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WO2024077197A1 - Panel de qpcr multiplex pour pathogènes gastro-intestinaux - Google Patents

Panel de qpcr multiplex pour pathogènes gastro-intestinaux Download PDF

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Publication number
WO2024077197A1
WO2024077197A1 PCT/US2023/076163 US2023076163W WO2024077197A1 WO 2024077197 A1 WO2024077197 A1 WO 2024077197A1 US 2023076163 W US2023076163 W US 2023076163W WO 2024077197 A1 WO2024077197 A1 WO 2024077197A1
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Prior art keywords
primer pair
probe
specific
sequences
campylobacter
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Inventor
Kelly Li
Ioanna PAGANI
Michael Tanner
Changfu WEI
Namrata PABBATI
Elvis HUARCAYA NAJARRO
Gregory Govoni
Elena INGERMAN
Daryn Kenny
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Life Technologies Corp
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Life Technologies Corp
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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
    • C12Q1/68—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
    • 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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • Example embodiments relate to nucleic acid-based kits, compositions, and methods for determining the presence or absence in a sample of diarrhea causing pathogens including the following: Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC).
  • pathogens including the following: Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC).
  • GI pathogens Bacterial gastrointestinal (GI) pathogens (such as Campylobacter, Salmonella and Shigella/Enteroinvasive Escherichia coli (EIEC)) are the leading causes of acute gastroenteritis, hemorrhagic colitis and fatal typhoid fever.
  • EIEC Bacterial gastrointestinal pathogens
  • the presence of GI pathogens is correlated to (a) plethora of highly impactful diseases and (b) an increase in antibiotic resistance. Accordingly, detection of the etiological agents of acute bacterial diarrhea is important for the management of the patient and for public health interventions, while key clinical decisions are driven by bacterial gastrointestinal identification.
  • Salmonella infection can cause a variable clinical disease starting from a mild, subclinical infection, or lead to severe systemic infection such as typhoid fever.
  • Salmonella sp. invades the host through the colonic epithelial cells, especially M cells using a type III secretion system. They are also able to survive within phagosomes of macrophages, and evade the host immune system by several ways. Campylobacter jejuni and coli are among the large Campylobacter family predominant human stool pathogens causing watery diarrhea, fever and typically hard abdominal pain; Campylobacter upsaliensis is the most important Campylobacter species after C. jejuni and C. coli. Shigella and EIEC are genetically closely related. Both of these organisms invade the colonic epithelium mediated by the genes located in virulence plasmid pINV coding e.g., Ipa proteins and their transcription regulator invE.
  • a method for determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample comprises the steps of producing at least one amplicon by subjecting a reaction mixture including the sample and at least five primer pairs to reaction conditions suitable to amplify targeted nucleic acids wherein the at least five primer pairs include at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome, at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome, at least one primer pair D that specifically hybridizes with a portion of Salmonella sp.
  • the at least five primer pairs include at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridize
  • At least one primer pair E that specifically hybridizes with a portion of Shigella sp./EIEC genome; and determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample based on the at least one amplicon.
  • the producing of the at least one amplicon includes performing PCR.
  • the at least one amplicon is one selected from an amplicon produced using the primer pair A and comprising a sequence selected from the sequences specific for Campylobacter coli in “Probe” column of Figure 1 , an amplicon produced using the primer pair B and comprising a sequence selected from the sequences specific for Campylobacter jejuni in “Probe” column of Figure 1, an amplicon produced using the primer pair C and comprising a sequence selected from the sequences specific for Campylobacter upsaliensis in “Probe” column of Figure 1, an amplicon produced using the primer pair D and comprising a sequence selected from the sequences specific for Salmonella sp. in “Probe” column of Figure 1, and/or an amplicon produced using the primer pair E and comprising a sequence selected from the sequences specific for Shigella sp./EIEC in “Probe” column of Figure 1.
  • the reaction mixture includes probes specific for the at least one amplicon.
  • the reaction mixture further comprises probes suitable for use with a specific forward and reverse primer pair, and the probes are selected from the probe sequences provided in Figure 1.
  • the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter coli in Figure 1
  • the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter jejuni in Figure 1
  • the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter upsaliensis in Figure 1
  • the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Salmonella sp. in Figure 1
  • the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Shigella sp./EIEC in Figure 1.
  • the reaction mixture further comprises probes suitable for use with said primer pairs in Figure 1.
  • the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 1 ;
  • the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 2;
  • the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 3;
  • the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 4;
  • the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 5.
  • the reaction mixture further contains a control sample and a primer pair F that specifically hybridizes with a portion of the control sample genome, and, optionally, a probe specific for target nucleic acid of the control sample.
  • the primer pair F comprises forward and reverse primer pair selected from Table 6, and the probe sequence is selected from Table 6.
  • the probes each include a fluorescent reporter.
  • the probes each includes a quencher.
  • each of the probes is labeled at or near the 5’ end with a dye selected from Alexa Fluor, ABY, VIC, JUN, FAM and 6FAM.
  • each of the probes is labeled at the 3’ end with a quencher selected from QSY, MGBNFQ, BHQ, and DFQ.
  • the reaction mixture comprises probes specific for amplicons produced using one or more of the at least one primer pair A, the at least one primer pair B and the at least one primer pair C, the probes being labeled with 6FAM, at least one probe specific for amplicons produced using the at least one primer pair D, the probes being labeled with VIC, and at least one probe specific for amplicons produced using the at least one primer pair E, the probe being labeled with ABY.
  • a composition for determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample comprises: at least five primer pairs, wherein the at least five primer pairs includes at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome, at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome, at least one primer pair D that specifically hybridizes with a portion of Salmonella sp. genome, and at least one primer pair E that specifically hybridizes with a portion
  • the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter coli in Figure 1
  • the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter jejuni in Figure 1
  • the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter upsaliensis in Figure 1
  • the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Salmonella sp. in Figure 1
  • the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Shigella sp./EIEC in Figure 1.
  • the composition further comprises probes specific for amplicons produced using one or more of the at least one primer pair A, the at least one primer pair B, the at least one primer pair C, the at least one primer pair D, and the at least one primer pair E.
  • the probes are suitable for use with a specific forward and reverse primer pair in Figure 1, and optionally, a probe in Figure 1.
  • the composition further comprises a polymerase, a buffer, and nucleotides.
  • the composition is suitable for producing at least one amplicon when included in a reaction mixture that is subjected to reaction conditions suitable to amplify target nucleic acid of a sample in the reaction mixture.
  • the composition further comprises a primer pair F that includes a forward and reverse primer pair selected from Table 6; and optionally, at least one probe selected from Table 6.
  • the at least one probe includes a fluorescent reporter.
  • the at least one probe includes a quencher.
  • the at least one probe is labeled at or near the 5’ end with a dye selected from Alexa Fluor, ABY, VIC, JUN, FAM and 6FAM.
  • the at least one probe is labeled at the 3’ end with a quencher selected from QSY, MGBNFQ, BHQ, and DFQ.
  • the probes specific for amplicons produced using one or more of the primer pairs A, B and C are labeled with 6FAM
  • the probe specific for amplicons produced using the primer pair D is labeled with VIC
  • the probe specific for amplicons produced using the primer pair E is labeled with ABY.
  • the probe specific for amplicons produced using the primer pair F is labeled with JUN.
  • a kit for determining the presence or absence of Campylobacter sp., Salmonella sp., and Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample comprising the composition described in the above embodiments.
  • the kit further comprises a control sample; a primer pair F that specifically hybridizes with a portion of control sample genome; and optionally, a probe that specifically hybridizes with target nucleic acid of a control sample.
  • the primer pair F comprises a forward and reverse primer pair selected from Table 6 and the probe sequence is selected from Table 6.
  • the at least five primer pairs and probe set comprise at least one set of the primer pairs and probe as provided in a Table 7.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 8.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 9.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 10.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 11.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 12.
  • FIG. 1 provides exemplary forward and reverse primer pairs, together with a probe that is used with that primer pair (that is, a primer pair/probe set), as well as, the pathogen (or control organism, in case of process control) that is detected with the primer pair/probe set (as can be determined by the column “pathogen or gene”).
  • a primer pair/probe set that is, a primer pair/probe set
  • pathogen or control organism, in case of process control
  • “Primer pair No.” denotes different primer pair/probe sets that can be used for detection of a particular pathogen.
  • the present invention provides a multiplex polymerase chain reaction (PCR) based assay method for detection of diarrhea causing pathogens, such as Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC).
  • PCR polymerase chain reaction
  • the present invention further provides materials such as primers, primer pairs and probes for use in the method of the invention.
  • the present application discloses primers and probes designed for target sequences and compatible for use in a multiplex qPCR determining the presence of multiple diarrhea causing pathogens.
  • Multiplex PCR presents a challenge for quantitation of the pathogen DNA: the different amplicons compete for the same PCR reaction components (such as e.g. DNA polymerase and MgC12) and this can compromise the quantitative comparison between samples. It is commonly known in the art that there is bias in the amplification efficiencies between different template amounts or lengths so that e.g. short amplicons are favored in the expense of longer ones. At the same time, undesired cross-reactions of multiplex set oligo combinations must be avoided.
  • primers and probes for detecting Salmonella sp. and Shigella sp./EIEC discloses primers and probes for detecting each of the Campylobacter jejuni, Campylobacter coli and Campylobacter upsaliensis.
  • the selected primers and probes of the assay do not cross-react with other closely-related pathogens.
  • the disclosed detection method, as well as detection assay may contain internal process control, such as Bacillus atrophaeus (BA).
  • the assay may have a real time PCR positive control.
  • the probes that target genomic regions of different pathogens utilize three distinct fluorophores, while a fourth fluorophore is designated for the process control detection.
  • composition e.g., the particular physical components of an assay such as primers and/or probes
  • kit e.g., primers and/or probes and additional buffers, reagents, etc.
  • method e.g., a process for detecting target nucleic acids
  • a method for determining the presence or absence of Campylobacter sp., Salmonella sp., and Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample comprising the steps of: (a) providing a reaction mixture containing the sample and at least 5 primer pair set; wherein the primer pair set comprises: at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome; at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome; at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome; at least one primer pair D that specifically hybridizes with a portion of Salmonella sp.
  • the primer pair set comprises at least one primers pair A, at least one primer pair B, at least one primer pair C, at least one primer pair D and at least one primer pair E. In some embodiments, the primer pair set further comprises at least one primer pair F. In some embodiments, the primer pairs are used with corresponding probes as set out in Figure 1. In some embodiments, the primer pair set comprises at least two primers pairs A, at least two primer pairs B, at least two primer pairs C, at least two primer pairs D and at least two primer pairs E.
  • the primer pair A comprises at least one of the primer pairs, in a combination with a corresponding probe sequence:
  • the primer pair B comprises at least one of the primer pairs, in a combination with a corresponding probe sequence:
  • the primer pair C comprises at least one of the primer pairs, in a combination with a corresponding probe sequence: Table 3 [0055] In some embodiments, the primer pair D comprises at least one of the primer pairs, in a combination with a corresponding probe sequence:
  • the primer pair E comprises at least one of the primer pairs, in a combination with a corresponding probe sequence:
  • the method further comprises at least one primer pair F and a probe for detection of control sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the method comprises the following primer pair and probe set for detection of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample:
  • the invention provides compositions, kits and method for the detection (i.e., the presence or absence) of diarrhea-causing pathogens including Salmonella sp., Campylobacter sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a specimen or a sample.
  • diarrhea-causing pathogens including Salmonella sp., Campylobacter sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a specimen or a sample.
  • the specimen/sample may comprise any number of things, including, but not limited to, include stool samples, rectal swabs or tissue samples (for example, taken during a biopsy) obtained from human patients; research samples; purified samples, such as purified genomic DNA, RNA, proteins, etc.; and raw samples (bacteria, virus, genomic DNA, etc.).
  • the specimen/sample type for diagnosis of diarrhea-causing pathogens is a stool sample or a rectal swab.
  • nucleic acid from the sample/specimen is isolated using known techniques.
  • the sample/specimen may be treated to lyse the cells, using known lysis buffers, sonication, electroporation, etc., with purification occurring as needed, as will be appreciated by those in the art.
  • the reactions outlined herein may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below.
  • the reaction may include a variety of other reagents that may be included in the assays.
  • reagents like salts, buffers, neutral proteins, e.g., albumin, detergents, etc., which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions.
  • reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample/specimen preparation methods and purity of the target diarrhea-causing pathogens.
  • the total nucleic acid extraction from a stool sample or a rectal swab is performed.
  • the specimen/sample may be collected and transported in the RemelTM Cary-Blair Transport Medium by ThermoFisher Scientific according to appropriate laboratory procedures.
  • RemelTM Cary-Blair Transport Medium is a semisolid medium recommended for use in the collection, transportation and preservation of sample/specimens, especially stool samples and rectal swabs.
  • Nucleic acids may be isolated and purified from the specimen/sample using a nucleic acid isolation, such as, e.g., the MagMAXTM Microbiome Ultra Nucleic Acid Isolation Kit with Bead Plate by Applied BiosystemsTM.
  • Nucleic acid extraction may be performed via an automated process using, e.g., the KingFisherTM Flex Purification System.
  • the RNA is reverse transcribed into cDNA.
  • the cDNA and genomic DNA are then subjected for amplification using the currently disclosed composition, method and kit.
  • Process control or Control organism or Control sample may further include a process control (“process control” is herein referred to, interchangeably, as “control organism” or “control sample”).
  • process control is an exogenous control that has the added advantage of being a bacterial lysis control in addition to being a nucleic acid extraction and recovery control.
  • Controls are treated and tested in parallel with target pathogen and are used to generate a predetermined expected result. When the expected result is reported, one or more aspects of the diagnostic test are confirmed to be working as intended, enabling the user of to verify the diagnostic test as valid.
  • the process control may be Bacillus atrophaeus, which is a grampositive, endospore forming bacterium.
  • the process control can function as a positive control for lysis, purification and amplification within the cartridges described herein.
  • One exemplified process control is lyophilized Bacillus atrophaeus, such as, e.g., the TaqManTM Universal Extraction Control Organism by ThermoFisher Scientific.
  • the process control may be supplied lyophilized in a quantity of 1 * IO copies/vial, and reconstituted in 200 pL of IX PBS, pH 7.4 to a final concentration 5 x I 6 copies/pL.
  • 10 pL of the process control is processed as a stand-alone sample in a background of universal transport media.
  • the process control can be added to a negative extraction control.
  • the process control may be added to one or more samples/ specimens at the start of the extraction process.
  • the process control is carried through the remainder of the workflow with the samples/specimens. It is recommended that at least one stand-alone control sample is run per extraction plate.
  • Another exemplified process control is a bacterial spore, such as a spore of & Bacillus species.
  • Suitable spores can be comprised of any species of Bacillus, including, e.g., Bacillus globgii, Bacillus atrophaeus, Bacillus subtilis, and Bacillus stearothermophilus .
  • the process control may be a Bacillus atrophaeus bacterial spore.
  • VOC Vicinal oxygen chelate
  • the disclosed method of detection may further include a positive control to determine the validity of the assay.
  • the positive control is a mixture of plasmids of the target pathogens.
  • an exemplified positive control is a mixture of plasmids of Salmonella sp., Campylobacter sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC).
  • Yet another exemplified positive control is a mixture of plasmids of Salmonella sp., Shigella sp./Enteroinvasive Escherichia coli (EIEC), Campylobacter coli (, Campylobacter jejuni and/or Campylobacter upsaliensis.
  • EIEC Enteroinvasive Escherichia coli
  • Campylobacter coli Campylobacter jejuni and/or Campylobacter upsaliensis.
  • reaction mixture refers to a mixture of components necessary to amplify at least one amplicon from nucleic acid templates.
  • the mixture may comprise nucleotides (dNTPs), a thermostable polymerase, primers, and a plurality of nucleic acid templates.
  • the mixture may further comprise a Tris buffer, a monovalent salt, and/or Mg2+.
  • the working concentration range of each component is well known in the art and can be further optimized as needed by an ordinary skilled artisan.
  • Amplification denotes the use of any amplification procedures to increase the concentration of a particular nucleic acid sequence within a mixture of nucleic acid sequences.
  • a sequence from a sample is amplified to produce a secondary target (e.g., an amplicon) that is detected, as outlined herein.
  • a secondary target e.g., an amplicon
  • Amplification involves the amplification (replication) of the sequence to be detected, such that the number of copies of the sequence is increased.
  • Suitable amplification techniques include, but are not limited to, the polymerase chain reaction (PCR), strand displacement amplification (SDA), transcription mediated amplification (TMA) and nucleic acid sequence-based amplification (NASBA).
  • PCR polymerase chain reaction
  • SDA strand displacement amplification
  • TMA transcription mediated amplification
  • NASBA nucleic acid sequence-based amplification
  • the amplification technique is PCR.
  • the polymerase chain reaction (PCR) is widely used and described, and involves the use of primer extension combined with thermal cycling to amplify a target sequence.
  • PCR refers to either singleplex or multiplex PCR assays, and can be real time or quantitative PCR (wherein detection occurs during amplification), end-point PCR (when detection occurs at the end amplification), or reverse transcription PCR, including but not limited to, “real-time PCR” or “quantitative PCR” or “qPCR”, “digital PCR” or “dPCR”, “reverse transcriptase PCR” or “RT-PCR”, “multiplex PCR”, “nested PCR”, “hot start PCR”, “long-range PCR”, “assembly PCR”, “asymmetric PCR”, “in situ PCR,” “single-cell PCR,” or “fast-cycling PCR,” among others.
  • Example embodiments of amplification are not limited to PCR.
  • signal amplification single base extension (SBE) or minisequencing, oligonucleotide ligation amplification (OLA) and/or rolling-circle amplification can be used for amplification.
  • amplification can include OLA followed by RCA.
  • exemplary systems include a real-time quantitative PCR (qPCR) instrument, including for example a QuantStudioTM Real-Time PCR system, such as the QuantStudioTM 5 Real-Time PCR System (QS5), QuantStudioTM 7 Real-Time PCR System (QS7), QuantStudioTM 12K Flex System (QS12K), QuantStudioTM DX Real-Time PCR System (QS Dx or QS5 Dx), or a 7500 Real-Time PCR system, such as the 7500 Fast Dx system, all from Applied BiosystemsTM - a ThermoFisher Scientific brand.
  • qPCR real-time quantitative PCR
  • amplified product or “amplicon” refer to a fragment of DNA amplified by a polymerase using a pair of primers in an amplification method such as PCR.
  • Probe is a non-extendable oli onucleotide attached to a fluorescent reporter dye and a quencher moiety.
  • Primer can refer to more than one primer and refers to an oligonucleotide, whether occurring naturally or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase, at a suitable temperature for a sufficient amount of time and in the presence of a buffering agent.
  • Such conditions can include, for example, the presence of at least four different deoxyribonucleoside triphosphates (such as G, C, A, and T) and a polymerization-inducing agent such as DNA polymerase or reverse transcriptase, in a suitable buffer (“buffer” includes substituents which are cofactors, or which affect pH, ionic strength, etc.), and at a suitable temperature.
  • the primer may be single-stranded for maximum efficiency in amplification.
  • the primers herein are selected to be substantially complementary to the different strands of each specific sequence to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands.
  • a non-complementary nucleotide fragment may be attached to the 5 '-end of the primer, with the remainder of the primer sequence being complementary, or partially complementary, to the target region of the target nucleic acid.
  • the primers are complementary, except when non-complementary nucleotides may be present at a predetermined sequence location, such as a primer terminus as described.
  • nucleic acid sequence refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5' end of one sequence is paired with the 3' end of the other, is in “antiparallel association.” Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases.
  • Dye or Detectable Label or Fluorescent Label
  • each fluorescent or detectable label may further comprise a fluorescent or other detectable label. It should be appreciated that when using multiple fluorescent or detectable labels, particularly in a multiplex format, each fluorescent or detectable label preferably differs in its spectral properties from the other detectable labels used therewith such that the labels may be distinguished from each other, or such that together the fluorescent or detectable labels emit a signal that is not emitted by either fluorescent or detectable label alone.
  • Exemplary fluorescent or detectable labels include, for instance, a fluorescent dye or fluorophore (e.g., a chemical group that can be excited by light to emit fluorescence or phosphorescence), “acceptor dyes” capable of quenching a fluorescent signal from a fluorescent donor dye, and the like, as described above.
  • a fluorescent dye or fluorophore e.g., a chemical group that can be excited by light to emit fluorescence or phosphorescence
  • acceptor dyes capable of quenching a fluorescent signal from a fluorescent donor dye, and the like, as described above.
  • Suitable fluorescent or detectable labels may include, for example, fluoresceins (e.g., 5-carboxy-2,7-dichlorofluorescein; 5- Carboxyfluorescein (5-FAM); 5-Hydroxy Tryptamine (5-HAT); 6-JOE; 6-carboxyfluorescein (6-FAM); Mustang Purple, VIC, ABY, JUN; FITC; 6-carboxy-4’,5’-dichloro-2’,7’- dimethoxy-'fluorescein (JOE)); 6-carboxy-l,4-dichloro-2’,7’-dichloro _, fluorescein (TET); 6- carboxy-l,4-dichloro-2’,4’,5’,7’-tetra-chlorofluorescein (HEX); Alexa Fluor fluorophores (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568, 594
  • EGFP blue fluorescent protein
  • EBFP blue fluorescent protein
  • CyPet yellow fluorescent protein
  • FRET donor/acceptor pairs e.g., fluorescein/fluorescein, fluorescein/tetramethylrhodamine, lAEDANS/fluorescein, EDANS/dabcyl, BODIPY FL/BODIPY FL, Fluorescein/QSY7 and QSY9
  • LysoTracker and LysoSensor e.g., LysoTracker Blue DND-22, LysoTracker Blue-White DPX, LysoTracker Yellow HCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensor Blue DND-
  • Exemplary fluorescent labels include but are not limited to 6FAM, ABY, VIC, JUN, and FAM.
  • the fluorescent labels include, for instance, 6FAM, VIC, ABY and JUN, or FAM, VIC, ABY and JUN.
  • the fluorescent labels include, for instance, FAM, HEX (JOE/VIC), Texas Red, and Cy5 dyes. Exemplary dye schemes are shown in Table 19.
  • the probes specific for amplicons produced using one or more primer pair A are labeled with FAM; the probes specific for amplicons produced using one or more primer pair B (target - C. upsaliensis) are labeled with FAM; the probes specific for amplicons produced using one or more primer pair C (target - C.
  • primers can be labeled and used to both produce amplicons and to detect the presence (or concentration) of amplicons produced in the reaction, and such may be used in addition to or as an alternative to labeled probes described herein.
  • primers may be labeled and utilized as described in Nazarenko et al. (Nucleic Acids Res. 2002 May 1 ; 30(9): e37), Hayashi et al. (Nucleic Acids Res. 1989 May 11 ; 17(9): 3605), and/or Neilan et al. (Nucleic Acids Res. Vol. 25, Issue 14, 1 July 1997, pp. 2938-39).
  • Those of skill in the art will also understand and be capable of utilizing the PCR processes (and associated probe and primer design techniques) described in Zhu et al. (Biotechniques. 2020 Jul: 10.2144/btn-2020-0057).
  • the primers and/or probes may further comprise a quencher.
  • Suitable quenchers include but are not limited to QSY (e.g., QSY7 and QSY21), BHQ (Black Hole Quencher) and DFQ (Dark Fluorescent Quencher).
  • the quencher is QSY7.
  • Detector probes may also include two probes, wherein, for example, a fluorophore is associated with one probe and a quencher is associated with a complementary probe such that hybridization of the two probes on a target quenches the fluorescent signal or hybridization on the target alters the signal signature via a change in fluorescence.
  • Detector probes may also include sulfonate derivatives of fluorescein dyes with SO3 instead of the carboxylate group, phosphoramidite forms of fluorescein, phosphoramidite forms of Cy5.
  • intercalating labels can be used such as ethidium bromide, SYBR Green I, SYBR GreenER, and PicoGreen (all products of Applied Biosystems - a brand of ThermoFisher Scientific), thereby allowing visualization in real-time, or end point, of an amplification product in the absence of a detector probe.
  • real-time visualization may include both an intercalating detector probe and a sequence-based detector probe.
  • the detector probe is at least partially quenched when not hybridized to a complementary sequence in the amplification reaction and is at least partially unquenched when hybridized to a complementary sequence in the amplification reaction.
  • probes may further comprise various modifications such as a minor groove binder (MGB) to further provide desirable thermodynamic characteristics.
  • MGB minor groove binder
  • the amplicon is labeled by incorporation of, or hybridization to labeled primer. In some embodiments, the amplicon is labeled by hybridization to a labeled probe. In some embodiments, the amplicon is labeled by binding of a DNA-binding dye. In some embodiments, the dye may be a single-strand DNA binding dye. In other embodiments, the dye may be a double-stranded DNA binding dye. In other embodiments, the amplicon is labeled via polymerization or incorporation of labeled nucleotides in a template-dependent (or template-independent) polymerization reaction.
  • the labeled nucleotide can be added after amplifying is completed.
  • the labeled amplicon (or labeled derivative thereof) can be detected using any suitable method such as, for example, electrophoresis, hybridization-based detection (e.g., microarray, molecular beacons, and the like), chromatography, NMR, and the like.
  • the labeled amplicon is detected using qPCR.
  • a plurality of different amplicons is formed, and optionally labeled, within a single reaction volume via a single amplification reaction.
  • a multiplex reaction e.g., 4-plex
  • a single tube or reaction vessel e.g., “single-tube” or “1-tube” or “single-vessel” reaction
  • the plurality of amplicons can be differentially labeled.
  • each of the plurality of amplicons produced during amplification is labeled with a different label.
  • say mixture or “assay mix” or “assay composition,” as used herein, include mixture containing the primer-probe pairs described above that are used in PCR.
  • PCR polymerase chain reaction
  • qPCR quantitative real-time polymerase chain reaction
  • the method includes: (i) contacting a sample comprising one or more target nucleic acid molecules with a) at least one probe, such as those described herein, being sequence specific for the target nucleic acid molecule, where the at least one probe undergoes a detectable change in fluorescence upon amplification of the one or more target nucleic acid molecules; and with b) at least one oligonucleotide primer pair; (ii) incubating the mixture of step (i) with a DNA polymerase under conditions sufficient to amplify one or more target nucleic acid molecules; and (iii) detecting the presence or absence or quantifying the amount of the amplified target nucleic acid molecules by measuring fluorescence of the probe.
  • the DNA polymerase comprises 5’ exonuclease activity. In some other embodiments, the DNA polymerase is a Thermus aquaticus (Taq) DNA polymerase. In some embodiments, the probe is a hydrolysis probe, such as a TaqMan probe.
  • Taq Thermus aquaticus
  • kits for PCR such as quantitative real-time polymerase chain reaction (qPCR) and reverse transcription polymerase chain reaction (RT- PCR).
  • the kit includes the assay mixture, the process control, and the positive control each described above, as well as a multiplex master mix.
  • the multiplex master mix is a RT-qPCR mix that provides for sensitive, reproducible detection of at least four different target pathogens in a single multiplex reaction.
  • the multiplex master mix may include an enzyme (for instance, DNA polymerase), a thermostable enzyme, enzyme cofactors, deoxynucleotide triphosphates (dNTPs) including dUTP, an enzyme inhibitor (for instance, RNase inhibitor), a dye and/or a buffer agent.
  • the multiplex master mix can be, for instance, TaqPathTM 1-step Multiplex Master Mix by Applied Biosystems - a brand of ThermoFisher Scientific.
  • the master mix may be concentrated.
  • the master mix may be provided at a 4X concentration.
  • the master mix is prepared such that it requires less than a 3X dilution prior to use in PCR, e.g., 2X dilution, 1.5X dilution, 1.2X dilution, etc.
  • the kit also includes instructions for conducting the PCR, and one or more of the following: a buffering agent, deoxynucleotide triphosphates (dNTPs), an organic solvent, an enzyme, enzyme cofactors, and an enzyme inhibitor.
  • dNTPs deoxynucleotide triphosphates
  • the kit for PCR comprises the described dye and/or quencher moiety, instructions for conjugating or labeling the dye and/or quencher moiety to a biomolecule, such as an oligonucleotide, instructions for conducting the PCR, and one or more of the following: a buffering agent, deoxynucleotide triphosphates (dNTPs), an organic solvent, an enzyme, enzyme cofactors, and an enzyme inhibitor.
  • a buffering agent deoxynucleotide triphosphates (dNTPs)
  • dNTPs deoxynucleotide triphosphates
  • an organic solvent an enzyme
  • enzyme cofactors enzyme inhibitors
  • the systems, compositions, methods, and devices used for nucleic acid amplification comprise a “point-of-service” (POS) system.
  • samples may be collected and/or analyzed at a “point-of-care” (POC) location.
  • POC point-of-care
  • analysis at a POC location typically does not require specialized equipment and has rapid and easy-to-read visual results.
  • analysis can be performed in the field, in a home setting, and/or by a lay person not having specialized skills.
  • the analysis of a small-volume clinical sample may be completed using a POS system in a short period of time (e.g., within hours or minutes).
  • a POS system is utilized at a location that is capable of providing a service (e.g., testing, monitoring, treatment, diagnosis, guidance, sample collection, verification of identity (ID verification), and other services) at or near the site or location of the subject.
  • a service may be a medical service, or it may be a non-medical service.
  • a POS system provides a service at a predetermined location, such as a subject's home, school, or work, or at a grocery store, a drug store, a community center, a clinic, a doctor's office, a hospital, an outdoor triage tent, a makeshift hospital, a border check point, etc.
  • a POS system can include one or more point of service devices, such as a portable virus/pathogen detector.
  • a POS system is a point of care system.
  • the POS system is suitable for use by non-specialized workers or personnel, such as nurses, police officers, civilian volunteers, or the patient.
  • a POC system is utilized at a location at which medical-related care (e.g., treatment, testing, monitoring, diagnosis, counseling, etc.) is provided.
  • a POC may be, e.g., at a subject's home, work, or school, or at a grocery store, a community center, a drug store, a doctor's office, a clinic, a hospital, an outdoor triage tent, a makeshift hospital, a border check point, etc.
  • a POC system is a system which may aid in, or may be used in, providing such medical-related care, and may be located at or near the site or location of the subject or the subject's health care provider (e.g., subject's home, work, or school, or at a grocery store, a community center, a drug store, a doctor's office, a clinic, a hospital, etc.).
  • the subject's health care provider e.g., subject's home, work, or school, or at a grocery store, a community center, a drug store, a doctor's office, a clinic, a hospital, etc.
  • a POS system is configured to accept a clinical sample obtained from a subject at the associated POS location. In embodiments, a POS system is further configured to analyze the clinical sample at the POS location. In embodiments, the clinical sample is a small volume clinical sample. In embodiments, the clinical sample is analyzed in a short period of time. In embodiments, the short period of time is determined with respect to the time at which sample analysis began. In embodiments, the short period of time is determined with respect to the time at which the sample was inserted into a device for the analysis of the sample. In embodiments, the short period of time is determined with respect to the time at which the sample was obtained from the subject.
  • a POS system or a POC system can include the amplification-based methods, compositions and kits disclosed herein, including any of the described assays and/or assay panels.
  • Such assays are contemplated for use with both thermal cycling amplification workflows and protocols, such as in PCR, as well as isothermal amplification workflows and protocols, such as in LAMP.
  • a POS or a POC system comprises self-collection of a biological sample, such as a stool sample or rectal swab.
  • the selfcollection may comprise the use of a self-collection kit and/or device, such as a swab or a tube (e g., a stool collection tube or similar sample collection device).
  • the self-collection kit comprises instructions for use, including collection instructions, sample preparation or storage instructions, and/or shipping instructions.
  • the selfcollection kit and/or device may be used by an individual, such as lay person, not having specialized skills or medical expertise.
  • self-collection may be performed by the patient themselves or by any other individual in proximity to the patient, such as but not limited to a parent, a care giver, a teacher, a friend, or other family member.
  • the nucleic acid amplification protocol can be configured for rapid processing (e.g., in less than about 45 minutes) and high throughput, allowing for a minimally invasive method to quickly screen large numbers of individuals in a scalable way. This can be particularly useful to perform asymptomatic testing (e.g., high frequency/widespread testing at schools, workplaces, conventions, sporting events, large social gatherings, etc.) or for epidemiological purposes.
  • the disclosed embodiments can also beneficially provide a lower cost sample collection system and method that enables selfcollection (reducing health care professional staffing needs) using a low-cost collection device. This eliminates the requirements for swabs, buffers, virus transmission media (or other specialized transport medium), and the like.
  • the disclosed embodiments also allow for a reduction in Personal Protective Equipment (PPE) requirements and costs. There is also a beneficial reduced dependence on supply-constrained items, and the compatibility of these methods and kit components with existing equipment improves the flexibility and simplicity of their implementation to the masses. Overall, such embodiments allow for a less expensive assay that can be accomplished more quickly from sample collection through result generation.
  • PPE Personal Protective Equipment
  • a method for determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample comprises the steps of producing at least one amplicon by subjecting a reaction mixture including the sample and at least five primer pairs to reaction conditions suitable to amplify targeted nucleic acids wherein the at least five primer pairs include at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome, at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome, at least one primer pair D that specifically hybridizes with a portion of Salmonella sp.
  • the at least five primer pairs include at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridize
  • At least one primer pair E that specifically hybridizes with a portion of Shigella sp./ElEC genome; and determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in the sample based on the at least one amplicon.
  • the producing of the at least one amplicon includes performing PCR.
  • the at least one amplicon is one selected from an amplicon produced using the primer pair A and comprising a sequence selected from the sequences specific for Campylobacter coli in “Probe” column of Figure 1, an amplicon produced using the primer pair B and comprising a sequence selected from the sequences specific for Campylobacter jejuni in “Probe” column of Figure 1, an amplicon produced using the primer pair C and comprising a sequence selected from the sequences specific for Campylobacter upsaliensis in “Probe” column of Figure 1, an amplicon produced using the primer pair D and comprising a sequence selected from the sequences specific for Salmonella sp. in “Probe” column of Figure 1, and/or an amplicon produced using the primer pair E and comprising a sequence selected from the sequences specific for Shigella sp./EIEC in “Probe” column of Figure 1.
  • the reaction mixture includes probes specific for the at least one amplicon.
  • the reaction mixture further comprises probes suitable for use with a specific forward and reverse primer pair, and the probes are selected from the probe sequences provided in Figure 1.
  • the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter coli in Figure 1
  • the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter jejuni in Figure 1
  • the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter upsaliensis in Figure 1
  • the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Salmonella sp. in Figure 1
  • the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Shigella sp./EIEC in Figure 1.
  • the reaction mixture further comprises probes suitable for use with said primer pairs in Figure 1.
  • the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 1;
  • the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 2;
  • the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 3;
  • the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 4; and/or the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the Table 5.
  • the reaction mixture further contains a control sample and a primer pair F that specifically hybridizes with a portion of control sample genome, and, optionally, a probe specific for target nucleic acid of the control sample.
  • the primer pair F comprises a forward and reverse primer pair selected from Table 6, and the probe specific for the target nucleic acid of the control sample is selected from Table 6.
  • the probes each include a fluorescent reporter.
  • the probes each includes a quencher.
  • each of the probes is labeled at or near the 5’ end with a dye selected from Alexa Fluor, ABY, VIC, JUN, FAM and 6FAM.
  • each of the probes is labeled at the 3’ end with a quencher selected from QSY, MGBNFQ, BHQ, and DFQ.
  • the reaction mixture comprises probes specific for amplicons produced using one or more of the at least one primer pair A, the at least one primer pair B and the at least one primer pair C, the probes being labeled with 6FAM, at least one probe specific for amplicons produced using the at least one primer pair D, the probes being labeled with VIC, and at least one probe specific for amplicons produced using the at least one primer pair E, the probe being labeled with ABY.
  • the probe specific for the target nucleic acid of the control sample is specific for amplicons produced using the primer pair F, the probe being labeled with JUN.
  • a composition for determining the presence or absence of Campylobacter sp., Salmonella sp., and/or Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample comprises: at least five primer pairs, wherein the at least five primer pairs include at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridizes with a portion of Campylobacter jejuni genome, at least one primer pair C that specifically hybridizes with a portion of Campylobacter upsaliensis genome, at least one primer pair D that specifically hybridizes with a portion of Salmonella sp. genome, and at least one primer pair E that specifically hybridizes with a portion of Shigella sp./EIEC genome.
  • the at least five primer pairs include at least one primer pair A that specifically hybridizes with a portion of Campylobacter coli genome, at least one primer pair B that specifically hybridizes
  • the at least one primer pair A comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter coli in Figure 1
  • the at least one primer pair B comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter jejuni in Figure 1
  • the at least one primer pair C comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Campylobacter upsaliensis in Figure 1
  • the at least one primer pair D comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Salmonella sp. in Figure 1
  • the at least one primer pair E comprises at least one forward and reverse primer pair selected from primer pair sequences selected from the sequences specific for Shigella sp./EIEC in Figure 1.
  • the composition further comprises probes specific for amplicons produced using one or more of the at least one primer pair A, the at least one primer pair B, the at least one primer pair C, the at least one primer pair D, and the at least one primer pair E.
  • the probes are suitable for use with a specific forward and reverse primer pair in Figure 1, and optionally, a probe in Figure 1.
  • the composition further comprises a polymerase, a buffer, and nucleotides.
  • the composition is suitable for producing at least one amplicon when included in a reaction mixture that is subjected to reaction conditions suitable to amplify target nucleic acid of a sample in the reaction mixture.
  • the composition further comprises a primer pair F that includes a forward and reverse primer pair selected from Table 6; and optionally, at least one probe selected from Table 6.
  • the at least one probe includes a fluorescent reporter.
  • the at least one probe includes a quencher.
  • the at least one probe is labeled at or near the 5’ end with a dye selected from Alexa Fluor, ABY, VIC, JUN, FAM and 6FAM.
  • the at least one probe is labeled at the 3’ end with a quencher selected from QSY, MGBNFQ, BHQ, and DFQ.
  • the probes specific for amplicons produced using one or more of the primer pairs A, B and C are labeled with 6FAM
  • the probe specific for amplicons produced using the primer pair D is labeled with VIC
  • the probe specific for amplicons produced using the primer pair E is labeled with ABY.
  • the probe specific for amplicons produced using the primer pair F is labeled with JUN.
  • a kit for determining the presence or absence of Campylobacter sp., Salmonella sp., and Shigella sp./Enteroinvasive Escherichia coli (EIEC) in a sample comprising the composition described in the above embodiments.
  • the kit further comprises a control sample; a primer pair F that specifically hybridizes with a portion of control sample genome; and optionally, a probe that specifically hybridizes with target nucleic acid of a control sample.
  • the primer pair F comprises a forward and reverse primer pair selected from Table 6 and the probe sequence is selected from Table 6.
  • the at least five primer pairs and probe set comprise at least one set of the primer pairs and probe as provided in a Table 7.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 8.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 9.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 10.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 11.
  • the at least five primer pairs and probe set comprise at least one set of primer pairs and probe as provided in a Table 12.

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Abstract

Procédé, composition et kit pour déterminer la présence ou l'absence de Salmonella sp., Shigella sp./Escherichia coli entéroinvasif (EIEC) et Campylobacter sp. dans un échantillon, comprenant la production d'un amplicon en soumettant un mélange réactionnel comprenant l'échantillon et cinq paires d'amorces à des conditions de réaction adaptées à l'amplification des acides nucléiques ciblés. Les cinq paires d'amorces comprennent la paire d'amorces A qui s'hybride spécifiquement avec une partie du génome de Campylobacter coli, la paire d'amorces B qui s'hybride spécifiquement avec une partie du génome de Campylobacter jejuni, la paire d'amorces C qui s'hybride spécifiquement avec une partie du génome de Campylobacter upsaliensis, la paire d'amorces D qui s'hybride spécifiquement avec une partie du génome de Salmonella sp. et la paire d'amorces E qui s'hybride spécifiquement avec une partie du génome de Shigella sp./EIEC ; et il est possible de déterminer la présence ou l'absence de Campylobacter sp, Salmonella sp. et/ou Shigella sp. /Escherichia coli entéroinvasif (EIEC) dans l'échantillon grâce à l'amplicon.
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