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WO2009132354A2 - Procédé de confirmation de la présence d'un analyte - Google Patents

Procédé de confirmation de la présence d'un analyte Download PDF

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Publication number
WO2009132354A2
WO2009132354A2 PCT/US2009/041839 US2009041839W WO2009132354A2 WO 2009132354 A2 WO2009132354 A2 WO 2009132354A2 US 2009041839 W US2009041839 W US 2009041839W WO 2009132354 A2 WO2009132354 A2 WO 2009132354A2
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WIPO (PCT)
Prior art keywords
sample
nucleic acid
contaminant
dna
multiplex pcr
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WO2009132354A3 (fr
Inventor
Mansour Samadpour
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IEH LABORATORIES AND CONSULTING GROUP
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IEH LABORATORIES AND CONSULTING GROUP
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Publication of WO2009132354A2 publication Critical patent/WO2009132354A2/fr
Publication of WO2009132354A3 publication Critical patent/WO2009132354A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction

Definitions

  • the invention relates to the field of microbiology and more specifically relates to methods, articles, and kits for confirming the presence of an analyte. More particularly, the invention relates to a method and system that employs means to corroborate the findings of an initial positive assay result by a second robust alternative assay protocol.
  • the present art of rapid screening suffers from the fact that either single signals are to be used to detect a given pathogen, in which case not all of the targets will be detected (they may not all have the target signal), or if multiple signals (multiplexes) are used to detect the pathogen/organism of interest, in matrixes such as food, water, environmental samples, and some clinical samples (such as urine and feces) where a diverse group of organisms are present, there is a distinct possibility that that the multiplex screen will detect a composite signal from more than one organism. For instance, if a food sample contains a non-toxigenic, eae negative E. coli 0157, an enteropathogenic E. coli, and an E.
  • the present invention allows for rapid confirmation of the initial analyte test results. Typically the process takes from about one to about three hours. Furthermore, in a preferred embodiment, the process will allow for confirmation of the presence of a given microbial target in a mixed culture, or a mixed enrichment media, even when the competing organisms in the mix belong to related species, or are various biotypes of the same species.
  • the methods of the present invention allow differentiation between E. coli 0157 and non-0157 Shiga toxin-producing E. coli in the same sample. Additionally, these methods can differentiate between Listeria monocytogenes and other Listeria species in the same sample. Also, these methods can identify multi-drug resistant salmonella from other Salmonella or other multi-drug resistant bacteria in the same sample.
  • the invention solves the problems of the prior art by providing protocols to meet the performance criteria and generate a result rapidly so that subsequent decisions can be made in a timely manner.
  • Various embodiments of the present invention are generally complementary to two broad classes of rapid screening methods which enrich a sample prior to application of an assay protocol.
  • 'enrich' is understood to consist of the steps of adding growth media to a sample and incubating the sample at an appropriate temperature and for an appropriate period of time, so that the target organism is given an opportunity to reproduce and reach a concentration level commensurate with the detecting ability of the assay protocol.
  • the first broad class of rapid screening methods which rely on enriching a sample use the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • DNA primer templates are added which are composed of specific nucleotide sequences that complement known portions of genes which are unique to the target analyte organism.
  • a DNA polymerase and nucleotides are added, and in vitro enzymatic replication of DNA occurs. As replicate DNA strands are generated, they serve as templates for the next cycle of replication.
  • cycles are defined by alternating heating and cooling of the sample to allow the DNA to physically separate and join, with the thermal cycling conditions providing another means of enhancing selectivity.
  • PCR With PCR it is possible to amplify a single or a few copies of a specific strand of DNA across several orders of magnitude, generating millions or more copies of the specific strand of DNA. Thereafter, the DNA is generally separated using agarose gel chromatography, and the DNA product, if present, is visualized by the application of a dye to the agarose gel.
  • the second broad class of rapid screening methods which rely on enriching a sample are immunoassays, which rely on reactions between antibodies and their corresponding antigens which are unique to the target analyte organism.
  • immunoassays which rely on reactions between antibodies and their corresponding antigens which are unique to the target analyte organism.
  • one group of methods are enzyme-linked immunosorbent assays (ELISAs) in which an antigen associated with the target analyte organism is affixed to a surface, and then an antibody specific to that antigen is washed over the surface so that it can bind to the antigen.
  • the antibody is linked to an enzyme which is capable of converting a substance that is added to some detectable signal.
  • a second group of methods are immunochromatographic assays (also called lateral flow devices, or LFDs) in which a test sample flows through a matrix via capillary action.
  • the sample encounters a visualizing reagent (e.g. colorimetric or fluorometric) which then migrates with the sample.
  • a visualizing reagent e.g. colorimetric or fluorometric
  • the sample encounters lines or zones which have been pretreated with either an antibody or antigen that binds to the target analyte, if present, and associated visualizing reagent.
  • the presumptive positive sample will be subjected to PCR reaction(s) which are designed to amplify multiple targets
  • the targets include at least two genes which are restricted mostly to or are unique in the target organism.
  • At least one PCR reaction should target the conserved regions of the molecule, and another PCR reaction should target the specific variation(s) which are unique to the target organism.
  • each PCR/multiplex confirmation reaction should be conducted with and without immunomagnetic separation, using specific antibodies to the target organism.
  • Additional target genes for the target organism may be included to increase the confidence in the confirmation, or better define the target organism.
  • each target gene two PCR reactions will be designed, each corresponding to different primer sequences. This will allow for redundancy and also protect against point mutation in primer sequences, and failure of one reaction.
  • a method of confirming the presence of contamination in a sample includes contacting a first portion of the sample with a magnetic bead coated with an antibody that specifically recognizes a microorganism and providing a second portion of the sample that has not been contacted with the magnetic bead coated with an antibody that has contacted the first portion of the sample and assaying the first portion of the sample for the presence of a contaminant microorganism by 4-band multiplex PCR assay using PCR primers complementary to four nucleic acid targets indicative of microbial contaminant, while separately assaying the first portion of the sample for the presence of a contaminant microorganism by 5 -band multiplex PCR assay using PCR primers complementary to four nucleic acid targets indicative of microbial contaminant and one nucleic acid target specific to a suspected microbe, while separately assaying the second portion of the sample for the presence of a contaminant microorganism by 4-band multiplex PCR assay using PCR primers
  • the sample may be contacted with a growth media to enrich the sample in a contaminant present in the sample.
  • This aspect of the invention may be conducted on a sample that has been analyzed for the presence of a contaminant microorganism by 4-band multiplex PCR assay using PCR primers complementary to four gene targets associated with the microbial contaminant, prior to contacting a first portion of the sample with a magnetic bead.
  • kits for performing an analytical test for detecting the presence of and distinguishing between E.Coli 0157 and non-0157 shinga toxin producing E. CoIi in a biological sample suspected of containing one or both.
  • the kit includes an analytical test device having a plurality of wells, wherein the wells do not communicate with each other (wherein each well includes a filter stack, the filter stack including (i) a porous membrane having an upper surface and a lower surface; and (ii) an absorbent material, wherein the lower surface of the porous membrane and the absorbent material are in physical contact and in fluid communication, and wherein upon contact of a biological fluid sample suspected of containing one or both of E.Coli 0157 and non-0157 shinga toxin producing E.
  • said fluid is able to flow through said membrane into said absorbent material, such that at least a portion of said E.Coli 0157 and non-0157 shinga toxin producing E. CoIi present in said biological fluid sample bind to said porous membrane); a first detection reagent capable of recognizing E.Coli 0157; a second detection reagent capable of recognizing non-0157 shinga toxin producing E. CoIi; and a reagent capable of generating a signal upon detection of one or both of E.Coli 0157 and non-0157 shinga toxin producing E. CoIi.
  • Another aspect of the invention provides a method for obtaining confirmation of the presence of a microbial analyte in less than three hours, by adding growth media to a sample of bacteria cells believed to contain a target analyte organism and incubating the sample at an appropriate temperature for an appropriate period of time, so that the target organism is given an opportunity to reproduce and to reach a concentration level sufficient to detect a sought after analyte; lysing bacterial cells to release the DNA contained therein, adding DNA primer templates composed of specific nucleotide sequences that complement known portions of genes which are unique to the target analyte organism; adding DNA polymerase and nucleotides to achieve in vitro enzymatic replication of desired DNA pieces; amplifying said DNA pieces by several orders of magnitude to generate at least a million copies of the DNA piece; separating said DNA pieces by using agarose gel chromatography, and adding a dye to the DNA product to determine visually the presence of said DNA.
  • Another aspect of the invention provides a method of detecting at least one substance of interest in a biological sample by providing a biological liquid sample suspected of comprising the substance(s) of interest; contacting the biological sample in a sufficient amount to with at least one specific binding pair member that is capable of binding, either directly or indirectly, the substance(s) of interest; and maintaining such contact for between about one and about 3 hours; and detecting the presence or absence of a complex comprising the specific binding pair member(s) and the substance(s) of interest, wherein the presence of at least one complex indicates the presence of at least one of the substances of interest in the biological sample.
  • Patent publication No. 20080096236 to Koulchin Patent publication No. 20080096189 to Boone; 20080038769 to Bernardi; 20080026953 to Gala, as well as US Patent Nos. 7,255,992 to Ecker, et al;
  • the present invention is drawn to methods of screeing and monitoring for microbial growth and contaminants including the use of secondary analysis methods needed for rapid screening and verification of primary or preliminary testing results.
  • an initial or preliminary testing result may be obtained by sampling and/or testing a subject or good by any one of several testing methodologies as described in U.S. Patent Publication No. 200/0115824, such as "presence/absence" tests or a plurality of the same.
  • Processes and systems to which the testing and verification methods of the instant application may be applicable include, but are not limited to: food production; manufacturing; processing; storage; transportation and distribution; with respect to microbial pathogens—process sanitation, environmental contaminants, and spoilage organisms; with respect to fermentation processes—determining purity of the seed stock and fermentation contaminants; aseptic processing (e.g., food and pharmaceutical; with respect to sterility and environmental control); water treatment (e.g., with respect to microbiological quality of the raw and treated water, and control of the organisms throughout the distribution system); wastewater treatment (e.g., with respect to microbiological quality of the treated wastewater and biosolids, control of the treatment process, control of the aerobic and anaerobic digestors, and assessment of the impact of the discharged wastewater and application of bio-solids on the receiving environments); control of microbial contaminants and assessment of their impact in the indoor environment and indoor air quality assessment studies; environmental microbiology (e.g., with respect to monitoring the microbiological quality of shellfish, shellfish beds and cultured aquatic organisms
  • test methods that may include, but are not limited to, multiplex PCR reaction(s), DNA chips, dot blots, multi- and single-target lateral flow devices, and combinations thereof.
  • assays suitable for detection of pathogenic or microbial contamination may include the use of immunoassays, nucleic acid amplification-based assays, PCR-based assays, nucleic acid hybridization- based assays, bio-sensor assays, immunostaining-microscopy-based assays, nucleic acid- array-based assays, DNA chip-based assays, bacteriophage-detection-based assays, classical microbiology-based assays, and chemical or biochemical assays based on the detection of compounds associated with particular target organisms or groups of target organisms, and combinations thereof.
  • a 4-band multiplex PCR assay is used as an initial, rapid screening method.
  • the four PCR assay targets are gene segments known to be associated with E. coli 0157. These assay targets are rfb, eae, stxl and stx2. After enrichment of a sample to allow the growth of the target organisms to the detection level, the enrichment is screened with the 4-band multiplex PCR assay.
  • PCR assay contains targets for stxl, stx2, rfb, eae (one universal one gamma).
  • targets for stxl, stx2, rfb, eae one universal one gamma.
  • One of the multiplex PCR assays also contains an additional gene unique to 0157. Molecular Confirmation is done using the two multiplexes, with and without immunomagnetic separation using magnetic beads coated with anti-0157 antibodies.
  • the key to separation of 0157 from non-0157 STEC and determination of whether there is a true 0157 versus a composite signal is determined by a comparison of the PCR assays with and without the beads and the fact that 0157 carries gamma eae. If a four band multiplex assay with the magnetic beads shows rfb, eae, and stx, this indicates that a true pathogenic 0157 E. coli in the enrichment, however if it only shows rfb, while the non-magnetic separated enrichment shows the rfb, eae, and stx, then we know that there is a non-toxigenic, eae-negative 0157 in the enrichment.
  • the same principle is used for Listeria monocytogenes vs. Listeria species in the same enrichment and for toxigenic Bacillus cereus versus other Bacilli in the same enrichment, and for Salmonella/multi-drug resistant Salmonella versus a multi-drug resistant bacteria in the same enrichment.
  • the same assay principles are used for other pathogenic, spoilage, indicators, biodegridative organisms, and pharmaceutical platform/producer strains of organisms.
  • Immunomagnetic Separation is used to purify and concentrate the sample prior to conducting a confirmation 3 -band multiplex PCR assay; however, the immunomagnetic beads in this case are coated with antibodies which bind specifically to Salmonella spp.
  • the pathogenic microbes Listeria spp. and Listeria monocytogenes are targeted using a 4-band multiplex PCR assay as a rapid screening method.
  • Two of the PCR assay targets are gene segments known to be associated with LM, while the remaining two are known to be associated with Listeria spp. If a presumptive positive result is obtained using the rapid screening method for LM, a second 2-band multipex PCR assay is used to corroborate the presumptive positive result and provide confirmation. If a presumptive positive result is obtained for Listeria spp. another independent 2-band multipex PCR assay is used to corroborate the presumptive positive result and provide confirmation
  • a seventh specific application again targets Listeria spp. and Listeria monocytogenes.
  • IMS is used to purify and concentrate the sample prior to applying one or both of the confirmation 2-band multiplex PCR assays; however, the immunomagnetic beads in this case are coated with antibodies which bind specifically to Listeria spp., of which LM is one type.
  • Another aspect of the present invention is directed to a rapid confirmation method for microorganisms, in mixed cultures or enrichment cultures.
  • the method allows for rapid confirmation of the presence of single or multiple target organisms in the same mixture.
  • the method has built in redundancy that increases the confidence in the results. It can also confirms and characterizes the organisms in a single step.
  • microorganisms may include a microbe or pathogen such as Escherichia coli O157:H7 (E. coli O157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichia coli (EIEC), enterpathogenic Escherichia coli (EPEC), Salmonella, Listeria, Yersinis, Campylobacter, Clostridial species, Staphylococcus spp.; frank and opportunistic bacterial, fungal, viral, parsitic pathogens; indicator organisms including heterotrophes, generic E.
  • a microbe or pathogen such as Escherichia coli O157:H7 (E. coli O157:H7), enterohemorrhagic Escherichia coli (EHEC), enterotoxigenic Escherichia coli (ETEC), enteroinvasive Escherichi
  • coli total and fecal coliforms and enterococcus
  • spoilage organisms including Pseudomonas
  • indicator molecules including glial fibillary acid protein (GFAP), transmissable spongiform encephalopathy (TSE) agents (prions), including bovine spongiform encephalopathy (BSE) agents, scrapie, chronic wasting disease; and combinations thereof.
  • Additional microbe sor pathogens are selected from the group consisting of Staph, aureus, Bacillus cereus, and Clostridium botulinum, Clostridium perfringes, Vibrio cholerae and V.
  • Organisms that are particularly suited for testing in one or more aspects of the present invention include the following: Bacillus anthracis Campylobacter coli Campylobacter jujuni Campylobacter lari Coliforms E. coli 0157
  • E. coli E. coli, Stx-producing (STEC) E. coli, Stx-producing with intimin E. coli, verotoxin producing

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des procédés et des nécessaires permettant une confirmation rapide du résultat d'un dosage initial concernant un analyte. Dans un mode de réalisation préféré, ce procédé permet d'obtenir confirmation de la présence d'une cible microbienne donnée dans une culture mixte ou dans un milieu d'enrichissement mixte, même lorsque les organismes concurrents présents dans le mélange appartiennent à des espèces apparentées ou correspondent à divers biotypes de la même espèce.
PCT/US2009/041839 2008-04-25 2009-04-27 Procédé de confirmation de la présence d'un analyte Ceased WO2009132354A2 (fr)

Applications Claiming Priority (2)

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US4799908P 2008-04-25 2008-04-25
US61/047,999 2008-04-25

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WO2009132354A2 true WO2009132354A2 (fr) 2009-10-29
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111133113A (zh) * 2017-09-21 2020-05-08 贝克顿·迪金森公司 有害污染物测试中的高动态范围测定物
USD1066729S1 (en) 2017-09-21 2025-03-11 Becton, Dickinson And Company Collection device
US12399088B2 (en) 2017-09-21 2025-08-26 Becton, Dickinson And Company Demarcation template for hazardous contaminant testing

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6620586B2 (en) * 2001-02-20 2003-09-16 Applied Gene Technologies, Inc. Methods and compositions for analyzing nucleic acids
CA2470774A1 (fr) * 2001-12-19 2003-06-26 Hvidovre Hospital Procede et kit de determination d'une numeration microbienne
DK1745139T3 (da) * 2004-04-15 2012-09-17 Inst Environmental Health Inc Trendanalyse og statistisk processtyring ved brug af multimålsat-screeningsassays
US7501253B2 (en) * 2004-09-21 2009-03-10 The Board Of Trustees Of The Leland Stanford Junior University DNA fingerprinting using a branch migration assay
US20060205040A1 (en) * 2005-03-03 2006-09-14 Rangarajan Sampath Compositions for use in identification of adventitious viruses
US20080003564A1 (en) * 2006-02-14 2008-01-03 Iquum, Inc. Sample processing

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111133113A (zh) * 2017-09-21 2020-05-08 贝克顿·迪金森公司 有害污染物测试中的高动态范围测定物
CN111133113B (zh) * 2017-09-21 2024-03-29 贝克顿·迪金森公司 有害污染物测试中的高动态范围测定物
USD1066729S1 (en) 2017-09-21 2025-03-11 Becton, Dickinson And Company Collection device
US12399088B2 (en) 2017-09-21 2025-08-26 Becton, Dickinson And Company Demarcation template for hazardous contaminant testing
US12436158B2 (en) 2017-09-21 2025-10-07 Becton, Dickinson And Company High dynamic range assays in hazardous contaminant testing

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Publication number Publication date
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