delivers a report to a recipient, wherein the report is based on the results of (a) .
  • the results stored by the memory unit may include one or more information selected from the group consisting of: (i) quantity and/or concentration of the at least one beneficial microbe and the at least one pathogenic microbe, and (ii) relative quantity and/or concentration of the at least one beneficial microbe and the at least one pathogenic microbe.
  • the system may further comprise one or more processors alone or in combination that are programmed to calculate a quantity or concentration of the at least one beneficial microbe and the at least one pathogenic microbe.
  • processors alone or in combination that are programmed to calculate a quantity or concentration of the at least one beneficial microbe and the at least one pathogenic microbe.
  • the report may be transmitted to the recipient at a local or remote location using any suitable communication medium including, for example, a network connection, a hard-wired connection, a wireless connection, a local intranet, or an internet connection.
  • the report can be sent to a recipient's device.
  • the recipient's device include personal computers (e.g., portable PC) , slate or tablet PC's (e.g., iPad, Galaxy Tab) , telephones, Smart phones (e.g., iPhone, Android-enabled device, ) , or personal digital assistants.
  • the system may comprise a computer and various other elements, some or all of which may be controlled by the computer.
  • Example computer systems are described above.
  • FIG. 1 illustrates a system in accordance with an embodiment.
  • the computer system can regulate various aspects of the methods of the present disclosure, such as, for example, sample acquisition, sample preparation, nucleic acid amplification, signal detection; signal transformation, data collection, and data processing.
  • the computer system can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the processor is a single core or multi core processor, or a plurality of processors for parallel processing.
  • the memory unit includes memory or memory location (e.g., random-access memory, read-only memory, flash memory) .
  • the system also includes an electronic storage unit (e.g., hard disk) , communication interface (e.g., network adapter) for communicating with one or more other systems, and peripheral devices, such as cache, other memory, data storage and/or electronic display adapters.
  • the memory unit, storage unit, interface and peripheral devices may be in communication with the one or more processors through a communication bus (solid lines) , such as a motherboard.
  • the storage unit can be a data storage unit (or data repository) for storing data.
  • the system can be operatively coupled to a computer network ( "network" ) with the aid of the communication interface.
  • the network can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network in some cases is a telecommunication and/or data network.
  • the network can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network in some cases with the aid of the computer system, can implement a peer-to-peer network, which may enable devices coupled to the computer system to behave as a client or a server.
  • the one or more processors can execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory.
  • the instructions can be directed to the one or more processors, which can subsequently program or otherwise configure the one or more processors to implement methods of the present disclosure. Examples of operations performed by the one or more processors of the present disclosure can include fetch, decode, execute, and writeback.
  • the one or more processors can be part of a circuit, such as an integrated circuit. One or more other components of the system can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC) .
  • ASIC application specific integrated circuit
  • the storage unit can store files, such as drivers, libraries and saved programs.
  • the storage unit can store user data, e.g., user preferences and user programs.
  • the computer system in some cases can include one or more additional data storage units that are external to the computer system, such as located on a remote server that is in communication with the computer system through an intranet or the Internet.
  • the system can include or be in communication with an electronic display that comprises a user interface (UI) for providing, for example, temperature levels, thermal cycling protocol conditions, and signal data from sample volumes.
  • UI user interface
  • Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • the system may comprise an output unit operatively connected to the memory unit.
  • the output unit may include input devices as described herein and/or may comprise input electronics for communication with the memory unit.
  • the output unit may be an electronic display, such as a display on a nucleic acid amplification device or a separate display device.
  • the electronic display may comprise a UI.
  • the output unit is a communication interface operatively coupled to a computer network such as, for example, the internet.
  • the output unit may transmit information to a recipient at a local or remote location using any suitable communication medium, including a computer network, a wireless network, a local intranet, or the internet.
  • the output unit is capable of analyzing data received from the memory unit.
  • the output unit may analyze information in real-time while signal detection and/or processing is occurring. Some data may be analyzed after the signal detection and/or processing has been completed.
  • the output unit includes a report generator capable of generating a report and transmitting the report as described herein to a recipient.
  • the output unit transmits information automatically in response to information received from the memory unit, such as in the form of raw data or data analysis performed by software included in the memory unit.
  • the output unit may transmit information after receiving instructions from a user. Information transmitted by the output unit may be viewed electronically or printed from a printer.
  • the system may further comprise an input unit that receives a user request to profile microbes (e.g., at least one beneficial microbe and at least one pathogenic microbe) present in a biological sample.
  • a user request to profile microbes (e.g., at least one beneficial microbe and at least one pathogenic microbe) present in a biological sample.
  • Any suitable module capable of accepting such a user request may be used.
  • the input unit may comprise, for example, a device that comprises one or more processors.
  • the input unit may be built into the system.
  • the input unit may be integrated into a housing of the system or accessible from outside the housing. Alternatively, the input unit may be separate or separable from the system.
  • the input unit may communicate with the system over a connection, such as those described elsewhere herein.
  • the input unit is configured to receive a user request to perform profiling of the microbes.
  • the input unit may receive the user request directly (e.g., by way of an input device such as a keyboard, mouse, or touch screen operated by the user) or indirectly (e.g., through a wired or wireless connection, including over the internet) . Via output electronics, the input unit may provide the user's request to the system.
  • an input unit may include a user interface (UI) , such as a graphical user interface (GUI) , that is configured to enable a user provide a request to profile the microbes.
  • UI user interface
  • GUI graphical user interface
  • a GUI can include textual, graphical and/or audio components.
  • a GUI can be provided on an electronic display, including the display of a system comprising a computer processor. Such a display may include a resistive or capacitive touch screen.
  • Non-limiting examples of users include the subject from which the biological sample was obtained, medical personnel, clinicians (e.g., doctors, nurses, laboratory technicians) , laboratory personnel (e.g., hospital laboratory technicians, research scientists, pharmaceutical scientists) , a clinical monitor for a clinical trial, or others in the health care industry.
  • clinicians e.g., doctors, nurses, laboratory technicians
  • laboratory personnel e.g., hospital laboratory technicians, research scientists, pharmaceutical scientists
  • the system can further comprise an amplification system that performs a nucleic acid amplification reaction on the sample or a portion thereof in response to the user request.
  • amplification system that performs a nucleic acid amplification reaction on the sample or a portion thereof in response to the user request.
  • a variety of methods of amplifying polynucleotides e.g., DNA and/or RNA
  • Amplification may be linear, exponential, or involve both linear and exponential phases in a multi-phase amplification process.
  • Amplification methods may involve changes in temperature, such as heat denaturation, or may be isothermal processes that do not require heat denaturation.
  • suitable amplification processes are described herein, such as with regard to any of the various aspects of the disclosure.
  • amplification comprises rolling circle amplification (RCA) .
  • a variety of systems for amplifying polynucleotides are available and may vary based on the type of amplification reaction to be performed.
  • the amplification system may comprise a thermocycler.
  • An amplification system can comprise a real-time amplification and detection instrument, such as systems manufactured by Applied Biosystems, Roche, and Strategene. Samples, polynucleotides, primers, polymerases, and other reagents can be any of those described herein, such as with regard to any of the various aspects. Systems can be selected and/or configured to execute any such methods.
  • Systems may further comprise a sequencing system that generates sequencing reads for polynucleotides amplified by the amplification system.
  • the sequencing system and the amplification system may be the same or comprise overlapping equipment.
  • both the amplification system and sequencing system may utilize the same thermocycler.
  • a variety of sequencing platforms for use in the system are available and may be selected based on the selected sequencing method. Examples of sequencing methods are described herein.
  • Amplification and sequencing may involve the use of liquid handlers. Several commercially available liquid handling systems can be utilized to run the automation of these processes (see for example liquid handlers from Perkin-Elmer, Beckman Coulter, Caliper Life Sciences, Tecan, Eppendorf, Apricot Design, Velocity 11 as examples) .
  • a variety of automated sequencing machines are commercially available, and include sequencers manufactured by Life Technologies (SOLiD platform, and pH-based detection) , Roche (454 platform) , Illumina (e.g., flow cell based systems, such as Genome Analyzer devices) . Transfer between 2, 3, 4, 5, or more automated devices (e.g., between one or more of a liquid handler and a sequencing device) may be manual or automated.
  • sequencing comprises a sequencing by synthesis process, where individual nucleotides are identified iteratively, as they are added to the growing primer extension product.
  • Pyrosequencing is an example of a sequence by synthesis process that identifies the incorporation of a nucleotide by assaying the resulting synthesis mixture for the presence of by-products of the sequencing reaction, namely pyrophosphate.
  • a primer/template/polymerase complex is contacted with a single type of nucleotide. If that nucleotide is incorporated, the polymerization reaction cleaves the nucleoside triphosphate between the a and ⁇ phosphates of the triphosphate chain, releasing pyrophosphate.
  • the presence of released pyrophosphate is then identified using a chemiluminescent enzyme reporter system that converts the pyrophosphate, with AMP, into ATP, then measures ATP using a luciferase enzyme to produce measurable light signals. Where light is detected, the base is incorporated, where no light is detected, the base is not incorporated. Following appropriate washing, the various bases are cyclically contacted with the complex to sequentially identify subsequent bases in the template sequence. See, e.g., U.S. Pat. No. 6,210,891. In related sequencing processes, the primer/template/polymerase complex is immobilized upon a substrate and the complex is contacted with labeled nucleotides.
  • the immobilization of the complex may be through the primer sequence, the template sequence and/or the polymerase enzyme, and may be covalent or noncovalent.
  • immobilization of the complex can be via a linkage between the polymerase or the primer and the substrate surface.
  • the nucleotides are provided with and without removable terminator groups. Upon incorporation, the label is coupled with the complex and is thus detectable. In the case of terminator bearing nucleotides, all four different nucleotides, bearing individually identifiable labels, are contacted with the complex. Incorporation of the labeled nucleotide arrests extension, by virtue of the presence of the terminator, and adds the label to the complex, allowing identification of the incorporated nucleotide.
  • the label and terminator are then removed from the incorporated nucleotide, and following appropriate washing, the process is repeated.
  • a single type of labeled nucleotide is added to the complex to determine whether it will be incorporated, as with pyrosequencing.
  • the various different nucleotides are cycled through the reaction mixture in the same process. See, e.g., U.S. Pat. No. 6,833,246.
  • the nucleic acids in the sample can be sequenced by ligation.
  • This method typically uses a DNA ligase enzyme to identify the target sequence, for example, as used in the polony method and in the SOLiD technology (Applied Biosystems, now Invitrogen) .
  • a DNA ligase enzyme to identify the target sequence, for example, as used in the polony method and in the SOLiD technology (Applied Biosystems, now Invitrogen) .
  • a pool of all possible oligonucleotides of a fixed length is provided, labeled according to the sequenced position. Oligonucleotides are annealed and ligated; the ligation by DNA ligase for matching sequences results in a signal corresponding to the complementary sequence at that position.
  • the sequencing system will typically comprise software for performing various operations in response to an input of sequencing data and input of parameters (e.g., selection of a reference genome) . Examples of alignment algorithms and aligners implementing these algorithms are described herein and may form part of the sequencing system.
  • the system can further comprise a report generator that sends a report to a recipient, wherein the report contains results for detection of the sequence variant.
  • a report may be in any suitable form described herein.
  • the system of the present disclosure may further comprise a biological sample treatment module that mixes the biological sample with the first buffer to obtain the mixture.
  • the biological sample treatment module may incubate the mixture.
  • the biological sample treatment module may comprise a heating block or an incubator that is capable of incubating the mixture of the biological sample with the first buffer at a temperature that is from about 10°C to 75°C, for example, at a temperature that is from about 10°C to 70°C, from about 15°C to 65°C, from about 15°C to 60°C, from about 15°C to 55°C, from about 20°C to 50°C, from about 20°C to 45°C, from about 20°C to 40°C, from about 20°C to 35°C, from about 20°Cto 30°C, from about 20°C to 25°C, or from about 25°C to 30°C.
  • the biological sample treatment module may comprise a heating block or an incubator that is capable of incubating the mixture of the biological sample with the first buffer at a temperature that is greater than or equal to 15°C, for example, at a temperature that is greater than or equal to about 20°C, greater than or equal to about 25°C, greater than or equal to about 30°C, greater than or equal to about 35°C, greater than or equal to about 40°C, greater than or equal to about 45°C, greater than or equal to about 50°C, greater than or equal to about 55°C, greater than or equal to about 60°C, greater than or equal to about 65°C, greater than or equal to about 70°C, greater than or equal to about 75°C, greater than or equal to about 80°C, greater than or equal to about 85°C, greater than or equal to about 90°C, greater than or equal to about 91°C, greater than or equal to about 92°C, greater than or equal to about 93°C, greater than or equal to about 94°C, greater than or
  • the biological sample treatment module may incubate the mixture of the biological sample with the first buffer for a period of time that is no more than about 20 minutes.
  • the period of time in (b) may be no more than about 19 minutes, no more than about 18 minutes, no more than about 17 minutes, no more than about 16 minutes, no more than about 15 minutes, no more than about 14 minutes, no more than about 13 minutes, no more than about 12 minutes, no more than about 11 minutes, no more than about 10 minutes, no more than about 9 minutes, no more than about 8 minutes, no more than about 7 minutes, no more than about 6 minutes, no more than about 5 minutes, no more than about 4 minutes, no more than about 3 minutes, no more than about 2 minutes, no more than about 1 minute, no more than about 50 seconds, no more than about 40 seconds, no more than about 30 seconds, no more than about 20 seconds, no more than about 15 seconds, no more than about 10 seconds, no more than about 5 seconds, no more than about 3 seconds, no more than about 2 seconds, or no more than about 1 seconds.
  • the biological sample treatment module may incubate the homogenized preparation at a temperature that is greater than or equal to about 40°C, for example, at a temperature that is greater than or equal to about 45°C, greater than or equal to about 50°C, greater than or equal to about 55°C, greater than or equal to about 60°C, greater than or equal to about 65°C, greater than or equal to about 70°C, greater than or equal to about 75°C, greater than or equal to about 80°C, greater than or equal to about 85°C, greater than or equal to about 90°C, greater than or equal to about 91°C, greater than or equal to about 92°C, greater than or equal to about 93°C, greater than or equal to about 94°C, greater than or equal to about 95°C, greater than or equal to about 96°C, greater than or equal to about 97°C, greater than or equal to about 98°C, greater than or equal to about 99°C, or greater than or equal to about 100°C.
  • the system of the present disclosure may further comprise an amplification module operatively coupled to the biological sample treatment module, wherein the amplification module (i) adds an amount of the mixture from the biological sample treatment module to the reaction vessel and (ii) subjects the reaction mixture in the reaction vessel to the primer extension reaction (s) to generate the amplified product that is indicative of a presence of the target nucleic acid molecule.
  • the mixture may be added to the reaction vessel manually, and the amplification module may subject the reaction mixture in the reaction vessel to the primer extension reaction (s) to generate the amplified product that is indicative of a presence of the target nucleic acid molecule.
  • the one or more computer processors may be individually or collectively programmed to suspend the biological sample in solution to obtain a homogenized preparation comprising the biological sample. This may be performed prior to mixing the biological sample with a first buffer.
  • the one or more computer processors may be individually or collectively programmed to subject the biological sample to centrifugation to yield a solution comprising the biological sample and a pellet, or to yield a pellet comprising the biological sample and a supernatant.
  • the one or more computer processors may be individually or collectively programmed to subject the mixture to centrifugation to yield a supernatant comprising the biological sample. Then, the supernatant may serve as the mixture for subsequent processing. For example, the supernatant may be added to a reaction vessel comprising reagents necessary for conducting nucleic acid amplification.
  • the one or more computer processors may be individually or collectively programmed to cultivate the biological sample for microbial proliferation.
  • the one or more computer processors prior to mixing the biological sample with a first buffer, may be individually or collectively programmed to subject the biological sample to enrichment culturing conditions for a culturing time period.
  • the enrichment culturing conditions may comprise culturing the biological sample in a suitable culture medium (e.g., tryptic soy broth, modified tryptic soy broth, tryptone, nutrient broth, L-broth, gram negative broth, peptone, tryptic soy broth with yeast, or Salmonella medium) at a suitable temperature (e.g., from 23°C to 40°C, such as 25°C, 30°C, 35°C, or 37°C) with or without shaking.
  • a suitable culture medium e.g., tryptic soy broth, modified tryptic soy broth, tryptone, nutrient broth, L-broth, gram negative broth, peptone, tryptic soy broth with yeast, or Salmonella medium
  • a suitable temperature e.g., from 23°C to 40°C, such as 25°C, 30°C, 35°C, or 37°C
  • the culture medium is a Salmonella medium which favors proliferation of Salmonella as compared to other bacteria.
  • Example Salmonella medium includes Bismuth Sulfite agar (BS) , xylose lysine deoxycholate agar (XLD) , selenite brilliant green sulfa medium (SBG) , but is not limited thereto.
  • the culturing time period may be from about 0.5 hour to 5 hours, e.g., greater than or equal to about 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours, 6.5 hours, 7 hours, 7.5 hours, 8 hours, 8.5 hours, 9 hours, 9.5 hours, or 10 hours.
  • the culturing time period is less than or equal to about 7 hours, e.g., less than or equal to about 6.5 hours, 6 hours, 5.5 hours, 5 hour, 4.5 hour, 4 hours, 3.5 hours, 3 hours 2.5 hours, 2 hours, 1.5 hours, 1 hour, or 0.5 hour.
  • the one or more computer processors are individually or collectively programmed to subject the biological sample to centrifugation to yield a solution comprising the biological sample and a pellet, or to yield a pellet comprising the biological sample and a supernatant.
  • the one or more computer processors are individually or collectively programmed to mix the biological sample with the first buffer without selective enrichment, plating on differential medium, and/or presumptive biomedical identification.
  • the one or more computer processors may be individually or collectively programmed to add a first buffer to the mixture.
  • the first buffer may be alkaline.
  • the first buffer comprises NaOH.
  • the first buffer may have a pH from about 7 to 14, such as from about 8 to 13, from about 9 to 12, from about 10 to 11.
  • the first buffer may have a pH greater than or equal to about 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, or 14.
  • the one or more computer processors may be individually or collectively programmed to add the mixture of the biological sample with the first buffer to the reaction vessel without being subject to DNA or RNA extraction. In some cases, the one or more computer processors may be individually or collectively programmed to add the mixture of the biological sample with the first buffer to the reaction vessel without being purified. In some cases, the one or more computer processors may be individually or collectively programmed to add the mixture of the biological sample with the first buffer to the reaction vessel without being subject to DNA or RNA concentration.
  • the reaction vessel may be one that comprises reagents necessary for conducting nucleic acid amplification.
  • the one or more computer processors may be individually or collectively programmed to provide a biological sample obtained from a subject or the mixture, supernatant, or homogenized preparation derived from the biological sample as described elsewhere herein with reagents necessary for nucleic acid amplification in a reaction vessel to obtain a reaction mixture.
  • the reaction vessel may be any reaction vessel as described herein.
  • the detection module may be an optical detection module.
  • the optical detection module may detect the optical signal produced during the amplification.
  • the optical signal may be an optical signal produced by any optically active dye as described herein.
  • the optical signal may be an optical signal accompanying the production of the amplified product (s) .
  • the optical signal may be an optical signal produced by an oligonucleotide probe upon breakdown.
  • the optical signal may be an optical signal produced when a molecular beacon hybridizes with the amplified product (s) .
  • the optical signal may be a fluorescent signal
  • the detection module may be a fluorescence detection module.
  • the fluorescence detection module may detect the fluorescent signal produced by any fluorescent dye as described herein.
  • the fluorescence detection module may detect the fluorescent signal produced by one or more fluorescent dyes selected from the group consisting of FAM, TET, ROX, JOE, HEX, TAMRA, VIC, NET, PET, Texas Red, and the like.
  • the detection module may detect a plurality of detectable signals in parallel.
  • the plurality of detectable signals is produced by a plurality of reporter agents, while the plurality of detectable signals produced by the plurality of reporter agents is distinct among one another.
  • Each of the plurality of detectable signals may be indicative of the presence or absence of the corresponding amplified product.
  • the intensity of each of the plurality of detectable signals may be proportional to the amount of the corresponding amplified product.
  • the detection module may detect FAM fluorescence and ROX fluorescence in parallel.
  • the detectable signal corresponding to one amplified product may be FAM fluorescence, while the detectable signal corresponding to another amplified product may be ROX fluorescence, thereby enabling the detection of distinct detectable signals in parallel.
  • Detection of distinct detectable signals in parallel may allow comparison of the presence and/or amount among different amplified products.
  • detection of distinct detectable signals in parallel may allow determination of the amount of the amplified product (s) relative to an internal reference.
  • the one or more computer processors may be individually or collectively programmed to preheat the biological sample at a preheating temperature between about 90°C to 100°C for a preheating duration less than or equal to about 10 minutes. This may be performed before conducting the primer extension reactions. In some embodiments, the preheating duration is less than or equal to about 1 minute.
  • Another aspect of the disclosure provides a method of assessing a susceptibility of a subject to a health condition associated with a plurality of microbes.
  • the method includes: (a) obtaining at least one biological sample of the subject; (b) assaying the at least one biological sample of the subject to assess a quantity or concentration of each of a plurality of microbes in the at least one biological sample, where the plurality of microbes comprises a first microbe and a second microbe, which first microbe has been identified to be beneficial with respect to a health of the subject and the second microbe has been identified to be harmful with respect to the health of the subject; (c) determining a quantity or relative concentration of each of the plurality of microbes relative to a reference (s) , to identify a dearth or abundance of each of the plurality of microbes; and (d) outputting a report indicative of the susceptibility of the subject to the health condition associated with the plurality of microbes, which susceptibility is determined based at least in part on
  • the plurality of types of microbes comprises at least two types of first microbes, at least three types of first microbes, at least four types of first microbes or at least five types of first microbes. In some embodiments, the plurality of types of microbes comprises at least two types of second microbes, least three types of second microbes, at least four types of second microbes, at least five types of second microbes.
  • the plurality of types of microbes include at least one microbe selected from the group consisting of: bacteria, viruses and fungi.
  • the plurality of types of microbes can also include any other type of microbe described herein.
  • bacteria include Escherichia, Paracolon, Enterobacter aerogenes, Proteus, Pseudomonas aeruginosa, Pneumobacillus, Bacillus, Bacteroides, Bifidobacterium, Clostridium, Collinsella, Faecalibacterium, Lactobacillus, Ruminococcus, Enterococcus, Dorea, Listeria, Streptococcus, Staphyloccocus, Corynebacterium, Propionibacterium, Clostridium butyricum, Campylobacter, Aeromonas, Plesiomonas shigelloides, Campylobacter, Clostridium difficile, Escherichia coli O157, Enteroaggregative E.
  • EAEC Enteropathogenic E. coli
  • EPEC Enteropathogenic E. coli
  • EHEC Enterohemorrhagic E. coli
  • ETEC Enterotoxigenic E. coli
  • EIEC Shiga-like Toxin producing E.
  • coli stx1/stx2, Salmonella, Shigella, Vibrio cholera 5, Yersinia enterocolitica, Klebsiella, Clostridium difficile, Listeria monocytogenes, Campylobacter jejuni, Enterobacter Sakazakii, Prevotella, Veillonella, Erwinia, Coprococcus, Roseburia, Campylobacter fetus and Lactobacillus rhamnosus GG strain. Additional examples of bacteria are provided elsewhere herein.
  • Non-limiting examples of viruses include Adenovirus 40/41, Norovirus GI/GII, Rotavirus, Rotavirus A, Astrovirus, Sapovirus, herpes simplex virus, hepatovirus, and CMV. Additional examples of viruses are provided elsewhere herein.
  • Non-limiting examples of fungi include Candida albicans, Hirsutellia, and Saccharomyces.
  • the plurality of types of microbes include at least one microbe selected from the group consisting of Lactobacillus, Bacteroides, Bifidobacterium, Escherichia, Faecalibacterium, Ruminococcus, and Lactobacillus rhamnosus GG strain.
  • the subject is up to 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 year (s) or 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 month (s) of age at a time at which the biological sample is obtained.
  • the subject is aged from six months to twelve years old. In some embodiments, the subject is aged from six months to six years old. In some embodiments, the subject is aged from six months to two years old. In some embodiments, the subject is aged from six months to one year old.
  • the reference comprises normal levels of the plurality of microbes, where a normal level of a first microbe comprises a first threshold and optionally, a second threshold, and a normal level of a second microbe comprises a second threshold and optionally, a first threshold, and where (c) comprises determining whether the quantity or concentration is above the first threshold and/or below the second threshold.
  • the first threshold is at most 10 15 , 10 14 , 10 13 , 10 13 , 10 12 , 10 11 , 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , or less of a given microbe of the plurality of types of microbes per gram of the biological sample.
  • the second threshold is at least 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or more copies of a given microbe of the plurality of types of microbes per gram of the biological sample.
  • the first threshold is 10 15 , 10 14 , 10 13 , 10 13 , 10 12 , 10 11 , 10 10 , 10 9 , 10 8 , 10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10 1 , or less colony-forming units (CFU) of a given microbe of the plurality of types of microbes per gram of the biological sample.
  • CFU colony-forming units
  • the second threshold is 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , or more CFU of a given microbe of the plurality of types of microbes per gram of the biological sample.
  • the method further comprises assessing susceptibility to a health condition of the subject if the quantity or concentration of at least one first microbe is lower than the first threshold and/or the quantity or concentration of the at least one second microbe is higher than the second threshold.
  • the condition is selected from the group consisting of a gut condition, diarrhea, jaundice, inflammatory bowel disease, Crohn's Disease, irritable bowel syndrome, a stomach ulcer, colitis, neonatal necrotizing enterocolitis, gastroesophageal reflux disease, constipation, functional bloating, gastritis, lactose intolerance, visceral hyperalgesia, colic, pouchitis, diverticulitis, diabetes, cholera, obesity, non-alcoholic steatohepatitis, non-alcoholic fatty liver disease, colorectal cancer, pathogen infection, purulent or suppurative inflammation, toxinosis, allergic diseases, typhoid fever, acute enteritis, sepsis,
  • the reference comprises normal levels of the plurality of microbes in a population to which the subject belongs. In some embodiments, the reference comprises normal levels of the plurality of microbes of the subject in a healthy state.
  • the assaying in (b) comprises conducting nucleic acid amplification reaction on nucleic acid molecules derived from the at least one biological sample.
  • the nucleic acid amplification reaction is selected from the group consisting of polymerase chain reaction (PCR) , real-time PCR, isothermal amplification, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, solid phase amplification, nucleic acid sequence-based amplification (NASBA) , linear amplification, digital PCR or any other type of amplification reaction described elsewhere herein.
  • the nucleic acid amplification is PCR.
  • the at least one biological sample is subjected to the PCR without purification. In some embodiments, the at least one biological sample is subjected to the PCR without deoxynucleic acid (DNA) or ribonucleic acid (RNA) enrichment. In some embodiments, the at least one biological sample is subjected to the PCR without pre-culturing. In some embodiments, the at least one biological sample is subjected to the PCR without non-selective enrichment. In some embodiments, the at least one biological sample is subjected to the PCR without selective enrichment. In some embodiments, the at least one biological sample is subjected to the PCR without plating on differential medium. In some embodiments, the at least one biological sample is subjected to the PCR without presumptive biomedical identification.
  • DNA deoxynucleic acid
  • RNA ribonucleic acid
  • the assaying in (b) comprises conducting sequencing on nucleic acid molecules derived from the at least one biological sample.
  • the quantity or concentration is a relative quantity or concentration of each of the plurality of types of microbes in the at least one biological sample.
  • the method further comprises determining a change in the quantity or concentration of at least a subset of the plurality of types of microbes in the at least one biological sample at multiple geographic locations.
  • the method further comprises determining a change in the quantity or concentration of at least a subset of the plurality of types of microbes in the at least one biological sample over a time period of at least 1 minute (min) .
  • the period of time is at least 10 min, 30 min, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or more than 1 year.
  • the biological sample is a stool sample.
  • the subject is aged from zero (0) to one (1) months old, zero (0) to two (2) months old, zero (0) to three (3) months old, zero (0) to four (4) months old, zero (0) to five (5) months old, zero (0) to six (6) months old, zero (0) to seven (7) months old, zero (0) to eight (8) months old, zero (0) to nine (9) months old, zero (0) to ten (10) months old, zero (0) to eleven (11) months old, zero (0) to twelve (12) months old at the time at which the stool sample is obtained.
  • the reference comprises normal levels of Lactobacillus, Escherichia, Bifidobacterium, Ruminococcus, Bacteroides, Faecalibacterium, and Lactobacillus rhamnosus GG strain corresponding to an amount of copies of bacteria in the stool sample of copies/g of stool sample, respectively.
  • subject is aged from six (6) months to twelve (1) year old, six (6) months to two (2) years old, six (6) months to three (3) years old, six (6) months to four (4) years old, six (6) months to five (5) years old, six (6) months to six (6) years old, six (6) months to seven (7) years old, six (6) months to eight (8) years old, six (6) months to nine (9) years old, six (6) months to ten (10) years old, six (6) months to eleven (11) years old, or six (6) months to twelve (12) years old at the time at which the stool sample is obtained.
  • the reference comprises normal levels of Lactobacillus, Escherichia, Bifidobacterium, Ruminococcus, Bacteroides, Faecalibacterium, and Lactobacillus rhamnosus GG strain corresponding to an amount of copies of bacteria in the stool sample of copies/g of stool sample, respectively.
  • (d) comprises outputting a report indicative of the susceptibility of the subject to allergy and/or eczema based at least in part on an abundance of E. coli and a dearth of Bacteroides. In some embodiments, (d) comprises outputting a report indicative of the susceptibility of the subject to enteritis and/or diarrhea based at least in part on an abundance of E. coli and a dearth of Faecalibacterium.
  • the method further comprises providing the subject with a questionnaire to access additional information before (d) , which additional information, when combined with an assessment of a dearth of the first microbe and/or abundance of the second microbe in the subject, is indicative of the susceptibility.
  • additional information when combined with an assessment of a dearth of the first microbe and/or abundance of the second microbe in the subject, is indicative of the susceptibility.
  • susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject, in combination with the additional information.
  • the additional information is selected from the group consisting of information regarding the subject, information regarding the mother of the subject, and information regarding living environment of the subject.
  • the information regarding the subject is selected from the group consisting of age, sex, ethnicity, height, weight, head circumference, mode of birth, gestational age at birth, geological location of birth, feeding status, prebiotic intake, probiotic intake, medical history, congenital disease, bowel movement, and stool appearance of the subject.
  • the feeding status includes breastfeeding, formula feeding, solid food feeding, and combinations thereof.
  • the mode of birth includes vaginal birth or caesarean section, presence or absence of labor induction, and combinations thereof.
  • the medical history includes prior or concurrent medication, prior or concurrent conditions, prior or concurrent symptoms, and combinations thereof.
  • the medication includes antibiotics.
  • the conditions include allergy, congenital diseases, gastrointestinal diseases, infectious disease, and combinations thereof.
  • the allergy is allergy to one or more allergens selected from the group consisting of milk, soy, wheat, lactose, and yogurt.
  • the stool appearance includes color, form, shape, consistency, fluidity, and combinations thereof.
  • the symptoms include constipation, diarrhea, jaundice, colic, itching, eczema and bloating.
  • the information regarding the mother of the subject is selected from age at birth, height, weight, medical history, allergy,
  • the medical history comprises the presence or absence of one or more diseases selected from the group consisting of urticarial, asthma, and gestational diabetes.
  • the allergy is allergy to one or more allergens selected from the group consisting of milk, soy, wheat, lactose, and yogurt.
  • the information regarding the living environment of the subject is selected from access to pets, sanitization of clothing and/or personal items, and sanitization of living environment.
  • the susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject at an accuracy of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%or higher.
  • An additional aspect of the disclosure provides a system for assessing a susceptibility of a subject to a health condition associated with a plurality of microbes.
  • the system includes: an input module that receives a user request to assess a susceptibility of a subject to a health condition associated with a plurality of microbes; an assay module that assays at least one biological sample of the subject to assess a quantity or concentration of each of a plurality of microbes in the at least one biological sample, where the plurality of microbes comprises a first microbe and a second microbe, which first microbe has been identified to be beneficial with respect to a health of the subject and the second microbe has been identified to be harmful with respect to the health of the subject; and one or more computer processor operatively coupled to the assay module.
  • the one or more computer processors can be individually or collectively programmed to: (a) determine a quantity or relative concentration of each of the plurality of microbes relative to a reference (s) , to identify a dearth or abundance of each of the plurality of microbes; and (b) output a report indicative of the susceptibility of the subject to the health condition associated with the plurality of microbes, which susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject at an accuracy of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%or higher.
  • the system further comprises a user interface for providing the subject with a questionnaire to access additional information, which additional information, when combined with an assessment of a dearth of the first microbe and/or abundance of the second microbe in the subject, is indicative of the susceptibility.
  • additional information when combined with an assessment of a dearth of the first microbe and/or abundance of the second microbe in the subject, is indicative of the susceptibility.
  • susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject, in combination with the additional information.
  • the additional information is selected from the group consisting of information regarding the subject, information regarding the mother of the subject, and information regarding living environment of the subject.
  • the information regarding the subject is selected from the group consisting of age, sex, ethnicity, height, weight, head circumference, mode of birth, gestational age at birth, geological location of birth, feeding status, prebiotic intake, probiotic intake, medical history, congenital disease, bowel movement, and stool appearance of the subject.
  • the feeding status includes breastfeeding, formula feeding, solid food feeding, and combinations thereof.
  • the mode of birth includes vaginal birth or caesarean section, presence or absence of labor induction, and combinations thereof.
  • the medical history includes prior or concurrent medication, prior or concurrent conditions, prior or concurrent symptoms, and combinations thereof.
  • the medication includes antibiotics.
  • the conditions include allergy, congenital diseases, gastrointestinal diseases, infectious disease, and combinations thereof.
  • the allergy is allergy to one or more allergens selected from the group consisting of milk, soy, wheat, lactose, and yogurt.
  • the stool appearance includes color, form, shape, consistency, fluidity, and combinations thereof.
  • the symptoms include constipation, diarrhea, jaundice, colic, itching, eczema and bloating.
  • the information regarding the mother of the subject is selected from age at birth, height, weight, medical history or an allergy.
  • the medical history comprises the presence or absence of one or more diseases selected from the group consisting of urticarial, asthma, and gestational diabetes.
  • the allergy is allergy to one or more allergens selected from the group consisting of milk, soy, wheat, lactose, and yogurt.
  • the information regarding the living environment of the subject is selected from access to pets, sanitization of clothing and/or personal items, and sanitization of living environment.
  • An additional aspect of the disclosure provides a non-transitory computer-readable medium comprising machine-executable code that, upon execution by one or more computer processors, implements a method of assessing a susceptibility of a subject to a health condition associated with a plurality of microbes.
  • Such a method can include: (a) obtaining at least one biological sample of the subject; (b) assaying the at least one biological sample of the subject to assess a quantity or concentration of each of a plurality of microbes in the at least one biological sample, where the plurality of microbes comprises a first microbe and a second microbe, which first microbe has been identified to be beneficial with respect to a health of the subject and the second microbe has been identified to be harmful with respect to the health of the subject; (c) determining a quantity or relative concentration of each of the plurality of microbes relative to a reference (s) , to identify a dearth or abundance of each of the plurality of microbes; and (d) outputting a report indicative of the susceptibility of the subject to the health condition associated with the plurality of microbes, which susceptibility is determined based at least in part on a dearth of the first microbe and/or abundance of the second microbe in the subject at an accuracy of at least about 50%, 55%, 60%, 65%
  • An additional aspect of the disclosure provides a method for determining and/or treating a health condition of a subject.
  • the method comprises: (a) determining at least one symptom from the subject; (b) performing an intestinal microflora detection and optionally a pathogen detection on the subject, where the intestinal microflora detection is conducted according to the method of the disclosure to assess the susceptibility of a subject to the health condition; and (c) upon determining that the at least one symptom of the subject matches the health condition of (b) , prescribing a drug and/or probiotic intervention to the subject.
  • the at least one symptom from the subject is determined by recording complaints from the subject or taking oral inquiry from the subject.
  • the method further comprises performing a second intestinal microflora detection on the subject, where the second intestinal microflora detection is conducted to assess the susceptibility of the subject to the health condition.
  • the method further comprises outputting a report indicative of recovery of the subject from the health condition if the susceptibility of the subject to the health condition has not been determined.
  • the method further comprises repeating (c) and (d) if the susceptibility of the subject to the health condition has been determined. In some embodiments, if the at least one symptom does not match the health condition of (b) , prior to (c) (i) taking a second inquiry from the subject based on the results of (b) ; (ii) optionally performing other detections.
  • the intestinal microflora detection and the second intestinal microflora detection are performed at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, or at least twelve months apart or at a greater interval.
  • An additional aspect of the disclosure provides a method for determining and/or treating a health condition of a subject suspected of dysbiosis of intestinal microflora.
  • the method comprises: (a) determining whether one or more symptoms of the dysbiosis are severe or mild; (b) if the one or more symptoms of the dysbiosis are mild, performing an intestinal microflora detection, where the intestinal microflora detection is conducted according to the method of the disclosure to assess a susceptibility of the subject to the health condition; if the one or more symptoms of the subject match the health condition of (b) , prescribing a probiotic intervention to the subject.
  • a mild of the dysbiosis is selected from the group consisting of loss of appetite, weakness, obesity, wasting, light diarrhea and constipation.
  • a severe symptom of the dysbiosis includes severe diarrhea and/or infectious diarrhea.
  • whether the one or more symptoms of the dysbiosis are severe or mild is determined by recording complaints from the subject or taking oral inquiry from the subject.
  • the method further comprises performing an additional intestinal microflora detection on the subject, where the additional intestinal microflora detection is conducted according to the method of the disclosure to assess the susceptibility of the subject to the health condition.
  • the method further comprises outputting a report indicative of recovery of the subject from the health condition if the susceptibility of the subject to the health condition is not determined. In some embodiments, the method further comprises repeating (c) if the susceptibility of the subject to the health condition is determined. In some embodiments, if the one or more symptoms of the dysbiosis are severe, the method further comprises performing a pathogen detection on the subject. In some embodiments, if the pathogen detection reveals an infectious disease, the method further comprises, performing a drug intervention on the subject. In some embodiments, the drug intervention comprises providing or recommending an antibiotic. In some embodiments, the method comprises proceeding to (b) after the drug intervention.
  • the method further comprises (i) taking a second inquiry from the; (ii) optionally performing one or more other detections; and (iii) proceeding to (c) .
  • the present disclosure provides a computer-readable medium.
  • the computer readable medium comprises code that, upon execution by one or more processors, implements a method of profiling a plurality of microbes in at least one biological sample.
  • the method comprises: (i) obtaining results of a nucleic acid amplification reaction comprising amplified nucleic acids from at least one beneficial microbe and at least one pathogenic microbe; (ii) determining, with one or more processors individually or collectively, (1) a quantity or concentration of the at least one beneficial microbe and at least one pathogenic microbe; and/or (2) relative quantity or concentration of the at least one beneficial microbe and at least one pathogenic microbe; and (iii) generating a report including results of (ii) .
  • the computer readable medium may take many forms, including but not limited to, a tangible (or non-transitory) storage medium, a carrier wave medium, or physical transmission medium.
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer (s) or the like, such as may be used to implement calculations, processing, etc.
  • Volatile storage media include dynamic memory, such as main memory of a computer.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system.
  • Carrier-wave transmission media can take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications.
  • RF radio frequency
  • IR infrared
  • Computer-readable media include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer can read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • FIG. 5 shows a computer system 1101 that is programmed or otherwise configured to implement the present disclosure.
  • the computer system 1101 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device.
  • the electronic device can be a mobile electronic device.
  • the computer system 1101 includes a central processing unit (CPU, also "processor” and “computer processor” herein) 1105, which can be a single core or multi core processor, or a plurality of processors for parallel processing.
  • the computer system 1101 also includes memory or memory location 1110 (e.g., random-access memory, read-only memory, flash memory) , electronic storage unit 1115 (e.g., hard disk) , communication interface 1120 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 1125, such as cache, other memory, data storage and/or electronic display adapters.
  • the memory 1110, storage unit 1115, interface 1120 and peripheral devices 1125 are in communication with the CPU 1105 through a communication bus (solid lines) , such as a motherboard.
  • the storage unit 1115 can be a data storage unit (or data repository) for storing data.
  • the computer system 1101 can be operatively coupled to a computer network ( "network" ) 1130 with the aid of the communication interface 1120.
  • the network 1130 can be the Internet, an internet and/or extranet, or an intranet and/or extranet that is in communication with the Internet.
  • the network 1130 in some cases is a telecommunication and/or data network.
  • the network 1130 can include one or more computer servers, which can enable distributed computing, such as cloud computing.
  • the network 1130 in some cases with the aid of the computer system 1101, can implement a peer-to-peer network, which may enable devices coupled to the computer system 1101 to behave as a client or a server.
  • the CPU 1105 can execute a sequence of machine-readable instructions, which can be embodied in a program or software.
  • the instructions may be stored in a memory location, such as the memory 1110.
  • the instructions can be directed to the CPU 1105, which can subsequently program or otherwise configure the CPU 1105 to implement methods of the present disclosure. Examples of operations performed by the CPU 1105 can include fetch, decode, execute, and writeback.
  • the CPU 1105 can be part of a circuit, such as an integrated circuit.
  • a circuit such as an integrated circuit.
  • One or more other components of the system 1101 can be included in the circuit.
  • the circuit is an application specific integrated circuit (ASIC) .
  • ASIC application specific integrated circuit
  • the storage unit 1115 can store files, such as drivers, libraries and saved programs.
  • the storage unit 1115 can store user data, e.g., user preferences and user programs.
  • the computer system 1101 in some cases can include one or more additional data storage units that are external to the computer system 1101, such as located on a remote server that is in communication with the computer system 1101 through an intranet or the Internet.
  • the computer system 1101 can communicate with one or more remote computer systems through the network 1130.
  • the computer system 1101 can communicate with a remote computer system of a user.
  • remote computer systems include personal computers (e.g., portable PC) , slate or tablet PC's (e.g., iPad, Galaxy Tab) , telephones, Smart phones (e.g., iPhone, Android-enabled device, ) , or personal digital assistants.
  • the user can access the computer system 1101 via the network 1130.
  • Methods as described herein can be implemented by way of machine (e.g., computer processor) executable code stored on an electronic storage location of the computer system 1101, such as, for example, on the memory 1110 or electronic storage unit 1115.
  • the machine executable or machine readable code can be provided in the form of software.
  • the code can be executed by the processor 1105.
  • the code can be retrieved from the storage unit 1115 and stored on the memory 1110 for ready access by the processor 1105.
  • the electronic storage unit 1115 can be precluded, and machine-executable instructions are stored on memory 1110.
  • the code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code or can be compiled during runtime.
  • the code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as-compiled fashion.
  • aspects of the systems and methods provided herein can be embodied in programming.
  • Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium.
  • Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.
  • “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming.
  • All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server.
  • another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links.
  • the physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software.
  • terms such as computer or machine "readable medium” refer to any medium that participates in providing instructions to a processor for execution.
  • a machine readable medium such as computer-executable code
  • a tangible storage medium such as computer-executable code
  • Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer (s) or the like, such as may be used to implement the databases, etc. shown in the drawings.
  • Volatile storage media include dynamic memory, such as main memory of such a computer platform.
  • Tangible transmission media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system.
  • Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications.
  • RF radio frequency
  • IR infrared
  • Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and/or data.
  • Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.
  • the computer system 1101 can include or be in communication with an electronic display 1135 that comprises a user interface (UI) 1140 for providing, for example.
  • UI user interface
  • Examples of UI's include, without limitation, a graphical user interface (GUI) and web-based user interface.
  • GUI graphical user interface
  • Methods and systems of the present disclosure can be implemented by way of one or more algorithms.
  • An algorithm can be implemented by way of software upon execution by the central processing unit 1105.
  • Example 1 Profiling Microbes in a Sample
  • a sample from a subject is obtained. DNA is extracted from the sample. Nucleic acid sequences are amplified. The relative quantity or relative concentration of at least one pathogenic microbe and at least beneficial microbe are determined. A health state of the subject from whom the sample was derived is determined based on the determined quantities or concentrations. If the at least one pathogenic microbe is above a reference threshold value, the at least one beneficial microbe is below a reference threshold value, or the ratio of the at least one pathogenic microbe to the at least one beneficial microbe is above a reference threshold value, the subject is characterized as having a disease or condition. Otherwise, the subject is characterized as normal or healthy.
  • a fecal sample is obtained from a subject.
  • To extract DNA 0.5 mL of a homogenized sample is added to 150 ⁇ L of acid phenol with 0.3 g of zirconium beads (diameter 0.1 mm) , and the suspension is treated at 5000 rpm for 3 minutes with a mini bead beater. The supernatant is further extracted with phenol-chloroform, treated with DNase-free RNase, and precipitated with ethanol. 16S rRNA gene sequences are amplified using primers that hybridize to conserved regions.
  • the primers may have sequences: CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGGAACGCGAAGAACCTT AC and CGGTGTACAAGACCC, and be used to amplify the V6 to V8 regions of bacterial 16S rRNA genes.
  • PCR is performed with a Taq DNA polymerase kit from ThermoFisher Scientific. Each PCR mixture contains dNTP, Taq polymerase, and both primers. Reactions are thermocycled as follows: 94°C for 5 minutes; 35 cycles consisting of 94°C for 30 seconds, 56°Cfor 20 seconds, and 68°C for 40 seconds; and then 68°C for 7 minutes.
  • Amplification products are sequenced using an Illumina HiSeq high-throughput sequencing system, according to manufacturer's protocol. Bacteria are identified taxonomically by aligning sequencing results against the GreenGenes database of bacterial 16S rRNA gene sequences (greengenes. lbl. gov/cgi-bin/nph-index. cgi) . Pathogenic and beneficial bacteria are quantified based on the sequencing and alignment data. The relative proportions of pathogenic bacteria (e.g., pathogenic E.
  • coli e.g., strain O157: H7
  • beneficial bacteria e.g., Bifidobacterium and Lactobacillus
  • a subject is diagnosed as having or likely to develop a condition if the pathogenic bacteria are above a threshold level and/or if the beneficial bacteria are below a threshold level (e.g., stomach ulcers with high H. Pylori, C. diff. colitis with high levels of C. diff, lactose intolerance with low levels of Lactobacillus, etc. ) .
  • a subject is treated to decrease the level of pathogenic bacteria and/or increase the level of beneficial bacteria based on the quantification.
  • Example 3 Profiling Bacteria in a Stool Sample
  • Various stool samples were obtained from human subjects aged from about 0 years old to about 12 years old.
  • the obtained stool samples were preserved in a stool preservation buffer prior to use.
  • the stool preservation buffer together with the stool sample was subjected to centrifugation. After centrifugation, the pellet was resuspended in a first buffer.
  • the stool preservation buffer together with the stool sample may be directly mixed with the first buffer.
  • the suspension or mixture was aliquoted into a fresh tube, followed by heating at 95°Cfor less than 15 minutes.
  • a lysis buffer containing NaOH was added and mixed thoroughly.
  • the mixture was subjected to centrifugation. After centrifugation, the supernatant was aliquoted for subsequent nucleic acid amplification.
  • the stool sample was used freshly.
  • the liquid or solid stool sample was mixed thoroughly with the first buffer, followed by centrifugation.
  • the supernatant was aliquoted into a fresh tube, followed by heating at 95°C for less than 5 minutes.
  • a lysis buffer containing NaOH was added and mixed thoroughly.
  • the mixture was subjected to centrifugation. After centrifugation, the supernatant was aliquoted for subsequent nucleic acid amplification.
  • nucleic acid amplification e.g., by polymerase chain reaction (PCR)
  • primers specific for 16S rDNA or recA sequences The specific primers used for amplification are shown in Table 1.
  • Amplification reaction mixtures included detection probes specific to the aforesaid rDNA or recA sequences, breakdown of which release fluorescent dyes that were used to quantify the number of copies of target nucleic acid in the stool samples, with values used to determine copy number of nucleic acid per gram of stool sample obtained.
  • the specific detection probe sequences used for this purpose are shown in Table 2.
  • ACGAGCGCAACCCTTACTGTTAGT ACGAGCGCAACCCTTACTGTTAGT TCTAGCAGGACTGCCGTTGACAAA TCTAGCAGGACTGCCGTTGACAAA ACTCTAAAGGGACTGCCGTTGACA TGAAAGCAGTTCATGGGTTGAGCC ACTGAGAGGTTGAACGGCCACATT CTAGTTGGTGAGGTAACGGCCCAC AACCTCAGTCCGGCTACCGATC CTAGTTGGTGAGGTAACGGCCCAC AAGTGACGGCTAACTACGTGCCAG AAGTGACGGCTAACTACGTGCCAG ATTACTGGGTGTAAAGGGAGCGCA AGCCGTCACTTCCTTGTTGAGTACC AACGTAGGTCACAAGCGTTGTCCG AAGTGACGGCTAACTACGTGCCAG ATTACTGGGTGTAAAGGGAGCGCA AGCCGTCACTTCCTTGTTGAGTACC AACGTAGGTCACAAGCGTTGTCCG AAGTGACGGCTAACTACGTGCCAG
  • Example 3 The profiles obtained in Example 3 were used to establish baseline level of Bacteroides, Bifidobacterium, Escherichia, Faecalibacterium, Lactobacillus, Ruminococcus, and Lactobacillus rhamnosus GG strain, which are regarded as beneficial bacteria, in human subjects. Baseline levels were determined for healthy subjects in age groups of 0-6 months and 6 months above. By performing metadata processing and other statistical analysis, baseline levels for healthy subjects ( "normal levels” ) in age groups of 0-6 months and 6 months above were established and shown in FIG. 3, and Table 3.
  • Stool samples from example subjects having different health conditions/disorders in different age groups were collected and processed as Example 3 and then subjected to 16S RNA amplification for the quantification of Bacteroides, Bifidobacterium, Escherichia, Faecalibacterium, Lactobacillus, Ruminococcus, and Lactobacillus rhamnosus GG strain. The results are as show in Table 5 and FIGs 4.1-4.9.
  • the quantities of the exemplified microbes have a close correlation with the health conditions or disorders of the subjects.
  • sequencing analyses were performed for V3 and V4 hypervariable regions of the 16S rRNA gene of gut fecal material to analyze the gut microbiome in subjects with or without eczema.
  • Fecal samples of the subjects were collected using sterile fecal sampling tubes (Germany Sarstedt) and then stored at -80°C until DNA extraction.
  • Genomic DNA of the samples was extracted by using TIANamp Stool DNA Kit (Tiangen biotech Beijing Co., LTD. ) according to the manufacturer's protocols. SDS-PAGE was used to test DNA purity and fluorimetry was used to measure DNA concentration. Sequencing analysis for V3-V4 hypervariable regions of the 16S rRNA gene was then performed to characterize the intestinal microbiome.
  • Sequencing libraries were prepared using a one-step polymerase chain reaction (PCR) in a 25 microliter reaction mixture containing 25 ng input DNA, 333 nmol each of forward and reverse primers and KAPA Hi-Fi PCR master mix (Kapa Biosystems, Boston, MA, USA) .
  • PCR was conducted with initial 3 min enzyme activation at 95°C followed by 20 cycles of 15s at 98°C, 30s at 50°C, 40s at 72°C and 10 min at 72°C.
  • the amplified products were then cooled down to 10°C before purification by Clean Beads.
  • Approximately 4.5 million reads of 16S rRNA V3-V4 amplicons were generated by Illumina HiSeq2500 instrument.
  • Primers for V3-V4 amplicons were CCTAYGGGRBGCASCAG (forward primer) and GGACTACNNGGGTATCTAAT (reverse primer) .
  • Beta-diversity analysis unweighted Unifrac distances matrices were based on a profiling table, followed by Unweighted Unifrac distances. Principal Coordinates Analysis (PCoA) was drawn based on these data. Inter-group T-test and LEfSe (Linear Discriminant Analysis Effect Size) were conducted to reveal biomarkers with statistical differences.
  • PCoA Principal Coordinates Analysis
  • Processing of data with the Random Forest algorithm included drawing ntree bootstrap samples from genus level OTUs. For each of the bootstrap samples, an unpruned classification was executed, or a regression tress was drawn. At each node, random sample mtry of the predictors was performed and the best split among those variables was chosen. Predictions were then made by aggregating the prediction of ntree trees.
  • PCoA analysis as shown in FIG. 6, suggested differences in microbiome composition and proportion between the healthy group and eczema group.
  • Taxonomic assignment showed that in the healthy group, the dominant bacteria in samples were Bacteroides, Bifidobacterium, Erwinia, Escherichia, Veillonella, and Faecalibacterium, which belong to bacteria families Bacteroidaceae, Bifidobacteriaceae, Enterobacteriaceae, Veillonellaceae, and Ruminococcaceae, respectively.
  • Bacteroides, Bifidobacterium, Erwinia, Escherichia, Veillonella, and Faecalibacterium which belong to bacteria families Bacteroidaceae, Bifidobacteriaceae, Enterobacteriaceae, Veillonellaceae, and Ruminococcaceae, respectively.
  • the samples also included high proportions of Bacteroides, Faecalibacterium, Veillonella and Escherichia, which is similar as that in the healthy group.
  • the abundance of Bifidobacterium was significantly lower (4.94%) in the eczem
  • FIG. 8 shows different bacterial compositions between eczema and health groups based on a T-test and LEfSe (Linear Discriminant Analysis Effect Size) .
  • T-test Bifidobacterium, Enterobacter, Roseburia and Erwinia genera were varied between healthy and eczema groups (FIG. 8 (panel (a) )
  • LEfSe Linear Discriminant Analysis Effect Size
  • Bifidobacterium genus (Animalis, Pseudolongum, Breve, Bifidum, Longum and Adolescentis) were evaluated between eczema and healthy groups.
  • Bifidum, Longum and Adolescentis (OTUs number >2000 in 49 eczema samples) were much more abundant than the others in the genus.
  • Five out of the six species showed decreased relative abundance in eczema samples compared to the healthy group, which is consistent with the dynamic of the genus. Breve, though, showed higher abundance in eczema samples.
  • 16S rRNA gene sequencing showed relative quantification of bacteria in each sample. According to 16S rRNA gene sequencing, Bifidobacterium had significantly lower abundance in eczema samples, whereas Bacteroides was a dominant bacteria in both healthy and eczema samples without significant differences between healthy and eczema groups.
  • qPCR was used for absolute quantification of both Bifidobacterium and Bacteroides in samples. Briefly, qPCR was conducted by using Direct Detect Seven Genus/Species Gut Microbes Detection Kit (PCR-Fluorescence Probe, Coyote Bioscience Inc. ) . The PCR program was set as 10 cycles of 5s at 50°C, 5s at 95°C, 40 cycles of 50s at 95°C and 30s at 60°C. The PCR amplification was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, USA) . Primers and probe for Bifidobacterium were:
  • Reverse primer GCGTGGACTACCAGGGTATCTAAT
  • Reverse primer CGCTCCCTTTAAACCCAATAA
  • the subjects were also sub-divided into four groups by their age (0-0.5-year, 0.5-1 year, 1-2 years and 2-3 years) .
  • the PCoA plot shown in FIG. 6 suggests persistent differences in intestinal microbiome compositions.
  • a slightly decreased diversity of microbiome was also observed for subjects of 0.5-1 years in age in the eczema group (FIG. 9 (panel c) ) .
  • Shannon diversity analysis suggested that decreased diversity in intestinal microbiome is associated with eczema of subjects 2-3 years in age and confirmed expansion in bacterial diversity during 0-3 years.
  • Taxonomic assignment provided the top 10 genera in each group, and Bacteroides was found to be the dominate bacteria in age 1-3 years samples (1-2 years: 41.53%in eczema group, 34.45%in healthy group; 2-3 years: 27.51%in eczema group, 29.84%in healthy group) , but it was not the most species in 0-0.5-and 0.5-1-year samples.
  • the inter-group dissimilarity was also assessed for different age groups. As shown in FIG. 7, analysis showed that the inter-group difference was larger than that of the intra-group in all of the four age groups. ANOSIM, MRPP, Adonis and Amova were applied to analyze the difference in bacterial composition among the age groups. Results from all of these statistical analyses showed significant differences between healthy and eczema samples (FIG. 7) . Data for all subjects across the total age range is also shown in FIG. 7.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés de profilage de microbes. Dans certains modes de réalisation, les procédés comprennent la mise en oeuvre d'un essai d'amplification d'acide nucléique afin d'évaluer la quantité ou la concentration d'au moins un type de microbe bénéfique ou d'au moins un type de microbe pathogène dans une pluralité de microbes. L'invention concerne également des systèmes et des compositions qui peuvent être utilisés dans les procédés.
PCT/CN2019/111447 2018-10-16 2019-10-16 Procédés et systèmes de profilage de microbes Ceased WO2020078378A1 (fr)

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CN112094934A (zh) * 2020-09-01 2020-12-18 江南大学 一种结合多种内标对样本中真菌进行绝对定量的方法
WO2022081815A1 (fr) * 2020-10-14 2022-04-21 Pickens Peter Vincent Détection optimisée de microbes véhiculés par le sang
CN114496088A (zh) * 2022-01-27 2022-05-13 中国农业科学院兰州兽医研究所 一种棘球蚴病微生物标志物及其筛选方法和应用
CN114496088B (zh) * 2022-01-27 2023-06-20 中国农业科学院兰州兽医研究所 一种棘球蚴病微生物标志物及其筛选方法和应用
CN117316292A (zh) * 2023-11-30 2023-12-29 北京大学 基于细菌-古菌-真菌共现网络评价抗生素生态效应的方法
CN117316292B (zh) * 2023-11-30 2024-01-30 北京大学 基于细菌-古菌-真菌共现网络评价抗生素生态效应的方法
CN118531104A (zh) * 2024-05-28 2024-08-23 江苏省家禽科学研究所 一种基于16S rRNA高通量测序技术对热应激环境下鸡肠道微生物的检测方法

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