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WO2018017880A1 - Kits and methods for detecting and treating gastrointestinal disorders and infections - Google Patents

Kits and methods for detecting and treating gastrointestinal disorders and infections Download PDF

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Publication number
WO2018017880A1
WO2018017880A1 PCT/US2017/043155 US2017043155W WO2018017880A1 WO 2018017880 A1 WO2018017880 A1 WO 2018017880A1 US 2017043155 W US2017043155 W US 2017043155W WO 2018017880 A1 WO2018017880 A1 WO 2018017880A1
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optionally
sample
dna
nucleic acid
interest
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PCT/US2017/043155
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French (fr)
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Nimita FIFADARA
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Management Revenue Group P.R., Llc
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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
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • 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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • 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
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6893—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for protozoa

Definitions

  • the invention generally relates to molecular biology, diagnostics and infectious diseases. More specifically, provided are products of manufacture, including kits, and methods for the detection and treatment of infectious diseases and conditions related to the presence of particular microbes, including infections, conditions and disorders affecting the gastrointestinal tract (GI), and in alternative embodiments, provided are diagnostic tests for non-viral GI disorders.
  • the GI infections, diseases and conditions detected by products of manufacture, including kits, and methods as provided herein include Crohn's disease, Inflammatory Bowel Disease (IBD), ulcers, celiac disease, intestinal disorders, and other gastrointestinal related functional or metabolic disorders.
  • Gastrointestinal diseases are extremely common throughout the world. For example, there are an estimated two billion cases of diarrheal disease every year which kill approximately 1.8 million people annually. Diarrheal disease is the second leading cause of death (ahead of malaria, measles and AIDS combined) and the leading cause of malnutrition in children under five years old. The impact on the health care system by diarrheal disease is significant, as is its rate of morbidity and mortality in certain populations. It can be difficult to differentiate among viral, bacterial and parasitic agents of diarrheal disease due to similarity between observed symptoms, and a large majority of cases go undiagnosed. As a result, very treatable conditions are untreated or improperly treated. SUMMARY
  • compositions including kits, and methods and uses for the detection of an infectious disease, an infection, or a condition related to the presence of a microbe in an individual in need thereof to provide individualized identification of a microbial profile in the individual, and for providing a guidance for a treatment regimen for the individual,
  • condition related to the presence of a microbe is a condition or disorder affecting the gastrointestinal (GI) tract
  • condition or disorder affecting the gastrointestinal (GI) tract is Crohn's disease, Inflammatory Bowel Disease (IBD), a gastric ulcer or a duodenal ulcer, celiac disease, or an intestinal disorder,
  • the microbe is a bacteria, a parasite, a protozoan, or a fungi
  • the infection is an infection by a parasite, a protozoa, a bacteria or a fungi
  • the sample is, or comprises, or is derived from a stool or a fecal sample, a colonoscopy sample (a sample derived from a colonoscopy), a tissue biopsy, a blood, a sputum or a urine sample;
  • H. pylori H. pylori
  • H. pylori Helicobacter pylori infection, or the presence of a gastric ulcer or a duodenal ulcer;
  • E. coli Escherichia coli
  • ETEC Enterotoxigenic E. coli
  • LT heat labile toxin
  • ETEC Enterotoxigenic E. coli
  • nimD nitroimidazole resistance
  • van A vancomycin resistance
  • vanB vancomycin resistance
  • any or all of (1) through (5) indicates a bacterial infection that is resistant to an antibiotic or presence of a bacteria, parasite, fungus or microbe in the individual that is resistant to an antibiotic, thereby indicating or assisting a treatment regimen that does not comprise use of an antibiotic to which the bacteria, parasite, fungus or microbe is resistant,
  • methods as provided herein further comprise determining the presence of a polypeptide biomarker in the sample
  • polypeptide biomarker is associated with the infectious disease, or the infection or the condition related to the presence of a microbe in the individual,
  • polypeptide biomarker complements a diagnosis or the detection of the infectious disease or the infection, or the condition related to the presence of a microbe, or the presence of an inflammation related to an infectious disease or condition, in the individual,
  • polypeptide biomarker is detected by a protocol or process comprising use of an enzyme-linked immunosorbent assay (ELISA), optionally a multiplexed ELISA,
  • ELISA enzyme-linked immunosorbent assay
  • biomarker is or comprises:
  • hemoglobin a hemoglobin, a myoglobin, or a heme or heme-containing globular protein (a globin), a lactoferrin,
  • the analyzing of the sample for the presence of the nucleic acid, optionally the DNA comprises:
  • amplification primer pairs optionally polymerase chain reaction primer (PCR) pairs, capable of amplifying a nucleic acid subsequence of interest, optionally adding a plurality of PCR amplification primer pairs capable of amplifying a plurality of corresponding sets of nucleic acid subsequences of interest, wherein optionally the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid
  • PCR polymerase chain reaction primer
  • subsequences of interest are DNA as set forth in claim 1, and amplifying the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid subsequences of interest, optionally by PCR or by multiplexed PCR;
  • step (c) immobilizing the amplified DNA of step (b);
  • sequence specific probe is biotinylated
  • detector material comprises a streptavidin
  • streptavidin is conjugated to a detectable moiety
  • detectable moiety comprises a fluorescent material
  • fluorescent material comprises a R-phycoerthrin (SAPE)
  • step (b) wherein optionally the amplified DNA of step (b) is immobilized by binding the amplified the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid subsequences of interest with or to a segregatable substrate,
  • the segregatable substrate comprises a magnetic nanoparticle
  • the amplified the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid subsequences of interest are bound to a nanoparticle specific nucleotide probe (NSNP)
  • NSNP nanoparticle specific nucleotide probe
  • condition related to the presence of a microbe is a condition or disorder affecting the gastrointestinal (GI) tract
  • condition or disorder affecting the gastrointestinal (GI) tract is Crohn's disease, Inflammatory Bowel Disease (IBD), a gastric ulcer or a duodenal ulcer, celiac disease, or an intestinal disorder, and optionally the microbe is a bacteria, a protozoa, a parasite, or a fungi,
  • the infection is an infection by a parasite, a protozoa, a bacteria or a fungi,
  • kits e.g., clinical diagnostic kits, and methods that will be effective to identify and quantify the presence of non-viral contributors to a wide range of gastrointestinal disorders, relatively quickly and with a limited number of separate test procedures. It is a further object of the present invention to determine resistance to various antibiotics frequently used in the treatment of gastrointestinal disorders.
  • products of manufacture including clinical diagnostic kits, as provided herein comprise components to practice a method as provided herein, including instructions for practicing a method as provided herein.
  • FIG. 1 is a schematic overview of an exemplary process as provided herein comprising: amplification and hybridization of a target gene using sequence specific primers and microsphere-conjugated probes, e.g., using nanoparticle specific nucleotide probes (NS Ps), for analysis by a multiplex reader or any sorter and bound substrate reader, as explained in further detail, below.
  • sequence specific primers and microsphere-conjugated probes e.g., using nanoparticle specific nucleotide probes (NS Ps)
  • NSPs nanoparticle specific nucleotide probes
  • compositions including kits, and methods and uses for the detection of an infectious disease, an infection, or a condition related to the presence of a microbe in an individual in need thereof to provide individualized identification of a microbial profile in the individual, and for providing a guidance for a treatment regimen for the individual, wherein optionally the condition related to the presence of a microbe is a condition or disorder affecting the gastrointestinal (GI) tract, and optionally the condition or disorder affecting the gastrointestinal (GI) tract is Crohn's disease, Inflammatory Bowel Disease (IBD), a gastric ulcer or a duodenal ulcer, celiac disease, or an intestinal disorder, and optionally the microbe is a bacteria, a parasite, a protozoan, or a fungi, and optionally the infection is an infection by a parasite, a protozoa, a bacteria or a fungi.
  • IBD Inflammatory Bowel Disease
  • the microbe is a bacteria, a parasite, a protozoan, or
  • kits including clinical diagnostic kits, for diagnosing and treating gastrointestinal (GI) disorders and conditions
  • the kits comprise a plurality of assay panels, each assay panel being effective to identify the presence and alternatively the amount of a plurality of relevant analytes, wherein the analytes are relevant in that they can diagnose the presence of a particular microbe, e.g., pathogen or bacteria, in a sample, e.g., a sample from a patient.
  • a particular microbe e.g., pathogen or bacteria
  • a sample e.g., a sample from a patient.
  • Table I lists an exemplary panel of analytes grouped into respective assay panels. For reference purposes, each panel is given a reference letter. It will be appreciated that the panels can be performed in any particular order, and that a given implementation may include all, several or just one assay panel, and/or may include an attempt to detect or measure the amount of every analyte listed therein, but can be used to detect or measure a subset, e.g., five of six analytes, four of six analytes, or three of six analytes, or 19 of 20 analytes, and the like.
  • Campylobacter C. jejuni, C. coli, C. lari
  • Escherichia coli E. coli
  • ETEC Enterotoxigenic E. coli
  • LT heat labile
  • Panel C for parasite/fungal/protozoan (other pathogen) detection
  • Panel D - detecting genes indicative of resistance to types of antibiotics
  • Panel E biomarker antibody target proteins
  • assay panels A, B, C and D employ multiplex polymerase chain reaction (PCR) and nanoparticle specific nucleotide probe (NS P) hybridization, or equivalents, to allow for rapid and accurate detection and/or measurement of (to quantify) several genes associated with the respective analytes.
  • PCR polymerase chain reaction
  • N P nanoparticle specific nucleotide probe
  • assay panel E employs an enzyme-linked immunosorbent assay (ELISA), e.g., a multiplex ELISA, or equivalents, to identify biomarkers associated with the analytes, or the analytes themselves; for example, the ELISA or equivalents can identify or quantify the amount of proteins associated with the analyte or antibodies produced in response thereto, i.e., antibodies that can specifically bind to the analyte or biomarker.
  • ELISA enzyme-linked immunosorbent assay
  • sample material for analysis can be in the form of any sample taken from an individual, e.g., a liquid or a solid sample, or a tissue or a microbial sample, e.g., a sample can be obtained from a stool or fecal sample, a colonoscopy sample (a sample derived from a colonoscopy), a tissue biopsy, a blood, a sputum or a urine sample, and the like.
  • appropriate positive and negative controls are used in each assay panel.
  • total (e.g., crude) DNA extracted from a fecal or stool sample, or other suitable sample is mixed with forward and reverse amplification (e.g., PCR) primers, allowing isolation and amplification of regions of target genes of interest.
  • forward and reverse amplification e.g., PCR
  • the amplified regions of the target genes are bound to NS Ps attached to segregatable substrates, allowing unbound DNA to be removed.
  • a detector material that binds to conserved, amplified regions of the DNA is then added, allowing the presence of the amplified regions to be detected and discriminated by a reader equipment operable to detect the substrates and the detectors.
  • the reverse primers are biotinylated, allowing the detectors to bind to and be conserved with the biotin of the amplified reverse primers.
  • detector binding is also employed in the ELISA panel (E).
  • fluorescing detectors are used.
  • the detector material can be streptavidin conjugated to R-phycoerthrin (SAPE).
  • SAPE streptavidin conjugated to R-phycoerthrin
  • a correlation curve is used to correlate MFI (or any other detector measurement) to the concentration of the analyte present.
  • MFI or any other detector measurement
  • These curves can be developed prior to clinical use of the assay panels by testing multiple samples with known concentrations of analyte present and charting the MFI values achieved. These correlation curves can also help validate the efficacy of positive and negative controls.
  • total DNA is extracted from the fecal, stool or other suitable sample.
  • the total DNA is added to a reaction volume with forward and reverse PCR primers for each analyte and other PCR mix components.
  • 10 ⁇ _ of the DNA sample is added along with the following mixture:
  • the 2.5 ⁇ _, of forward and reverse PCR primers are taken from the following mixture: d. 3800 ⁇ , of Tris-EDTA (TE) buffer, pH 8.0; e. 375 ⁇ _, of ureA forward primer (100 ⁇ );
  • cagA forward primer 100 ⁇
  • Table II includes exemplary sequence information for the forward and reverse primers for each panel A analyte (other sequences capable of specifically amplifying a subsequence of the target DNA also can be used):
  • thermocycler e.g., using a thermocycler
  • NSNPs are attached to substrates, with a different substrate being used for each target analyte.
  • substrates include magnetic COOH microspheres (Luminex Corporation, Austin, TX).
  • 10 ⁇ . of the amplified DNA is combined with a 40 ⁇ _, mixture including:
  • TMAC tetramethyl ammonium chloride
  • NSNPs w/ substrates (probes bound to magnetic nanoparticles).
  • Exemplary conditions for pooling and hybridizing the probes and substrates to the amplified PCR products include the following, optionally also performed using a
  • thermocycler
  • the amplified regions from the target analytes are now bound to the segregatable substrates NSNP (e.g., magnetic nanoparticles).
  • NSNP e.g., magnetic nanoparticles
  • the sample is then placed on a magnet so remaining DNA not bound to the substrates can be removed, and the only conserved DNA will be the amplified DNA of interest.
  • Detector material is then added to the sample containing only the conserved, amplified regions of DNA bound to the substrates by the NSNPs. For example, a mixture of the following is used:
  • the sample is heated (on the thermocycler) at 42° C for another 5 minutes, to bind the SAPE to the amplified and conserved biotinylated reverse primers, and the sample is now ready for analysis in the reader - in an alternative embodiment, a MAGPixTM machine (Luminex Corporation).
  • the reader indicates MFI values associated with each type of microsphere. A positive indication for each analyte is indicated below in Table IV. Further quantification can be performed using a correlation curve, as described above:
  • total DNA is extracted from the fecal, stool or other suitable sample.
  • the total DNA is added to a reaction volume with forward and reverse PCR primers for each analyte and other PCR mix components.
  • 10 ⁇ _ of the DNA sample is added along with the following mixture:
  • BSA bovine serum albumin
  • the 7 ⁇ _, of forward and reverse PCR primers are taken from the following mixture of ⁇ stock primers: dd. 1340 ⁇ L ⁇ of TE buffer, pH 8.0;
  • Table V includes exemplary sequence information for the forward and reverse primers for each panel B analyte (other sequences capable of specifically amplifying a subsequence of the target DNA also can be used):
  • thermocycler e.g., using a thermocycler
  • Exemplary conditions for pooling and hybridizing the probes and substrates to the amplified PCR products include the following - as well as detector (SAPE) binding, optionally also performed using a thermocycler:
  • the amplified regions from the target analytes are now bound to the segregatable substrates (e.g., magnetic nanoparticles).
  • the sample is then placed on a magnet so remaining DNA not bound to the substrates can be removed, and the only conserved DNA will be the amplified DNA of interest.
  • Detector material is then added to the sample containing only the conserved, amplified regions of DNA bound to the substrates by the NSNPs. For example, a mixture of the following is used:
  • 10 ⁇ _, of the amplified DNA is combined with:
  • SAPE and reporter buffer mix is taken from a solution of 0.4 ⁇ _, SAPE (0.22 mg/mL) with 75 ⁇ _, IX reporter buffer.
  • Table VI lists exemplary NSNP sequences for each target analyte:
  • the sample is heated (e.g., on the thermocycler) at 42° C for another 5 minutes, to bind the SAPE to the amplified and conserved biotinylated reverse primers, and the sample is now ready for analysis in the reader -in this specific embodiment, a MAGPixTM (Luminex Corporation).
  • Panel C for parasite/fungal/parasite (other pathogen) detection
  • total DNA is extracted from the fecal, stool or other suitable sample.
  • the total DNA is added to a reaction volume with forward and reverse PCR primers for each analyte and other PCR mix components.
  • 10 ⁇ _, of the DNA sample is added along with the 12.5 ⁇ _, of Apex-SupermixTM.
  • Blastocystis forward primer (0.5 ⁇ );
  • Candida reverse primer (0.25 ⁇ );
  • Trichomonas reverse primer (0.5 ⁇ );
  • Table VII includes exemplary sequence information for the forward and reverse primers for each panel C analyte (other sequences capable of specifically amplifying a subsequence of the target DNA also can be used):
  • thermocycler e.g., using a thermocycler
  • the sample is subjected to NSNP hybridization, in order to bind the amplified regions of the target gene analytes to the NSNPs.
  • the NSNPs are attached to substrates, with a different substrate being used for each target analyte. Suitable substrates include magnetic COOH microspheres (Luminex Corporation, Austin, TX).
  • 10 ⁇ _, of the amplified DNA is combined with:
  • NSNPs w/ substrates (probes bound to magnetic microspheres).
  • Exemplary conditions for pooling and hybridizing the probes and substrates to the amplified PCR products include the following, optionally using a thermocycler:
  • the amplified regions from the target analytes are now bound to the segregatable substrates (e.g., magnetic microspheres).
  • the sample is then placed on a magnet so remaining DNA not bound to the substrates can be removed, and the only conserved DNA will be the amplified DNA of interest.
  • Detector material is then added to the sample containing only the conserved, amplified regions of DNA bound to the substrates by the 10 NSNPs. For example, a mixture of the following is used:
  • the sample is heated (e.g., on a thermocycler) at 52° C for another 5 minutes, to bind the SAPE to the amplified and conserved biotinylated reverse primers, and the sample is now 15 ready for analysis in the reader - optionally using a MAGPixTM machine (Luminex
  • Panel D looks specifically for genes indicative of resistance to certain types of antibiotics that would be used to treat various forms of gastrointestinal disease, and consequently only needs to be run where positive results on other panels would indicate treatment with antibiotics. Sequence specific primers are utilized that recognize highly
  • total DNA is extracted from the fecal, stool or other suitable sample.
  • the total DNA is added to a reaction volume with forward and reverse PCR primers for each analyte and other PCR mix components.
  • 10 ⁇ _ of the DNA sample is added along with the following:
  • the forward and reverse PCR primers are taken from the following mixture:
  • nimA forward primer (2.5 ⁇ );
  • nimA reverse primer 2.5 ⁇
  • vanB reverse primer (2.5 ⁇ ).
  • Table X includes exemplary sequence information for the forward and reverse primers for each panel D analyte (other sequences capable of specifically amplifying a subsequence of the target DNA also can be used):
  • vanB GTC GCA ATC ATC TTC GGC GG /5Biosg/TAT CGC AGC GTT TAG TTC
  • thermocycler e.g., using a thermocycler
  • the sample is subjected to NSNP hybridization, in order to bind the amplified regions of the target gene analytes to the NSNPs.
  • the NSNPs are attached to substrates, with a different substrate being used for each target analyte. Suitable substrates include magnetic COOH microspheres (Luminex Corporation, Austin, TX).
  • 10 ⁇ _, of the amplified DNA is combined with:
  • TMAC tetramethyl ammonium chloride
  • Table XI lists exemplary NSNP sequences for each target analyte:
  • Exemplary conditions for pooling and hybridizing the probes and substrates to the amplified PCR products include the following, preferably also performed using a
  • thermocylcer
  • the amplified regions from the target analytes are now bound to the segregatable substrates (e.g., magnetic nanoparticles).
  • the sample is then placed on a magnet so remaining DNA not bound to the substrates can be removed, and the only conserved DNA will be the amplified DNA of interest.
  • Detector material is then added to the sample containing only the conserved, amplified regions of DNA bound to the substrates by the NSNPs. For example, a mixture of the following is used:
  • the sample is heated (e.g., on a thermocycler) at 52° C for another 5 minutes, to bind the SAPE to the amplified and conserved biotinylated reverse primers, and the sample is now ready for analysis in the reader, optionally using a MAGPix machine (Luminex Corporation, Austin, TX).
  • the reader indicates MFI values associated with each type of microsphere.
  • a positive indication for each analyte is an MFI of > or + 350. Further quantification can be performed using a correlation curve, as described above.
  • Panel E capture antibodies specific to biomarker target proteins
  • Panel E lists exemplary biomarker polypeptides (proteins) that are targeted by (specifically bound by) antibodies, including polyclonal or monoclonal (mAb) antibodies.
  • assays to detect the biomarker polypeptides of panel E comprise use of an enzyme-linked immunosorbent assay (ELISA), e.g., a multiplex ELISA, or equivalents.
  • ELISA enzyme-linked immunosorbent assay
  • biomarkers are associated with the analytes, or the analytes themselves; for example, the ELISA or equivalents can identify or quantify the amount of proteins associated with the analyte or antibodies produced in response thereto, i.e., antibodies that can specifically bind to the analyte or biomarker.
  • monoclonal or polyclonal capture antibodies specific to biomarker target proteins from samples are detected in an ELISA, e.g., a multiplex ELISA.
  • the antibodies are biotinylated to bind to the detector material, and segregatable substrates (nanoparticle probes) are used that will capture the antibodies. Identification and quantification are then performed in a similar manner to what is described above for the other assay panels.
  • appropriate standards and controls are used to determine biomarkers for secretory IgAl, sIgA2,
  • Lactoferrin, Elastase, transglutaminase and FIT Fecal immunochemical test implied to leaky guts, infection, inflammation, exocrine pancreatic issue, gluten sensitivity and colon cancer respectively.
  • Example 1 Exemplary protocol for the detection of antibiotic resistance
  • This example describes exemplary procedures for the detection of antibiotic resistance from genomic DNA of a patient's stool, biopsy and/or colonoscopy sample; where in some embodiments, the patient has been diagnosed positive for bacterial pathogens. Data derived from this assay gives information to a physician that includes detected reservoirs of antibiotic resistant genes from the patient's gastrointestinal (GI) samples and allows them to make a timely decision for treatment with correct antimicrobial therapy.
  • GI gastrointestinal
  • Methods provided herein can be performed directly with a stool, a GI biopsy and colonoscopy specimens - obviating the need for isolation of the organism by culture. Methods provided herein can lessen the biohazard risk which may occur due to propagation of culture.
  • Stool samples are collected by the patient or via colonoscopy and transported in the appropriate transport container at room temperature (RT).
  • RT room temperature
  • SAPE (lmg/mL streptavidin-phycoerythrin)
  • Cut-off values for identifying positive gene resistance is set at 349 MFI (Median Fluorescence Intensity). Any MFI above this cut-off value is considered positive for expressing resistance to that gene. All the data is screened and MFI values are electronically entered, e.g., into an LFMS (Laboratory Information Management System), for reporting.
  • LFMS Laboratory Information Management System
  • Example 2 Exemplary protocol for the detection of gastrointestinal pathogens
  • This example describes exemplary procedures for the detection of gastrointestinal pathogens.
  • GFP Gastrointestinal Pathogen Panel
  • MCT microcentrifuge tube
  • Example 3 Exemplary protocol for the detection of detection of Candida species.
  • This example describes exemplary procedures for the detection of Candida species, Trichomonas species and Blastocystis hominis from genomic DNA of patient's stool, and colonoscopy samples.
  • MCT microcentrifuge tube
  • Cut-off values for identifying shift in Candida spps is set at 999 MFI (Median Fluorescence Intensity), Trichomonas spps is 500 MFI, and for ?, hominis is 349. Any MFI above this cut-off value is considered positive.
  • Example 4 Exemplary protocol for the detection of detection of Helicobacter pylori
  • This example describes exemplary procedures for the detection of Helicobacter pylori housekeeping genes (ureA and 23 S rRNA) as well as pathogenicity genes (cagA, iceA, vacA and oipA) and single nucleotide polymorphisms (S Ps) associated with clarithromycin resistance using, e.g., PCR, e.g., multiplex PCR, and 2% agarose gel electrophoresis.
  • ureA and 23 S rRNA pathogenicity genes
  • S Ps single nucleotide polymorphisms
  • cagA - cytotoxicity associated immunodominant antigen island protein alpha gene encodes for a protein associated with higher pathogenic strains of H. pylori that can often lead to PUD and/or gastric cancer.
  • Probe control beads • H. pylori multiplex (HPM) primer mix ( ⁇ stock of each of the following primers to create working solution of each forward and reverse primers as explained in detail on page 2.
  • HPM H. pylori multiplex
  • MCT microcentrifuge tube
  • nuclease-free water as the negative control for first tube in sequence (sample 1).
  • An ATCC genomic DNA of H. pylori for strain 26695 (Sample 2) and strain J99 (Sample3) is used as the positive control.
  • the second last sample is designated for Internal control and the last sample is set for probe control.
  • thermocycler Place the strip tubes in a thermocycler.

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Abstract

Provided are methods for the detection of an infectious disease, an infection, or a condition related to the presence of a microbe in an individual in need thereof to provide individualized identification of a microbial profile in the individual, and for providing a guidance for a treatment regimen for the individual. In alternative embodiments, the GI infections, diseases and conditions detected by products of manufacture, including kits, and methods as provided herein include Crohn's disease, Inflammatory Bowel Disease (IBD), ulcers, celiac disease, intestinal disorders, and other gastrointestinal related functional or metabolic disorders.

Description

KITS AND METHODS FOR DETECTING AND TREATING
GASTROINTESTINAL DISORDERS AND INFECTIONS
RELATED APPLICATIONS
This application claims the benefit of priority to U.S. Provisional Patent Application Serial No. (USSN) 62/364,461, filed July 20, 2016. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.
TECHNICAL FIELD
The invention generally relates to molecular biology, diagnostics and infectious diseases. More specifically, provided are products of manufacture, including kits, and methods for the detection and treatment of infectious diseases and conditions related to the presence of particular microbes, including infections, conditions and disorders affecting the gastrointestinal tract (GI), and in alternative embodiments, provided are diagnostic tests for non-viral GI disorders. In alternative embodiments, the GI infections, diseases and conditions detected by products of manufacture, including kits, and methods as provided herein include Crohn's disease, Inflammatory Bowel Disease (IBD), ulcers, celiac disease, intestinal disorders, and other gastrointestinal related functional or metabolic disorders. In alternative embodiments, provided are methods for the detection of an infectious disease, an infection, or a condition related to the presence of a microbe in an individual in need thereof to provide individualized identification of a microbial profile in the individual, and for providing a guidance for a treatment regimen for the individual..
BACKGROUND
Gastrointestinal diseases are extremely common throughout the world. For example, there are an estimated two billion cases of diarrheal disease every year which kill approximately 1.8 million people annually. Diarrheal disease is the second leading cause of death (ahead of malaria, measles and AIDS combined) and the leading cause of malnutrition in children under five years old. The impact on the health care system by diarrheal disease is significant, as is its rate of morbidity and mortality in certain populations. It can be difficult to differentiate among viral, bacterial and parasitic agents of diarrheal disease due to similarity between observed symptoms, and a large majority of cases go undiagnosed. As a result, very treatable conditions are untreated or improperly treated. SUMMARY
In alternative embodiment, provided are compositions, including kits, and methods and uses for the detection of an infectious disease, an infection, or a condition related to the presence of a microbe in an individual in need thereof to provide individualized identification of a microbial profile in the individual, and for providing a guidance for a treatment regimen for the individual,
wherein optionally the condition related to the presence of a microbe is a condition or disorder affecting the gastrointestinal (GI) tract,
and optionally the condition or disorder affecting the gastrointestinal (GI) tract is Crohn's disease, Inflammatory Bowel Disease (IBD), a gastric ulcer or a duodenal ulcer, celiac disease, or an intestinal disorder,
and optionally the microbe is a bacteria, a parasite, a protozoan, or a fungi, and optionally the infection is an infection by a parasite, a protozoa, a bacteria or a fungi,
the method comprising:
(a)
(A) providing a sample from the individual,
wherein optionally the sample is, or comprises, or is derived from a stool or a fecal sample, a colonoscopy sample (a sample derived from a colonoscopy), a tissue biopsy, a blood, a sputum or a urine sample;
(B) analyzing the sample for the presence of a nucleic acid, optionally a DNA, from, derived from or characteristic of:
(i)
(1) a Helicobacter pylori (H. pylori) urea,
(2) a H pylori virulence factor cagA,
(3) a pylori virulence factor vacA,
(4) a H pylori virulence factor iceA,
(5) a pylori Clarithromycin resistance/23 s, or
(6) a combination of any of (1) through (5), or all of (1) through (5),
wherein the presence of any of all of (1) through (5) detects the presence of a
Helicobacter pylori (H. pylori) infection, or the presence of a gastric ulcer or a duodenal ulcer;
(ϋ) (1) a Clostridium difficile (C difficile) toxin A,
(2) a C. difficile toxin B,
(3) a Campylobacter (optionally a C jejuni, C coli, C lari),
(4) an Escherichia coli (E. coli),
(5) an Enterotoxigenic E. coli (ETEC) heat labile toxin (LT),
(6) an Enterotoxigenic E. coli (ETEC) heat stable toxin (ST),
(7) a Salmonella spp.,
(8) a Shiga-like toxin producing E. coli (STEC) stxl toxin, or a Shigella stxl toxin,
(9) a Shiga-like toxin producing E. coli (STEC) stx2 toxin, or a Shigella stx2 toxin,
(10) a Shigella spp., optionally an S. dysenteriae,
(11) an Entamoeba histolytica,
(12) a Giardia spp.,
(13) a Listeria monocytogenes, or
(14) an Aspergillus spps.,
wherein the presence of any or all of (1) through (14) detects the presence of a microbial pathogen or a microbial infection;
(iii)
(1) a Blastocystis hominis,
(2) a Candida spp.,
(3) a Trichomonas spp., or
(4) a Cryptosporidium spps (optionally a C. parvum or a C. hominis),
wherein the presence of any of all of (1) through (4) detects the presence of a parasite or a fungal infection; and
(C) analyzing the sample for the presence of a gene indicative of or characteristic of a resistance to an antibiotic,
where optionally the gene indicative of or characteristic of a resistance to an antibiotic comprises:
(1) a β-lactamase resistance (bl2b-TEM) gene,
(2) a nitroimidazole resistance (nimD) gene,
(3) a fluoroquinoline resistance (qnrA) gene,
(4) a tetracycline resistance (tetA(P)) gene,
(5) an imidazole resistance (nimA) gene,
(6) a vancomycin resistance (van A) gene, (7) a vancomycin resistance (vanB) gene, or
(8) a combination of any of (1) through (5), or all of (1) through (5),
wherein the presence of any or all of (1) through (5) indicates a bacterial infection that is resistant to an antibiotic or presence of a bacteria, parasite, fungus or microbe in the individual that is resistant to an antibiotic, thereby indicating or assisting a treatment regimen that does not comprise use of an antibiotic to which the bacteria, parasite, fungus or microbe is resistant,
thereby providing an individualized identification of the microbial profile in the individual, and providing a guidance for a treatment and antibiotic regimen for the individual.
In alternative embodiment, methods as provided herein further comprise determining the presence of a polypeptide biomarker in the sample,
wherein the polypeptide biomarker is associated with the infectious disease, or the infection or the condition related to the presence of a microbe in the individual,
and the presence of the polypeptide biomarker complements a diagnosis or the detection of the infectious disease or the infection, or the condition related to the presence of a microbe, or the presence of an inflammation related to an infectious disease or condition, in the individual,
and optionally the polypeptide biomarker is detected by a protocol or process comprising use of an enzyme-linked immunosorbent assay (ELISA), optionally a multiplexed ELISA,
and optionally the biomarker is or comprises:
a secretory IgAl or sIgA2 immunoglobulin (antibody),
a fecal blood,
a hemoglobin, a myoglobin, or a heme or heme-containing globular protein (a globin), a lactoferrin,
an elastase, or
a transglutaminase.
In alternative embodiment, the analyzing of the sample for the presence of the nucleic acid, optionally the DNA, comprises:
(a) extracting or purifying DNA, or total DNA, found in the sample;
(b) adding amplification primer pairs, optionally polymerase chain reaction primer (PCR) pairs, capable of amplifying a nucleic acid subsequence of interest, optionally adding a plurality of PCR amplification primer pairs capable of amplifying a plurality of corresponding sets of nucleic acid subsequences of interest, wherein optionally the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid
subsequences of interest are DNA as set forth in claim 1, and amplifying the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid subsequences of interest, optionally by PCR or by multiplexed PCR;
(c) immobilizing the amplified DNA of step (b);
(d) washing away or removing all or substantially all of the non-amplified DNA;
(e) hybridizing to the immobilized DNA a sequence specific probe capable of specifically binding to or hybridizing to an amplified nucleic acid of interest; and
(f) detecting the presence of the amplified nucleic acid of interest by detecting the sequence specific probe specifically bound or hybridized to the amplified nucleic acid of interest, wherein optionally the detecting comprises use of a detector material bound to the sequence specific probe,
wherein optionally the sequence specific probe is biotinylated, and optionally the detector material comprises a streptavidin, and optionally the streptavidin is conjugated to a detectable moiety, and optionally the detectable moiety comprises a fluorescent material, and optionally the fluorescent material comprises a R-phycoerthrin (SAPE),
wherein optionally the amplified DNA of step (b) is immobilized by binding the amplified the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid subsequences of interest with or to a segregatable substrate,
wherein optionally the segregatable substrate comprises a magnetic nanoparticle, and optionally the amplified the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid subsequences of interest are bound to a nanoparticle specific nucleotide probe (NSNP), and the NSNP is bound to the segregatable substrate, and optionally the NSNP is bound to one of primer of each amplification primer pair.
In alternative embodiment, provided are methods for treating or ameliorating an infectious disease, an infection, or a condition related to the presence of a microbe, in an individual in need thereof,
wherein optionally the condition related to the presence of a microbe is a condition or disorder affecting the gastrointestinal (GI) tract,
and optionally the condition or disorder affecting the gastrointestinal (GI) tract is Crohn's disease, Inflammatory Bowel Disease (IBD), a gastric ulcer or a duodenal ulcer, celiac disease, or an intestinal disorder, and optionally the microbe is a bacteria, a protozoa, a parasite, or a fungi,
and optionally the infection is an infection by a parasite, a protozoa, a bacteria or a fungi,
the method comprising:
administering to the individual in need thereof an antibiotic or drug capable of treating or ameliorating the infectious disease, infection, or condition related to the presence of a microbe as detected by or diagnosed using a method of any of the preceding claims, and optionally administering to the individual in need thereof an antibiotic or a drug to which the microbe has not been found to be resistant using a method as provided herein.
In alternative embodiments, provided are products of manufacture, e.g., clinical diagnostic kits, and methods that will be effective to identify and quantify the presence of non-viral contributors to a wide range of gastrointestinal disorders, relatively quickly and with a limited number of separate test procedures. It is a further object of the present invention to determine resistance to various antibiotics frequently used in the treatment of gastrointestinal disorders.
In alternative embodiments, products of manufacture, including clinical diagnostic kits, as provided herein comprise components to practice a method as provided herein, including instructions for practicing a method as provided herein.
These and other objects, aspects and advantages of the present invention will be better appreciated in view of the drawings and following detailed description of preferred embodiments.
Details of one or more embodiments as provided herein are set forth in the accompanying drawings and in the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.
BRIEF DESCRIPTION OF THE DRAWINGS
The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
FIG. 1 is a schematic overview of an exemplary process as provided herein comprising: amplification and hybridization of a target gene using sequence specific primers and microsphere-conjugated probes, e.g., using nanoparticle specific nucleotide probes (NS Ps), for analysis by a multiplex reader or any sorter and bound substrate reader, as explained in further detail, below.
Like reference symbols in the various drawings indicate like elements.
Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.
DETAILED DESCRIPTION
In alternative embodiments, provided are compositions, including kits, and methods and uses for the detection of an infectious disease, an infection, or a condition related to the presence of a microbe in an individual in need thereof to provide individualized identification of a microbial profile in the individual, and for providing a guidance for a treatment regimen for the individual, wherein optionally the condition related to the presence of a microbe is a condition or disorder affecting the gastrointestinal (GI) tract, and optionally the condition or disorder affecting the gastrointestinal (GI) tract is Crohn's disease, Inflammatory Bowel Disease (IBD), a gastric ulcer or a duodenal ulcer, celiac disease, or an intestinal disorder, and optionally the microbe is a bacteria, a parasite, a protozoan, or a fungi, and optionally the infection is an infection by a parasite, a protozoa, a bacteria or a fungi.
In alternative embodiments, provided are products of manufacture, e.g., kits, including clinical diagnostic kits, for diagnosing and treating gastrointestinal (GI) disorders and conditions, wherein the kits comprise a plurality of assay panels, each assay panel being effective to identify the presence and alternatively the amount of a plurality of relevant analytes, wherein the analytes are relevant in that they can diagnose the presence of a particular microbe, e.g., pathogen or bacteria, in a sample, e.g., a sample from a patient. For example, in alternative embodiments, using only five (5) assay panels, up to forty (40) analytes can be detected. In alternative embodiments, products of manufacture, e.g., kits and methods for using them, as provided herein allow the quantification of some or all of the detected analytes without additional testing.
Table I, below, lists an exemplary panel of analytes grouped into respective assay panels. For reference purposes, each panel is given a reference letter. It will be appreciated that the panels can be performed in any particular order, and that a given implementation may include all, several or just one assay panel, and/or may include an attempt to detect or measure the amount of every analyte listed therein, but can be used to detect or measure a subset, e.g., five of six analytes, four of six analytes, or three of six analytes, or 19 of 20 analytes, and the like.
Table I: Analytes and Assay Panels
Panel A - for Helicobacter pylori (H. pylori) detection
1. Helicobacter pylori (H. pylori) ureA
2. H. pylori virulence factor cagA
3. H. pylori virulence factor vacA
4. H. pylori virulence factor iceA
5. H. pylori virulence factor oipA
6. H. pylori Clarithromycin resistance/23 s
Panel B - for microbial pathogen detection
7. Clostridium difficile (C. difficile) toxin A
8. C. difficile toxin B
9. Campylobacter (C. jejuni, C. coli, C. lari)
10. Escherichia coli (E. coli)
11. Enterotoxigenic E. coli (ETEC) heat labile (LT)
12. ETEC heat stable (ST)
13. Salmonella spp.
14. <S¾/'go-like toxin producing E. coli (STEC) stxl
15. STEC stx2
16. Shigella spp.
17. Entamoeba histolytica
18. Giardia spp.
19. Listeria monocytogenes
20. Aspergillus spps.
Panel C - for parasite/fungal/protozoan (other pathogen) detection
21. Blastocystis hominis
22. Candida spp.
23. Trichomonas spp.
24. Cryptosporidium spps (C. parvum and C. hominis)
Panel D - detecting genes indicative of resistance to types of antibiotics
25. β-lactamase resistance (bl2b-TEM) gene 26. Nitroimidazole resistance (nimD) gene
27. Fluoroquinoline resistance (qnrA) gene
28. Tetracycline resistance (tetA(P)) gene
29. Imidazole resistance (nimA) gene
30. Vancomycin resistance (vanA) gene
31. Vancomycin resistance (vanB) gene
Panel E - biomarker antibody target proteins
32. Anti-Gliadin IgA
33. Calprotectin
34. Elastase
35. Fecal Fat
36. Fecal occult blood (FOBT)
37. Lactoferrin
38. Secretory (slgAl)
39. Secretory (slgA2)
40. H. pylori stool antigen
In alternative embodiments, assay panels A, B, C and D employ multiplex polymerase chain reaction (PCR) and nanoparticle specific nucleotide probe (NS P) hybridization, or equivalents, to allow for rapid and accurate detection and/or measurement of (to quantify) several genes associated with the respective analytes.
In alternative embodiments, assay panel E employs an enzyme-linked immunosorbent assay (ELISA), e.g., a multiplex ELISA, or equivalents, to identify biomarkers associated with the analytes, or the analytes themselves; for example, the ELISA or equivalents can identify or quantify the amount of proteins associated with the analyte or antibodies produced in response thereto, i.e., antibodies that can specifically bind to the analyte or biomarker.
In alternative embodiments, for all panels, sample material for analysis can be in the form of any sample taken from an individual, e.g., a liquid or a solid sample, or a tissue or a microbial sample, e.g., a sample can be obtained from a stool or fecal sample, a colonoscopy sample (a sample derived from a colonoscopy), a tissue biopsy, a blood, a sputum or a urine sample, and the like. In alternative embodiments, appropriate positive and negative controls are used in each assay panel.
Referring to Figure 1 and the exemplary assay illustrated therein, for each of the multiplex PCR and nanoparticle specific nucleotide probe (NSNP) hybridization assay panels, including panels A, B, C and/or D, as described above, total (e.g., crude) DNA extracted from a fecal or stool sample, or other suitable sample, is mixed with forward and reverse amplification (e.g., PCR) primers, allowing isolation and amplification of regions of target genes of interest. The amplified regions of the target genes are bound to NS Ps attached to segregatable substrates, allowing unbound DNA to be removed.
A detector material that binds to conserved, amplified regions of the DNA is then added, allowing the presence of the amplified regions to be detected and discriminated by a reader equipment operable to detect the substrates and the detectors. In alternative embodiments, the reverse primers are biotinylated, allowing the detectors to bind to and be conserved with the biotin of the amplified reverse primers. In alternative embodiments, detector binding is also employed in the ELISA panel (E).
Quantification of the results will depend on the type of detector and corresponding reader used. In one embodiment, fluorescing detectors are used. For example, the detector material can be streptavidin conjugated to R-phycoerthrin (SAPE). When each assay panel is read, there will be a mean fluorescence intensity (MFI) value for each analyte detected.
In alternative embodiments, for each analyte, a correlation curve is used to correlate MFI (or any other detector measurement) to the concentration of the analyte present. These curves can be developed prior to clinical use of the assay panels by testing multiple samples with known concentrations of analyte present and charting the MFI values achieved. These correlation curves can also help validate the efficacy of positive and negative controls.
Panel A - for Helicobacter pylori (H. pylori) detection
Exemplary protocols and sequences
In alternative embodiments, as with the other PCR samples, total DNA is extracted from the fecal, stool or other suitable sample. The total DNA is added to a reaction volume with forward and reverse PCR primers for each analyte and other PCR mix components. In an exemplary embodiment 10 μΙ_, of the DNA sample is added along with the following mixture:
a. 0.50 μΙ_, of nuclease-free water;
b. 12.5 μΙ_, of Apex PCR Supermix™ (a suitable example being Taq 2. OX Hot Start Master Mix™ (Apex BioResearch; Genesee Scientific); and c. 2.5 μΙ_, of the forward and reverse PCR primers.
The 2.5 μΙ_, of forward and reverse PCR primers are taken from the following mixture: d. 3800 μΐ, of Tris-EDTA (TE) buffer, pH 8.0; e. 375 μΙ_, of ureA forward primer (100 μΜ);
f. 375 μL of ureA reverse primer (100 μΜ);
g. 375 μL of cagA forward primer (100 μΜ);
h. 375 μΙ_, of cagA reverse primer (100 μΜ);
i. 200 μΙ_, of vac A forward primer (100 μΜ);
j . 200 μΙ_, of vac A reverse primer (100 μΜ);
k. 375 μΙ_, of iceA forward primer (100 μΜ);
1. 375 μL of iceA reverse primer (100 μΜ);
m. 375 μΙ_, of oipA forward primer (100 μΜ);
n. 375 μΙ_, of oipA reverse primer (100 μΜ);
o. 200 μΙ_, of 23 s forward primer (100 μΜ); and
p. 200 μL of 23s reverse primer (100 μΜ).
Table II, below, includes exemplary sequence information for the forward and reverse primers for each panel A analyte (other sequences capable of specifically amplifying a subsequence of the target DNA also can be used):
Figure imgf000013_0001
The mixture and sample volume are then subjected to the following cycles (e.g., using a thermocycler):
q. an initial 95° C hold for 15 minutes; r. 40 cycles of a three step PCR:
i. denaturation at 95° C for 30 seconds;
ii. primer annealing at 55° C for 30 seconds; and
iii. primer extension 68° C for 1 minute; and
s. a final elongation step at 68° C for 10 minutes.
After the amplification (PCR) process, the sample is subjected to NSNP
hybridization, in order to bind the amplified regions of the target gene analytes to the NSNPs. The NSNPs are attached to substrates, with a different substrate being used for each target analyte. Exemplary suitable substrates include magnetic COOH microspheres (Luminex Corporation, Austin, TX). In alternative embodiments, 10 μΐ. of the amplified DNA is combined with a 40 μΙ_, mixture including:
t. 32 μΙ_, of 1.5M tetramethyl ammonium chloride (TMAC) hybridization buffer; u. 8 μΐ, of IX TE buffer, pH 8.0; and
v. NSNPs w/ substrates (probes bound to magnetic nanoparticles).
Table III, below, lists exemplary NSNP sequences for each target analyte:
Figure imgf000014_0001
Exemplary conditions for pooling and hybridizing the probes and substrates to the amplified PCR products include the following, optionally also performed using a
thermocycler:
w. denaturation of dsDNA at 60° C for 3 minutes; and
x. hybridization at 42° C for 40 minutes.
The amplified regions from the target analytes are now bound to the segregatable substrates NSNP (e.g., magnetic nanoparticles). The sample is then placed on a magnet so remaining DNA not bound to the substrates can be removed, and the only conserved DNA will be the amplified DNA of interest. Detector material is then added to the sample containing only the conserved, amplified regions of DNA bound to the substrates by the NSNPs. For example, a mixture of the following is used:
y. 1.0M TMAC reporter buffer; and
z. SAPE (lmg/mL).
The sample is heated (on the thermocycler) at 42° C for another 5 minutes, to bind the SAPE to the amplified and conserved biotinylated reverse primers, and the sample is now ready for analysis in the reader - in an alternative embodiment, a MAGPix™ machine (Luminex Corporation). The reader indicates MFI values associated with each type of microsphere. A positive indication for each analyte is indicated below in Table IV. Further quantification can be performed using a correlation curve, as described above:
Figure imgf000015_0001
Panel B - for microbial pathogen detection
Exemplary protocols and sequences
In alternative embodiments, as with the other PCR samples, total DNA is extracted from the fecal, stool or other suitable sample. The total DNA is added to a reaction volume with forward and reverse PCR primers for each analyte and other PCR mix components. In an exemplary embodiment 10 μΙ_, of the DNA sample is added along with the following mixture:
aa. 12.5 μΙ_, of Apex-Supermix™;
bb. 7 μΙ_, of the forward and reverse PCR primers; and
cc. 0.5 μΙ_, of 100X molecular grade bovine serum albumin (BSA).
The 7 μΙ_, of forward and reverse PCR primers are taken from the following mixture of ΙΟΟμΜ stock primers: dd. 1340 μL· of TE buffer, pH 8.0;
ee. 100 μΐ- of C. difficile A;
ff. 100 μΐ, of C. difficile B;
gg- 100 μΐ. of Campylobacter;
hh. 100 μΐ, of E. coli (0157);
ii. 80 μΐ, of E. coli LT;
jj - 100 μΐ, of E. coli ST;
kk. 100 μΐ. of Salmonella;
11. 100 μΐ, of stxl;
mm. 70 μΐ^ of stx2;
nn. 100 μΐ, of Shigella;
oo. 100 μΐ. of E. histolytica;
pp. 120 μΐ. of Giardia;
qq. 100 μΐ. of Listeria monocytogi
rr. 70 μΐ. of Aspergillus spps.
Table V, below, includes exemplary sequence information for the forward and reverse primers for each panel B analyte (other sequences capable of specifically amplifying a subsequence of the target DNA also can be used):
Figure imgf000016_0001
Sal ttrA CGG AAG AAT ACC GCG ACA A (SEQ /5Biosg/GGA GGT CAA CAG TTC GGT ID NO:31) AAG (SEQ ID NO:32)
PAS stxl CCT TCC GAT ATA CCT AAC GCT AAC /5Biosg/CCA TGT GCA ATA TGC AAC
(SEQ ID NO:33) TAC TG (SEQ ID NO:34)
stx2 GAA CGT TCC GGA ATG CAA ATC /5Biosg/GGA TGC ATC TCT GGT CAT
(SEQ ID NO:35) TGT A (SEQ ID NO: 36)
invH AGG TAT GGG AGA GAG CGT AAT C /5Biosg/GAA GGT GCT GCA ACA GAA
(SEQ ID NO:37) GA (SEQ ID NO:38)
HM-1 GTTGACATCGTTTATAGTTAGGACTAC /5Biosg/AGCGAAAGCATTTCACTCAAT
(SEQ ID NO:39) (SEQ ID NO:40)
NADP- CTT CCG TGT CCA GTA CAA CTC (SEQ /5Biosg/CGA AAC CGA GGA ACT TGA GDH ID NO:41) GAA (SEQ ID NO:42)
lmo2234 CCG CTG ATC GGA TTG TTT CT (SEQ /5Biosg/ACC GTA TCT CCG TAT CCT
ID NO:43) TCT C (SEQ ID NO:44)
Asp ATG GCC GTT CTT AGT TGG TG (SEQ /5Biosg/CTC GGC CAA GGT GAT GTA
18SrRNA ID NO:45) CT (SEQ ID NO:46)
The mixture and sample volume are then subjected to the following cycles (e.g., using a thermocycler):
ss. an initial 95° C hold for 15 minutes;
tt. 40 cycles of a three step PCR:
i. denaturation at 95° C for 30 seconds;
ii. primer annealing at 55° C for 30 seconds; and
iii. primer extension 68° C for 1 minute; and
uu. a final elongation step at 68° C for 10 minutes.
Exemplary conditions for pooling and hybridizing the probes and substrates to the amplified PCR products include the following - as well as detector (SAPE) binding, optionally also performed using a thermocycler:
vv. denaturation of dsDNA at 60° C for 3 minutes; and
ww. hybridization at 42° C for 40 minutes.
The amplified regions from the target analytes are now bound to the segregatable substrates (e.g., magnetic nanoparticles). The sample is then placed on a magnet so remaining DNA not bound to the substrates can be removed, and the only conserved DNA will be the amplified DNA of interest. Detector material is then added to the sample containing only the conserved, amplified regions of DNA bound to the substrates by the NSNPs. For example, a mixture of the following is used:
xx. 1.0M TMAC reporter buffer (75 ; and
yy. SAPE (lmg/mL) (Life Technologies, Carlsbad, CA).
According to a preferred embodiment, 10 μΙ_, of the amplified DNA is combined with:
zz. 6 μΙ_, NSNPs w/ substrates (probes bound to magnetic microspheres); and aaa. 75 μΙ_, SAPE and reporter buffer mix.
The SAPE and reporter buffer mix is taken from a solution of 0.4 μΙ_, SAPE (0.22 mg/mL) with 75 μΙ_, IX reporter buffer. Table VI, below, lists exemplary NSNP sequences for each target analyte:
Figure imgf000018_0001
The sample is heated (e.g., on the thermocycler) at 42° C for another 5 minutes, to bind the SAPE to the amplified and conserved biotinylated reverse primers, and the sample is now ready for analysis in the reader -in this specific embodiment, a MAGPix™ (Luminex Corporation). The reader indicates MFI values associated with each type of microsphere. A positive indication for each analyte is > or = 350. Further quantification can be performed using a correlation curve, as described above.
Panel C - for parasite/fungal/parasite (other pathogen) detection
Exemplary protocols and sequences
In alternative embodiments, as with the other PCR samples, total DNA is extracted from the fecal, stool or other suitable sample. The total DNA is added to a reaction volume with forward and reverse PCR primers for each analyte and other PCR mix components. In an exemplary embodiment, 10 μΙ_, of the DNA sample is added along with the 12.5 μΙ_, of Apex-Supermix™.
Forward and reverse PCR primers are added to obtain final concentration for each reaction mixture to be:
bbb. Blastocystis forward primer (0.5 μΜ);
ccc. Blastocystis reverse primer (0.5 μΜ);
ddd. Candida forward primer (0.25 μΜ);
eee. Candida reverse primer (0.25 μΜ);
fff Trichomonas forward primer (0.5 μΜ);
ggg. Trichomonas reverse primer (0.5 μΜ);
hhh. Cryptosporidium forward primer (0.5 μΜ); and
iii. Cryptosporidium reverse primer (0.5 μΜ).
Table VII, below, includes exemplary sequence information for the forward and reverse primers for each panel C analyte (other sequences capable of specifically amplifying a subsequence of the target DNA also can be used):
Figure imgf000019_0001
Trichol 8SrR CAT TGG TGC CTT TCG GTA /5Biosg/CTG CGC TGA GTC ATT NA CT (SEQ ID NO:65) CAT GT (SEQ ID NO:66)
Cryptosporidi AGATGGACTGTTGAAGAAG /5Biosg/GCCCATCTCAAATTTCGT um spps GAA (SEQ ID NO:67) TTGT (SEQ ID NO:68)
The mixture and sample volume are then subjected to the following cycles (e.g., using a thermocycler):
jjj . an initial 95° C hold for 15 minutes;
kkk. 40 cycles of a three step PCR:
i. denaturation at 95° C for 30 seconds;
ii. primer annealing at 55° C for 30 seconds; and
iii. primer extension 68° C for 1 minute; and
111. a final elongation step at 68° C for 10 minutes.
After the amplification (e.g., PCR) process, the sample is subjected to NSNP hybridization, in order to bind the amplified regions of the target gene analytes to the NSNPs. The NSNPs are attached to substrates, with a different substrate being used for each target analyte. Suitable substrates include magnetic COOH microspheres (Luminex Corporation, Austin, TX). In one embodiment, 10 μΙ_, of the amplified DNA is combined with:
mmm. 32 μΙ_, 1.5M TMAC hybridization buffer;
nnn. 6 μΐ, IX TE buffer, pH 8.0; and
ooo. 4 μΙ_, NSNPs w/ substrates (probes bound to magnetic microspheres).
Table VIII, below, lists exemplary NSNP sequences for each target analyte:
Figure imgf000020_0001
Exemplary conditions for pooling and hybridizing the probes and substrates to the amplified PCR products include the following, optionally using a thermocycler:
ppp. denaturation of dsDNA at 95° C for 5 minutes; and qqq. hybridization at 52° C for 15 minutes.
5 The amplified regions from the target analytes are now bound to the segregatable substrates (e.g., magnetic microspheres). The sample is then placed on a magnet so remaining DNA not bound to the substrates can be removed, and the only conserved DNA will be the amplified DNA of interest. Detector material is then added to the sample containing only the conserved, amplified regions of DNA bound to the substrates by the 10 NSNPs. For example, a mixture of the following is used:
rrr. 75 μL· 1.0M TMAC reporter buffer; and
sss.0.2 μΐ, SAPE (lmg/mL).
The sample is heated (e.g., on a thermocycler) at 52° C for another 5 minutes, to bind the SAPE to the amplified and conserved biotinylated reverse primers, and the sample is now 15 ready for analysis in the reader - optionally using a MAGPix™ machine (Luminex
Corporation, Austin, TX). The reader indicates MFI values associated with each type of microsphere. A positive indication for each analyte is indicated below in Table IX. Further quantification can be performed using a correlation curve, as described above:
Figure imgf000021_0001
20 Panel D - genes indicative of resistance to antibiotics
Panel D looks specifically for genes indicative of resistance to certain types of antibiotics that would be used to treat various forms of gastrointestinal disease, and consequently only needs to be run where positive results on other panels would indicate treatment with antibiotics. Sequence specific primers are utilized that recognize highly
25 conserved regions of several genes (vanA, vanB, nimA, nimD, bl2b-tem2, tetA(P), and qnrA) associated with antibiotic resistance (vancomycin, metronidazole, beta-lactam, tetracycline, and fluoroquinolone, respectively) in several of the most common pathogens and microbiota of the gastrointestinal tract. Thus, Panel D gives results that are primarily patient specific, as opposed to pathogen specific.
Exemplary protocols and sequences
As with the other PCR samples, total DNA is extracted from the fecal, stool or other suitable sample. The total DNA is added to a reaction volume with forward and reverse PCR primers for each analyte and other PCR mix components. In an exemplary embodiment 10 μΙ_, of the DNA sample is added along with the following:
ttt. 12.5 iL oi Apex-Supermix™;
uuu. 2.0 μΙ_, of the forward and reverse PCR primers. The forward and reverse PCR primers are taken from the following mixture:
vvv. bl2b-TEM forward primer (2.5 μΜ);
www. bl2b-TEM reverse primer (2.55 μΜ);
XXX. nimD forward primer (2.5 μΜ);
yyy- nimD reverse primer (2.5 μΜ);
zzz. qnrA forward primer (2.5 μΜ);
aaaa. qnrA reverse primer (2.5 μΜ);
bbbb. tetA(P) forward primer (2.5 μΜ);
cccc. tetA(P) reverse primer (2.5 μΜ);
dddd. nimA forward primer (2.5 μΜ);
eeee. nimA reverse primer (2.5 μΜ);
ffff vanA forward primer (2.5 μΜ);
gggg- vanA reverse primer (2.5 μΜ);
hhhh. vanB forward primer (2.5 μΜ); and
. vanB reverse primer (2.5 μΜ).
Table X, below, includes exemplary sequence information for the forward and reverse primers for each panel D analyte (other sequences capable of specifically amplifying a subsequence of the target DNA also can be used):
Figure imgf000022_0001
nimD CAA GAG TGT GAT TTG CAG /5Biosg/CTT TGC CGT AAC GCT GTC AGG AC (SEQ ID NO:75) AAT C (SEQ ID NO: 76) qnrA CAG GGA GTG CGA TCT CAA /5Biosg/CAA GGG TTC CAG CAG
GGG (SEQ ID NO: 77) TTG CTC C (SEQ ID NO:78)
tetA TTC TGT TAG TGG GGC ATT /5Biosg/CTA ACA ACA CCA CAA
TGA TTA C (SEQ ID NO: 79) GCG AAC TC (SEQ ID NO: 80) nimA CAC GCG CGT TGT TTC CAA TC /5Biosg/CAA GTT TCA AGA CGT
(SEQ ID NO: 81) GGG TAC G (SEQ ID NO: 82) vanA GGG GTT GCT CAG AGG AGC /5Biosg/CGC CGG CTT AAC AAA
(SEQ ID NO: 83) AAC AGG (SEQ ID NO: 84)
vanB GTC GCA ATC ATC TTC GGC GG /5Biosg/TAT CGC AGC GTT TAG TTC
(SEQ ID NO: 85) TTC CG (SEQ ID NO: 86)
The mixture and sample volume are then subjected to the following cycles (e.g., using a thermocycler):
jjjj . an initial 95° C hold for 15 minutes;
kkkk. 37 cycles of a three step PCR:
i. denaturation at 95° C for 30 seconds;
ii. primer annealing at 55° C for 30 seconds; and
iii. primer extension 68° C for 30 seconds; and
1111. a final elongation step at 68° C for 7 minutes.
After the amplification (e.g., PCR) process, the sample is subjected to NSNP hybridization, in order to bind the amplified regions of the target gene analytes to the NSNPs. The NSNPs are attached to substrates, with a different substrate being used for each target analyte. Suitable substrates include magnetic COOH microspheres (Luminex Corporation, Austin, TX). In one embodiment, 10 μΙ_, of the amplified DNA is combined with:
mmmm. 31 μΙ_, of 1.5M tetramethyl ammonium chloride (TMAC) hybridization buffer;
nnnn. 8 μΐ. of 1 X ΊΈ buffer, pH 8.0; and
oooo. 1 μΙ_, of NSNPs w/ substrates (probes bound to magnetic
microspheres).
Table XI, below, lists exemplary NSNP sequences for each target analyte:
I Table XI: Exemplary SSOP Sequences I Gene Target Probe (5' to 3 ')
B12b-tem2 / 5 AmMC 12/T AT TGC TGA TAA ATC TGG AGC CGG (SEQ ID NO:87) nimD / 5 AmMC 121 ATA CTG ATG CGC CAT TAC AGT AGC C (SEQ ID
NO:88)
qnrA /5AmMC12/TTC CTT GAT GGG CTC AGA TCT CAG C (SEQ ID
NO:89)
tetA /5AmMC12/GTG TTT TAT ATC GCT TTT CAA GTG ATT TCT ATT
CTC ATT CA (SEQ ID NO:90)
nimA /5AmMC12/GCG AAA GGC ATT GGA ACT TCT GCT TGA TAA
ATA TTC TCC AG (SEQ ID NO:91)
vanA / 5 AmMC 12/TT A ATA AAG ATG ATA GGC CGG TGG C (SEQ ID
NO: 92)
vanB /5AmMC12/TGG AC A AAT CAC TGG CCT AC A TTC T (SEQ ID
NO:93)
Exemplary conditions for pooling and hybridizing the probes and substrates to the amplified PCR products include the following, preferably also performed using a
thermocylcer:
pppp. denaturation of dsDNA at 72° C for 3 minutes; and
qqqq. hybridization at 52° C for 30 minutes.
The amplified regions from the target analytes are now bound to the segregatable substrates (e.g., magnetic nanoparticles). The sample is then placed on a magnet so remaining DNA not bound to the substrates can be removed, and the only conserved DNA will be the amplified DNA of interest. Detector material is then added to the sample containing only the conserved, amplified regions of DNA bound to the substrates by the NSNPs. For example, a mixture of the following is used:
rrrr. 1.0M TMAC reporter buffer; and
ssss. SAPE (lmg mL).
The sample is heated (e.g., on a thermocycler) at 52° C for another 5 minutes, to bind the SAPE to the amplified and conserved biotinylated reverse primers, and the sample is now ready for analysis in the reader, optionally using a MAGPix machine (Luminex Corporation, Austin, TX). The reader indicates MFI values associated with each type of microsphere. A positive indication for each analyte is an MFI of > or + 350. Further quantification can be performed using a correlation curve, as described above.
Panel E - capture antibodies specific to biomarker target proteins
Panel E lists exemplary biomarker polypeptides (proteins) that are targeted by (specifically bound by) antibodies, including polyclonal or monoclonal (mAb) antibodies. In alternative embodiments, assays to detect the biomarker polypeptides of panel E comprise use of an enzyme-linked immunosorbent assay (ELISA), e.g., a multiplex ELISA, or equivalents. These biomarkers are associated with the analytes, or the analytes themselves; for example, the ELISA or equivalents can identify or quantify the amount of proteins associated with the analyte or antibodies produced in response thereto, i.e., antibodies that can specifically bind to the analyte or biomarker.
Exemplary protocols and sequences
In Panel E, monoclonal or polyclonal capture antibodies specific to biomarker target proteins from samples, e.g., fecal or stool supernatants, are detected in an ELISA, e.g., a multiplex ELISA. In alternative embodiments, the antibodies are biotinylated to bind to the detector material, and segregatable substrates (nanoparticle probes) are used that will capture the antibodies. Identification and quantification are then performed in a similar manner to what is described above for the other assay panels. In alternative embodiments, appropriate standards and controls are used to determine biomarkers for secretory IgAl, sIgA2,
Lactoferrin, Elastase, transglutaminase and FIT (Fecal immunochemical test) implied to leaky guts, infection, inflammation, exocrine pancreatic issue, gluten sensitivity and colon cancer respectively.
Each of the above assays has been determined to be highly sensitive for each target gene or other biomarker, and affords highly accurate and reproducible results. Simultaneous testing for multiple causes of gastrointestinal disorders, as well multiple types of potentially relevant antibiotic resistance, is thereby made possible within a matter of hours, without the time consuming and expensive need for growing multiple cultures or running separate tests for each target gene or biomarker.
In general, the foregoing description is provided for exemplary and illustrative purposes; the present invention is not necessarily limited thereto. Rather, those skilled in the art will appreciate that additional modifications, as well as adaptations for particular circumstances, will fall within the scope of the invention as herein shown and described. The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.
EXAMPLES
Example 1 : Exemplary protocol for the detection of antibiotic resistance
This example describes exemplary procedures for the detection of antibiotic resistance from genomic DNA of a patient's stool, biopsy and/or colonoscopy sample; where in some embodiments, the patient has been diagnosed positive for bacterial pathogens. Data derived from this assay gives information to a physician that includes detected reservoirs of antibiotic resistant genes from the patient's gastrointestinal (GI) samples and allows them to make a timely decision for treatment with correct antimicrobial therapy. Compared to conventional antibiotic susceptibility methods, antibiotic gene identification as provided herein, including gene detection, e.g., based on PCR, can provide a more rapid and reliable assessment of antimicrobial resistance. Methods provided herein can be performed directly with a stool, a GI biopsy and colonoscopy specimens - obviating the need for isolation of the organism by culture. Methods provided herein can lessen the biohazard risk which may occur due to propagation of culture.
Stool samples are collected by the patient or via colonoscopy and transported in the appropriate transport container at room temperature (RT). Recommended samples size, or recommended minimum weight requirements, are:
· Colonoscopy; Colonoscopy transport tube minimum weight of tube with sample 20.69 grams (g),
• Patient collected: Stool transport tube (saline solution) minimum weight of tube with sample 23.29 g,
• Patient collected: Stool transport tube (Cary -Blair) minimum weight of tube with sample 25.96 g,
• Biopsy- Received within 7 days of collection date.
Exemplary Sample preparation
1. Thaw all DNA samples (control DNAs and patient test DNAs), ARM primer mix and the 2X Hot Start Taq
2. PCR Master Mix in the clean room dead air box.
3. After reagents thaw place on cold block. 4. Label 8-well strip tubes with the sample numbers and check DNA samples against the batch worksheet.
5. Vortex and aliquot ΙΟμΙ of the DNA sample into the appropriate tube of the 8-well strip tubes.
6. Aliquot 10 ml of positive control last to minimize possible contamination.
7. Prepare the master mix in a 1.5mL Microcentrifuge tube using the volumes for each reagent shown on the batch sheet.
a. 12.5μ/ reaction 2X Hot Start Taq PCR Master Mix
b. 2.0μ1/ reaction ARM primer mix (From stock ΙΟΟμΜ primer mix)
8. Vortex the master mix and aliquot 15μ1 into each tube of the 8-well strip tubes.
9. Close each strip tube with the strip caps.
10. Pulse centrifuge each strip of tubes.
Exemplary Ampli fication protocol
1. Place the strip tubes in a thermocycler.
2. Amplify the reactions with the "AR amplification" program on the thermocycler. a. 95°C for 15 minutes
b. 37 cycles of 95°C for 30 sec, 55°C for 30 sec, 68°C for 30 sec
c. 68°C for 7 minutes
d. 4°C hold
3. Return reagents and DNA Isolation to designated area.
4. Decontaminate area with 30% bleach.
Hybridization
• Following PCR, prepare a 96-well PCR.
• Using the ARM worksheet, create the first hybridization mix in a 15 mL conical tube and vortex thoroughly:
1.5M TMAC hybridization Buffer, 31 μL/ reaction
IX Tris-EDTA (TE) Buffer, pH 8.0, 8 μΙ7 reaction
1. Use a sharpie to mark the wells used on a 96 well plate. For example, if you have 23 wells in your assay, draw a line to mark off 23 wells on your plate.
2. Add 1 μΐ, of ARM beads to each well. It is important to vortex the beads frequently to ensure that the AR beads do not aggregate at the bottom of the tube and distribute evenly in the wells.
3. Add 39 μΐ^ of hyb mix to each well, vortexing the conical after every 8 wells 4. Add ΙΟμΙ. of amplified DNA to each well using the multichannel pipette, pipetting up and down to mix
5. Place a plate sealer over the plate and move to the Thermocycler
6. Hybridize the reactions with the "AR Hyb" program on the thermocycler.
a. 95°C for 5 minutes
b. 52°C for 15 minutes
7. Remove from the Thermocycler and place on the magnet for 5 minutes
8. Keep the plate covered as hybridization beads are light sensitive
9. Prepare the second hybridization buffer while the plate sits on the magnet
10. 1.0M TMAC Reporter Buffer
11. SAPE (lmg/mL streptavidin-phycoerythrin)
12. After 5 minutes, remove the plate cover and pick up the magnet containing the PCR plate and empty the plate contents into the biohazard box
13. Leave the plate on the magnet to prevent loss of AR beads.
14. Pat the plate onto a napkin to clean up the plate before adding 75μΙ^ of your second hybridization buffer, using the multichannel pipette.
15. Cover using Plate cover
16. Place back on Thermocycler
17. 52°C for 5 minutes
18. After 5 minutes, remove the plate seal and place on a Luminex™ or equivalent. Reading on the Luminex
1. On the computer with Luminex™ (Luminex Magpix™ Serial No.13283702), open up the Xponent icon,
a. Before running any assays, make sure the "daily fluidics prep" under the maintenance tab in the top right has been completed
2. Ej ect the Multiplex Reader tray
3. Make sure the solution wells are full
4. Well 1. Deionized HiO
5. Well 2. 70% EtOH
6. Well 3. NaOH
7. Well 4. Empty
8. Start Fluidics prep
9. Under the "Maintenance" tab click "Probe & Heater" a. Turn heater "ON" and set temperature to 52°C
10. Create a Batch Sheet
a. Click the "Batches" tab on the top right
11. "Create a new batch from existing protocol"
12. "Name Batch" ARM-ACC year (XXXX)month(XX)day(XX).
13. Click AR Protocol
14. Next
15. Select the number of wells you are testing and click the yellow "unknown" button in the bottom box
a. Minimize the Luminex window
b. Open excel batch sheet
c. Copy/Paste patient accessions on the excel batch sheet onto a notepad window and save in the folder "Patient Load List" as ARM-ACC
year(XXXX)month(XX)day(XX)
d. Re-open Luminex and upload Patient Load list using "Import List" on the right side of the screen
e. Double -check and when done click "Save"
16. Place the AR plate onto the Luminex™ and click "Run".
Interpretation:
Cut-off values for identifying positive gene resistance is set at 349 MFI (Median Fluorescence Intensity). Any MFI above this cut-off value is considered positive for expressing resistance to that gene. All the data is screened and MFI values are electronically entered, e.g., into an LFMS (Laboratory Information Management System), for reporting.
Example 2: Exemplary protocol for the detection of gastrointestinal pathogens
This example describes exemplary procedures for the detection of gastrointestinal pathogens.
Stool samples are collected by the patient or via colonoscopy and transported in the appropriate transport container at room temperature. Samples are recommended to meet the minimum weight requirements as listed:
Colonoscopy transport tube minimum weight of tube with sample 20.69 g.
• Patient collected: Stool transport tube (saline solution) minimum weight of tube with sample 23.29 g. • Patient collected: Stool transport tube (Cary -Blair) minimum weight of tube with sample
25.96 g.
• Biopsy transport received within 7 days from collection date.
MATERIALS:
· Isolated DNA samples
• Gastrointestinal Pathogen Panel (GPP) primer mix (16μΙ. of each of the following primers added to 250 iL IX TE buffer, pH 8.0)
• xTAG Salmonella
• xTAG Giardia
· xTAG ShigaToxin 1
• xTAG ShigaToxin 2
• xTAG Shigella
• xTAG E. Histolytica
• xTAG Campylobacter
· xTAG Escherichia Coli 0157
• xTAG Escherichia Coli LT
• xTAG Escherichia Coli ST
• xTAG C. Difficile A
• xTAG C. Difficile B
· Aspergillus spps
• Listeria monocytogenes
• 250μΙ. IX TE buffer, pH 8.0- Sigma cat. No. 93283
• 2. OX Hot Start Master Mix
• Nuclease-free water
· GPP Hybridization beads
• Streptavidin Phycoerythrin (SAPE) (0.22 mg/mL)
• xTAG Reporter Buffer
• xTAG 100X Bovine Serum Albumin (BSA)
• Micropipettor tips (10-200μ1, 2-20μ1, 1-10μ1)
· 1.5mL microcentrifuge tube (MCT), sterile
• TE buffer
• 1.5 X TMAC • 1.0 X TMAC
• 15 ml conical tube
• PCR plate
• Sealer
• 25 ml Reservoir
Control Preparation
1. Generate a new GI Pathogen Panel batch sheet
2. Input "Negative Control" as sample 1.
3. Input "Positive Control" as sample 2.
4. Input the accession number for each of the DNA samples to be tested beginning with well 3.
5. Input "Negative Control" after the last test DNA sample.
6. Name the batch with the following naming format: GPP-ACC- year(XXXX)month(XX)day(XX) .
7. Save the file using the batch name and print a copy of the completed batch sheet.
8. Create a sample list by selecting all of the sample IDs, copy all of the sample IDs on a new Excel Batch Sheet using the latest previous batch sheet as a template.
9. Go to sample log using batch review sheet that contains the positive patients' samples data and mark all accessions for GPP and HP testing with the positive and negative data in each column under each accession column.
Controls
1. Nuclease-free water is used as the negative control.
2. A combination of ATCC cell lines and known positive patient samples are used as the positive controls.
Sample Preparation
1. Thaw all DNA samples (control DNAs and test DNAs), GPP primer mix and the 2X Hot Start™ Taq PCR Master Mix in the clean room dead air box.
2. After reagents thaw place on cold block.
3. Label 8-well strip tubes with the sample numbers.
4. Vortex and aliquot ΙΟμΙ of the DNA sample into the appropriate tube of the 8-well strip tubes.
5. Aliquot 10 ml of positive control last to minimize possible contamination. 6. Prepare the master mix in a 1.5mL MCT using the volumes for each reagent shown on the batch sheet. :
• List of Master mix components:
• 0.58 μΐ/ reaction of nuclease-free water.
• 5 μΐ/ reaction Apex-Supermix™.
• 1.67 μΐ/reaction GPP primers.
• 0.25 μΐ/reaction lOOx BSA.
7. Vortex the master mix and aliquot 15μ1 into each tube of the 8-well strip tubes.
8. Close each strip tube with the strip caps.
9. Pulse centrifuge each strip of tubes.
10. Place the strip tubes in a thermocycler.
11. Amplify the reactions by selecting a GI PCR DNA thermocycler program.
• 95°C for 15 minutes.
• 40 cycles of 95°C for 30 sec, 55°C for 30 sec, 68°C for 1 min.
• 68°C for 10 minutes
• 4°C hold
12. Return reagents and DNA Isolation to designated area.
13. Decontaminate area with 30% bleach.
Luminex™ Hybridization
1. Following PCR, Prepare a 96-well PCR plate by writing GPP-ACC
year(XXXX)month(XX)day(XX).
2. Vortex the GI pathogen panel premixed bead aliquot for 15 seconds.
3. Mix bead premix by pipetting several times and aliquot 12 μΐ bead premix into each well.
4. Prepare SAPE working solution by combining 0.6 μΐ SAPE (0.22 mg/mL) with 74 μΐ xTAG lx Reporter buffer based on the volumes shown on the GPP batch sheet.
5. Add 74.6μΙ. of SAPE + Reporter Buffer mix to each well
6. Add 5μΙ_, of DNA to each well using the multichannel pipette, pipetting up and down to mix
7. Place a plate sealer over the plate and move to the Thermocycler
8. Hybridize the reactions with the "GI Hyb" program on the thermocycler.
a. 60°C for 3 minutes
b. 45°C for 45 minutes c. 45°C hold
9. Remove the plate seal and place on the Luminex
Reading on the Luminex™
1. On the computer, open up the Luminex icon
a. Before running any assays, make sure the "daily fluidics prep" under the maintenance tab in the top right is completed.
i . Ej ect Multiplex Reader Tray
ii. Make sure the solution wells are full
1. Well 1. Deionized H20
2. Well 2. 70% EtOH
3. Well 3. NaOH
4. Well 4. Empty
iii. Start Fluidics prep
2. Under the "Maintenance" tab click "Probe & Heater"
a. Turn heater "ON" and set temperature to 45°C
Reference Ranges
• Salmonella- 10-5000 MFI
• Giardia- 10-5000 MFI
• ShigaToxin 1 - 10-5000 MFI
• ShigaToxin 2- 10-5000 MFI
• Shigella- 10-5000 MFI
• E. histolytica- 10-5000 MFI
• Campylobacter- 10-5000 MFI
• Escherichia Coli 0157- 10-5000 MFI
• Escherichia Coli LT- 10-5000 MFI
• Escherichia Coli ST- 10-5000 MFI
• C. Difficile A- 10-5000 MFI
• C. Difficile B- 10-5000 MFI
• Aspergillus spps. 10-10000 MFI
• Listeria monocytogenes 1-10000 MFI
Interpretation/Critical Results
All analytes
• < 350 = Negative • > 350 = Positive
• Except Aspergillus and Listeria monocytogenes < 500 = Negative
> 500 = Positive
From the GPP bacterial analytes, positive accessions based upon pre-determined cutoff values are write down in Batch sheet for further antibiotic resistance profiling.
Example 3 : Exemplary protocol for the detection of detection of Candida species.
Trichomonas species and Blastocystis hominis
This example describes exemplary procedures for the detection of Candida species, Trichomonas species and Blastocystis hominis from genomic DNA of patient's stool, and colonoscopy samples.
Sample requirements
Stool samples are collected by the patient or via colonoscopy and transported in the appropriate transport container at room temperature. It is recommended that the samples meet the minimum weight requirements as listed:
• Colonoscopy; Colonoscopy transport tube minimum weight of tube with sample 20.69 g-
• Patient collected: Stool transport tube (saline solution) minimum weight of tube with sample 23.29 g.
• Patient collected: Stool transport tube (Cary-Blair) minimum weight of tube with sample 25.96 g.
Materials
• Isolated DNA samples stored @ -20° C
• Fungal primer mix as follows:
Can forward 2.5μΜ
Can reverse biotinylated 2.5μΜ
Blasto forward 5μΜ
Blasto reverse biotinylated 5μΜ
Tricho forward 5μΜ
Tricho reverse biotinylated 5μΜ
2. OX Hot Start Master Mix™ (Apex BioResearch) • Nuclease-free water
• Fungal Hybridization beads
• 1.5M TMAC (Tetram ethyl Ammonium Chloride) hybridization Buffer
• 1.0X Reporter Buffer
• IX Tris-EDTA (TE) Buffer, pH 8.0
• 0.5mL thin wall PCR 8-well strip tubes and caps
• 1.5mL microcentrifuge tube (MCT), sterile
• Negative control
• Positive controls (ATCC C. albicans, C. tropicalis, T. vaginalis and B. hominis) Amplification
1. Generate a new Fungal batch sheet by opening the "Fungal batch sheet template"
2. Input "Negative Control" as sample 1
3. Input "Positive Control" as sample 2
4. Input the accession number for each of the DNA samples to be tested in the subsequent samples.
5. Input "Negative Control" after the last test DNA sample.
6. Name the batch with the following naming format: Fungal-ACC
year(XXXX)month(XX)day(XX).
7. Save the file using the batch name and print a copy of the completed batch sheet.
8. Create a sample list by selecting all of the sample IDs, copy all of the
sample IDs on a new Excel Batch Sheet using the latest previous batch sheet as a template.
9. Thaw all DNA samples (control DNAs and test DNAs),
10. Thaw Fungal primer mix and the 2X Hot Start Taq PCR Master Mix in the clean room dead air box.
11. Label 8-well strip tubes with the sample numbers.
12. Vortex patient sample and then aliquot ΙΟμΙ of the DNA sample into the appropriate tube of the 8-well strip tubes.
13. Prepare the master mix in a 1.5mL Microcentrifuge tube using the volumes for each reagent shown on the batch sheet. 14. Vortex the master mix and aliquot 15μ1 into each tube of the 8-well strip tubes.
15. Close each strip tube with the strip caps.
16. Pulse centrifuge each strip of tubes.
17. Place the strip tubes in a thermocycler.
18. Amplify the reactions with the "Fungal amplification" program on the thermocycler.
a. 95°C for 15 minutes
b. 40 cycles of 95°C for 30 sec, 55°C for 30 sec, 68°C for 60 sec c. 68°C for 10 minutes
d. 4°C hold
19. Then proceed to Hybridization
20.
Hybridization
1. Following PCR( Amplification), Prepare a 96-well PCR plate by writing Fungal-ACC year(XXXX)month(XX)day(XX) on the side of plate.
2. Add 2 ul of Fungal beads to each well. After every 4 wells close and vortex the bead mix.
3. Using the Fungal worksheet, create the first hybridization mix in a 15mL conical tube a. 32 μΙ_, 1.5M TMAC hybridization Buffer per reaction
b. 6 μΐ, IX Tris-EDTA (TE) Buffer, pH 8.0 per reaction
c. Vortex first hybridization mix
4. Add 38 μΙ_, of hyb mix to each well, vortexing the conical tube after every 8 wells
5. Remove 8-well strip tubes from the thermal cycler and spin the tubes.
6. Add ΙΟμΙ. of DNA to each well using the multichannel pipette, pipetting up and down to mix DNA well.
7. Change tips after each dispense of DNA into the 96-well plate.
8. Place a plate seal over the plate and transfer the plate to the Thermocycler
9. Hybridize the reactions with the "Fungal Hyb" program on the thermocycler.
a. 95°C for 5 minutes
b. 52°C for 15 minutes
10. Remove from the Thermocycler and place on the magnet for 5 minutes
a. Keep the plate covered as hybridization beads are light sensitive 11. Prepare the second hybridization buffer while the plate sits on the magnet a. Using the Fungal worksheet, create the second hybridization mix in a 15mL conical tube
b. 75 μΐ. 1.0M TMAC Reporter Buffer per reaction
c. 0.2 μΐ. SAPE (lmg/mL; streptavidin-phycoerythrin) per reaction d. Vortex second hybridization mix
12. After 5 minutes, remove the plate seal and pick up the magnet containing the PCR plate and empty the plate contents into the biohazard box
a. Leave the plate on the magnet to prevent loss of fungal beads.
13. Pat the plate onto a napkin to clean up the plate
14. Remove plate from magnet
15. Add 75μΙ^ of your second hybridization buffer, using the multichannel pipette.
16. Change tips after each dispense of second hybridization buffer
17. Cover using Plate seal
18. Place plate back on Thermocycler
a. 52°C for 5 minutes
19. After 5 minutes, remove the plate seal and place on the Luminex
Reading on the Luminex™
1. On the computer with Luminex™ (Genomic Luminex #1), open up the Xponent™ icon
a. Before running any assays, make sure the "daily fluidics prep" under the maintenance tab in the top right has been completed
2. Ej ect the Multiplex Reader
3. Make sure the solution wells are full
a. Well 1. Deionized HiO
b Well 2. 70% EtOH
c Well 3. NaOH
d Well 4. Empty
2 Under the "Maintenance" tab click "Probe & Heater
a. Turn heater "ON" and set temperature to 52°C
3 Create a Batch
Interpretation: Cut-off values for identifying shift in Candida spps is set at 999 MFI (Median Fluorescence Intensity), Trichomonas spps is 500 MFI, and for ?, hominis is 349. Any MFI above this cut-off value is considered positive.
Example 4: Exemplary protocol for the detection of detection of Helicobacter pylori
housekeeping genes
This example describes exemplary procedures for the detection of Helicobacter pylori housekeeping genes (ureA and 23 S rRNA) as well as pathogenicity genes (cagA, iceA, vacA and oipA) and single nucleotide polymorphisms (S Ps) associated with clarithromycin resistance using, e.g., PCR, e.g., multiplex PCR, and 2% agarose gel electrophoresis.
Following amplification, detection and quantitation of multiple H pylori genes, hybridization is performed using Luminex™.
• cagA - cytotoxicity associated immunodominant antigen island protein alpha gene, encodes for a protein associated with higher pathogenic strains of H. pylori that can often lead to PUD and/or gastric cancer.
Sample requirements
Stool samples are collected by the patient or via colonoscopy and transported in the appropriate transport container at room temperature. It is recommended that the samples meet the minimum weight requirements as listed:
• Colonoscopy transport tube minimum weight of tube with sample 20.69 g.
• Patient collected: Stool transport tube (saline solution) minimum weight of tube with sample 23.29 g.
• Patient collected: Stool transport tube (Cary-Blair) minimum weight of tube with sample
25.96 g.
• Biopsy- Received within 7 days of collection date.
Materials
• Isolated DNA samples
• ATCC Helicobacter pylori genomic DNA strain J99
• ATCC Helicobacter pylori genomic DNA strain 26695
• Internal control (IC) DNA
• Probe control beads • H. pylori multiplex (HPM) primer mix (ΙΟΟμΜ stock of each of the following primers to create working solution of each forward and reverse primers as explained in detail on page 2.
ureA forward
ureA reverse biotinylated
cagA forward
cagA reverse biotinylated
vacA forward
vacA reverse biotinylated
iceA forward
iceA reverse biotinylated
oipA forward
oipA reverse biotinylated
23 S rRNA forward
23 S rRNA reverse biotinylated
IC forward
IC reverse biotinylated
• 2. OX Hot Start Master Mix
• Nuclease-free water
• HP (HP mix Beads) Hybridization beads
• 1.5M TMAC (Tetram ethyl Ammonium Chloride) hybridization Buffer
• 1.0X Reporter Buffer
• IX Tris-EDTA (TE) Buffer, pH 8.0
• 0.5mL thin wall PCR 8-well strip tubes and caps
• 1.5mL microcentrifuge tube (MCT), sterile
• Sealer
Procedure
Making Primer Preparation for H. pylori primer mix (5,000 μΐ = 2,000 reactions).
1. Take sterilize 15 ml conical tube and add primers below under the dead air box hood: a. 3800 μΐ IX TE buffer, pH 8.0
b. 375 μΐ ureA-F primer (100 μΜ)
c. 375 μΐ ureA-R_biotin primer (100 μΜ)
d. 375 μΐ CagA-F primer (100 μΜ) e. 375 μΐ Cag-R_biotin primer (100 μΜ)
f. 200 μΐ VacA-F primer (100 μΜ)
g. 200 μΐ VacA-R_biotin primer (100 μΜ)
h. 200 μΐ 23s-F primer (100 μΜ)
i. 200 μΐ 23s-R_biotin primer (100 μΜ)
375 μΐ iceA-F primer (100 μΜ)
j . 375 μΐ iceA-R_biotin primer (100 μΜ)
k. 375 μΐ oipA-F primer (100 μΜ)
1. 375 μΐ oipA-R_biotin primer (100 μΜ)
2. Vortex for 15 seconds.
3. Aliquot into 1.5 ml microcentrifuge tubes of 1500 μΐ each. Label each tube with HP Primer mix',
preparation date, initials of preparer.
Note: Store in HP Stock Box in -20°C in pre-PCR freezer and label box with lot number. Internal control primers are kept separately and to be used only for that well with 0.5 μΐ of forward and reverse each.
Sample Preparation
Pre-PCR- (Batch sheet creation)
1. Generate a new HPM Panel batch sheet by opening the "HPM batch sheet template"
2. Input "Negative Control" as sample 1 (nucl ease-free water) on the HPM batch sheet.
3. Input "Positive Control" as sample 2 & 3 (ATCC genomic DNA) on the HPM batch sheet.
4. Input the accession number for each of the DNA samples to be tested beginning with well 3.
5. Input "Negative Control" after the last test DNA sample.
6. Name the batch with the following naming format: HPM-ACC- year(XXXX)month(XX)day(XX) and initials .
7. Save the file using the batch name and print a copy of the completed batch sheet. 9. Create a sample list by selecting all of the sample IDs from the daily sample log, copy all of the sample IDs on a new Excel Batch Sheet using the latest previous batch sheet as a template. 10. Go to sample log using batch review sheet that contains the positive patients' samples data and mark all accessions for HP testing with the positive and negative data in each column under each accession column.
Controls
Use nuclease-free water as the negative control for first tube in sequence (sample 1). An ATCC genomic DNA of H. pylori for strain 26695 (Sample 2) and strain J99 (Sample3) is used as the positive control. The second last sample is designated for Internal control and the last sample is set for probe control.
Amplification
Thaw all DNA samples (control DNAs and test DNAs), HP primer mix and the 2X Hot
Start™ Taq PCR Master Mix in the clean room dead air box.
After reagents thaw place on cold block.
Label 8-well strip tubes with the sample numbers.
Vortex and aliquot ΙΟμΙ of the DNA sample into the appropriate tube of the 8-well strip tubes.
Aliquot 10 μΐ of positive control last to minimize possible contamination.
Prepare the master mix in a 1.5mL MCT using the volumes for each reagent shown on the batch sheet. :
• List of Master mix components:
• 0.50 μΐ/ reaction of nuclease-free water.
• 12.5 μΐ/ reaction Apex-Supermix™.
• 2.5 μΐ/reaction HP primer mix from stock box.
Vortex the master mix and aliquot 15.5 μΐ into each tube of the 8-well strip tubes. Close each strip tube with the strip caps.
Pulse centrifuge each strip of tubes.
Place the strip tubes in a thermocycler.
Amplify the reactions with the "HPM amplification" program on the thermocycler.
• 95°C for 15 minutes.
• 40 cycles of 95°C for 30 sec, 55°C for 30 sec, 68°C for 1 min.
• 68°C for 10 minutes
• 4°C hold
Return reagents and DNA Isolation to designated area.
Decontaminate area with 30% bleach. Hybridization
1. Following PCR, Prepare a 96-well PCR plate by writing HPM-ACC
year(XXXX)month(XX)day(XX).
2. Using the HPM worksheet, create the first hybridization mix in a 15mL conical tube and vortex thoroughly
a. 1.5M TMAC hybridization Buffer
b. IX Tris-EDTA (TE) Buffer, pH 8.0
c. HPM beads
3. Add 40μΕ of hyb mix to each well, vortexing the conical after every 8 wells
a. Note :I s important to vortex frequently to ensure that the HP beads do not aggregate at the bottom of the tube
4. Add ΙΟμΙ. of DNA to each well using the multichannel pipette, pipetting up and down to mix
5. Place a plate sealer over the plate and move to the Thermocycler
6. Hybridize the reactions with the "HP Hyb" program on the thermocycler.
a. 60°C for 3 minutes
b. 42°C for 40 minutes
c. Immediately proceed to the Pre-Reading prep on Luminex step.
Pre-Reading Prep on the Luminex™
Recommended to complete Pre-Reading Prep process within 15 to 20 minutes.
1. On the computer, open up the Luminex™ icon
a. Note: Before running any assays, make sure the "daily fluidics prep" under the maintenance tab in the top right has been completed.
2. Eject the Multiplex Reader by clicking the eject button in the Luminex™ software.
3. Fill each well with assigned solutions (See below):
1. Well 1. Deionized HiO
2. Well 2. 70% EtOH
3. Well 3. NaOH
4. Well 4. Empty
4. Under the "Maintenance" tab click "Probe & Heater".
5. Turn heater "ON" and set temperature to 42°C.
6. Next Create a Batch Sheet
a. Click the "Batches" tab on the top right i. → "Create a new batch from existing protocol"
ii. → "Name Batch" HPM-ACC year(XXXX)month(XX)day(XX). iii. Click HP Protocol
7. Next Select the number of wells you are testing and click the yellow "unknown"
button in the bottom box.
8. Minimize the Luminex window.
9. Open excel batch sheet.
10. Copy/Paste patient accessions on the excel batch sheet onto a notepad window and save in the folder "Patient Load List" as HPM-ACC
year(XXXX)month(XX)day(XX).
1 1. Re-open Luminex and upload Patient Load list using "Import List" on the right side of the screen.
12. Then Double -check and when done click "Save".
Note: Proceed to next step below after completing the Pre Reading Prep on Luminex process. Continue Hybridization (Steps 7-13) After 40 minutes at 42°C is complete :
Remove from the Thermocycler and place on the magnet for 5 minutes
a. Keep the plate covered as hybridization beads are light sensitive
Prepare the second hybridization buffer while the plate sits on the magnet
a. 1.0M TMAC Reporter Buffer
b. SAPE (lmg/mL)
After 5 minutes, remove the plate cover and pick up the magnet containing the PCR plate and empty the plate contents into the biohazard box
a. Leave the plate on the magnet to prevent loss of HP beads.
Pat the plate onto a napkin to clean up the plate before adding 75μΙ^ of your second hybridization buffer, using the multichannel pipette.
Cover using Plate cover
Place back on Thermocycler
a. 42°C for 5 minutes
After 5 minutes, remove the plate seal and place on the Luminex.
Next proceed to Luminex reading. A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. A method for the detection of an infectious disease, an infection, or a
condition related to the presence of a microbe in an individual in need thereof to provide individualized identification of a microbial profile in the individual, and for providing a guidance for a treatment regimen for the individual,
wherein optionally the condition related to the presence of a microbe is a condition or disorder affecting the gastrointestinal (GI) tract,
and optionally the condition or disorder affecting the gastrointestinal (GI) tract is Crohn's disease, Inflammatory Bowel Disease (IBD), a gastric ulcer or a duodenal ulcer, celiac disease, or an intestinal disorder,
and optionally the microbe is a bacteria, a parasite, a protozoan, or a fungi, and optionally the infection is an infection by a parasite, a protozoa, a bacteria or a fungi,
the method comprising:
(a)
(A) providing a sample from the individual,
wherein optionally the sample is, or comprises, or is derived from a stool or a fecal sample, a colonoscopy sample (a sample derived from a colonoscopy), a tissue biopsy, a blood, a sputum or a urine sample;
(B) analyzing the sample for the presence of a nucleic acid, optionally a DNA, fr m, derived from or characteristic of:
Figure imgf000045_0001
(1) a Clostridium difficile (C difficile) toxin A,
(2) a C. difficile toxin B,
(3) a Campylobacter (optionally a C jejuni, C coli, C lari),
(4) an Escherichia coli (E. coli),
(5) an Enterotoxigenic E. coli (ETEC) heat labile toxin (LT),
(6) an Enterotoxigenic E. coli (ETEC) heat stable toxin (ST),
(7) a Salmonella spp.,
(8) a Shiga-like toxin producing E. coli (STEC) stxl toxin, or a Shigella stxl toxin,
(9) a Shiga-like toxin producing E. coli (STEC) stx2 toxin, or a Shigella stx2 toxin,
(10) a Shigella spp., optionally an S. dysenteriae,
(11) an Entamoeba histolytica,
(12) a Giardia spp.,
(13) a Listeria monocytogenes, or
(14) an Aspergillus spps.,
wherein the presence of any or all of (1) through (14) detects the presence of a microbial pathogen or a microbial infection;
(iii)
(1) a Blastocystis hominis,
(2) a Candida spp.,
(3) a Trichomonas spp., or
(4) a Cryptosporidium spps (optionally a C. parvum or a C. hominis), wherein the presence of any of all of (1) through (4) detects the presence of a parasite or a fungal infection; and
(C) analyzing the sample for the presence of a gene indicative of or characteristic of a resistance to an antibiotic,
where optionally the gene indicative of or characteristic of a resistance to an antibiotic comprises:
(1) a β-lactamase resistance (bl2b-TEM) gene,
(2) a nitroimidazole resistance (nimD) gene,
(3) a fluoroquinoline resistance (qnrA) gene,
(4) a tetracycline resistance (tetA(P)) gene, (5) an imidazole resistance (nimA) gene,
(6) a vancomycin resistance (van A) gene,
(7) a vancomycin resistance (vanB) gene, or
(8) a combination of any of (1) through (5), or all of (1) through (5), wherein the presence of any or all of (1) through (5) indicates a bacterial infection that is resistant to an antibiotic or presence of a bacteria, parasite, fungus or microbe in the individual that is resistant to an antibiotic, thereby indicating or assisting a treatment regimen that does not comprise use of an antibiotic to which the bacteria, parasite, fungus or microbe is resistant,
thereby providing an individualized identification of the microbial profile in the individual, and providing a guidance for a treatment and antibiotic regimen for the individual.
2. The method of claim 1, further comprising determining the presence of a polypeptide biomarker in the sample,
wherein the polypeptide biomarker is associated with the infectious disease, or the infection or the condition related to the presence of a microbe in the individual, and the presence of the polypeptide biomarker complements a diagnosis or the detection of the infectious disease or the infection, or the condition related to the presence of a microbe, or the presence of an inflammation related to an infectious disease or condition, in the individual,
and optionally the polypeptide biomarker is detected by a protocol or process comprising use of an enzyme-linked immunosorbent assay (ELISA), optionally a multiplexed ELISA,
and optionally the biomarker is or comprises:
a secretory IgAl or sIgA2 immunoglobulin (antibody),
a fecal blood,
a hemoglobin, a myoglobin, or a heme or heme-containing globular protein (a globin),
a lactoferrin,
an elastase, or
a transglutaminase.
3. The method of any of the preceding claims, wherein the analyzing of the sample for the presence of the nucleic acid, optionally the DNA, in step 1(b) of claim 1 comprises:
(a) extracting or purifying DNA, or total DNA, found in the sample;
(b) adding amplification primer pairs, optionally polymerase chain reaction primer (PCR) pairs, capable of amplifying a nucleic acid subsequence of interest, optionally adding a plurality of PCR amplification primer pairs capable of amplifying a plurality of corresponding sets of nucleic acid subsequences of interest, wherein optionally the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid subsequences of interest are DNA as set forth in claim 1, and amplifying the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid subsequences of interest, optionally by PCR or by multiplexed PCR;
(c) immobilizing the amplified DNA of step (b);
(d) washing away or removing all or substantially all of the non-amplified
DNA;
(e) hybridizing to the immobilized DNA a sequence specific probe capable of specifically binding to or hybridizing to an amplified nucleic acid of interest; and
(f) detecting the presence of the amplified nucleic acid of interest by detecting the sequence specific probe specifically bound or hybridized to the amplified nucleic acid of interest, wherein optionally the detecting comprises use of a detector material bound to the sequence specific probe,
wherein optionally the sequence specific probe is biotinylated, and optionally the detector material comprises a streptavidin, and optionally the streptavidin is conjugated to a detectable moiety, and optionally the detectable moiety comprises a fluorescent material, and optionally the fluorescent material comprises a R- phycoerthrin (SAPE),
wherein optionally the amplified DNA of step (b) is immobilized by binding the amplified the nucleic acid subsequence of interest or the plurality of
corresponding sets of nucleic acid subsequences of interest with or to a segregatable substrate,
wherein optionally the segregatable substrate comprises a magnetic nanoparticle, and optionally the amplified the nucleic acid subsequence of interest or the plurality of corresponding sets of nucleic acid subsequences of interest are bound to a nanoparticle specific nucleotide probe (NSNP), and the NS P is bound to the segregatable substrate,
and optionally the NSNP is bound to one of primer of each amplification primer pair.
4. A method for treating or ameliorating an infectious disease, an infection, or a condition related to the presence of a microbe, in an individual in need thereof,
wherein optionally the condition related to the presence of a microbe is a condition or disorder affecting the gastrointestinal (GI) tract,
and optionally the condition or disorder affecting the gastrointestinal (GI) tract is Crohn's disease, Inflammatory Bowel Disease (IBD), a gastric ulcer or a duodenal ulcer, celiac disease, or an intestinal disorder,
and optionally the microbe is a bacteria, a protozoa, a parasite, or a fungi, and optionally the infection is an infection by a parasite, a protozoa, a bacteria or a fungi,
the method comprising:
administering to the individual in need thereof an antibiotic or drug capable of treating or ameliorating the infectious disease, infection, or condition related to the presence of a microbe as detected by or diagnosed using a method of any of the preceding claims,
and optionally administering to the individual in need thereof an antibiotic or a drug to which the microbe has not been found to be resistant using a method of any of the preceding claims.
5. A product of manufacture comprising components to practice a method of any of the preceding claims, wherein optionally the product of manufacture comprises instructions for practicing a method of any of the preceding claims.
6. The product of manufacture of claim 5, wherein the product of manufacture is a kit, optionally a clinical diagnostic kit.
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WO2020148610A1 (en) * 2019-01-15 2020-07-23 3M Innovative Properties Company Loop-mediated isothermal amplification primers for shiga toxin producing e. coli (stec) detection
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