WO2018014614A1 - 一种同时鉴别8种牛病原体的引物组合及GeXP检测方法 - Google Patents

一种同时鉴别8种牛病原体的引物组合及GeXP检测方法 Download PDF

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WO2018014614A1
WO2018014614A1 PCT/CN2017/081056 CN2017081056W WO2018014614A1 WO 2018014614 A1 WO2018014614 A1 WO 2018014614A1 CN 2017081056 W CN2017081056 W CN 2017081056W WO 2018014614 A1 WO2018014614 A1 WO 2018014614A1
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virus
primer
primer pair
tested
bovine
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French (fr)
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谢芝勋
范晴
谢志勤
邓显文
谢丽基
黄莉
罗思思
黄娇玲
张艳芳
曾婷婷
王盛
刘加波
庞耀珊
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广西壮族自治区兽医研究所
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Priority to US15/567,612 priority Critical patent/US20180291473A1/en
Publication of WO2018014614A1 publication Critical patent/WO2018014614A1/zh

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    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the invention relates to a primer combination for simultaneously identifying eight bovine pathogens and a GeXP detection method.
  • FMDV Foot and Mouth Disease Virus
  • BTV Bluetongue Virus
  • VSV Vesicular Stomatitis Virus
  • BVDV Bovine Viral Diarrheal Virus
  • BTEC Bovine Rotavirus
  • IBRV Infectious Bovine Rhinotracheitis Virus
  • PPRV Peste des Petits Ruminants Virus
  • Bovine foot-and-mouth disease bovine vesicular stomatitis caused by VSV and bovine bluetongue caused by BTV are highly acute infectious diseases of cattle, which are generally fulminant. Clinically, lesions occur in the mouth, hooves and breasts. Similar indistinguishable, high mortality, classified by the World Organisation for Animal Health (OIE) as a Class A infectious disease.
  • OIE World Organisation for Animal Health
  • BVDV, BRV and ETEC are also the main pathogens causing bovine diarrhea, and BVDV is present in asymptomatic forms of persistent infection. A significant proportion of cattle in the herd are carriers of these pathogens, and often with other outbreaks, symptoms of sick cattle For acute watery diarrhea, rapid weight loss.
  • IBRV immunosuppressive disease virus, which can be followed by bacterial infection after infection.
  • the small ruminant disease caused by PPRV is also a new foreign disease that has appeared in recent years.
  • OIE lists it as an animal disease that must be reported, and it is classified as a kind of animal disease in China. These diseases are a huge potential for the cattle industry. Once an outbreak occurs, it will cause huge economic losses. Therefore, it is imperative to carry out research on rapid detection technology of bovine infectious diseases.
  • the rapid and accurate detection of infectious diseases is the premise and basis for effective prevention and control.
  • the main methods used to differentially diagnose these bovine infectious diseases include pathogen isolation and serological tests, but these methods are often limited by the freshness, contamination level or serum titer of clinical disease materials, resulting in incorrect results and time consuming. Power consumption has certain limitations in practical applications.
  • molecular biology diagnostic methods based on PCR technology have been widely used in the detection and diagnosis of infectious diseases, including PCR, fluorescent PCR and LAMP, but these methods can only be used for single or 2 to The detection of four pathogens cannot achieve high-throughput detection of multiple pathogens in the true sense.
  • the GeXP Multi-Gene Expression Analysis System is a novel high-throughput gene detection technology that combines multiplex PCR and capillary electrophoresis techniques with fluorescently labeled universal primers and specific chimeric primers (ie, gene-specific primers 5).
  • the combination of 'end-connected universal primers' triggers the amplification of multiplex PCR systems, enabling simultaneous detection and analysis of up to 30 genes of interest, enabling the real high-throughput assay to identify multiple pathogens.
  • the present invention provides a primer combination consisting of primer pair I, primer pair II, primer pair III, primer For IV, primer pair V, primer pair VI, primer pair VII and primer pair VIII;
  • the primer pair I consists of a primer FMDV-F and a primer FMDV-R;
  • the primer FMDV-F is as follows (a1) or (a2) or (a3):
  • (a3) a DNA molecule having (a1) or (a2) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a1) or (a2);
  • the primer FMDV-R is as follows (a4) or (a5) or (a6):
  • (a6) a DNA molecule having (a4) or (a5) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a4) or (a5);
  • the primer pair II is composed of a primer BTV-F and a primer BTV-R;
  • the primer BTV-F is as follows (a7) or (a8) or (a9):
  • the primer BTV-R is as follows (a10) or (a11) or (a12):
  • Sequence 4 of the Sequence Listing is a single-stranded DNA molecule represented by nucleotides 20 to 37 at the 5' end;
  • (a12) a DNA molecule having (a10) or (a11) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a10) or (a11);
  • the primer pair III consists of a primer VSV-F and a primer VSV-R;
  • the primer VSV-F is as follows (a13) or (a14) or (a15):
  • the primer VSV-R is as follows (a16) or (a17) or (a18):
  • (a18) a DNA molecule having (a16) or (a17) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a16) or (a17);
  • the primer pair IV is composed of a primer BVDV-F and a primer BVDV-R;
  • the primer BVDV-F is as follows (a19) or (a20) or (a21):
  • (a21) a DNA molecule having (a19) or (a20) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a19) or (a20);
  • the primer BVDV-R is as follows (a22) or (a23) or (a24):
  • the primer pair V consists of a primer BRV-F and a primer BRV-R;
  • the primer BRV-F is as follows (a25) or (a26) or (a27):
  • (a27) a DNA molecule having (a25) or (a26) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a25) or (a26);
  • the primer BRV-R is as follows (a28) or (a29) or (a30):
  • the primer pair VI consists of the primer ETEC-F and the primer ETEC-R:
  • the primer ETEC-F is as follows (a31) or (a32) or (a33):
  • (a33) a DNA molecule having (a31) or (a32) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a31) or (a32);
  • the primer ETEC-R is as follows (a34) or (a35) or (a36):
  • (a36) a DNA molecule having (a34) or (a35) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a34) or (a35);
  • the primer pair VII is composed of a primer IBRV-F and a primer IBRV-R;
  • the primer IBRV-F is as follows (a37) or (a38) or (a39):
  • (a39) a DNA molecule having (a37) or (a38) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a37) or (a38);
  • the primer IBRV-R is as follows (a40) or (a41) or (a42):
  • (a42) a DNA molecule having (a40) or (a41) substituted and/or deleted and/or added by one or several nucleotides and having the same function as (a40) or (a41);
  • the primer pair VIII consists of the primer PPRV-F and the primer PPRV-R:
  • the primer PPRV-F is as follows (a43) or (a44) or (a45):
  • the primer PPRV-R is as follows (a46) or (a47) or (a48):
  • the use of the primer combination is any one of the following (b1) to (b6):
  • (b3) Check whether the pathogen to be tested is foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhea virus, bovine rotavirus, enterotoxin-producing Escherichia coli, bovine infectious rhinotracheitis virus or small ruminant Animal disease virus
  • (b4) Preparation for detecting whether the pathogen to be tested is foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhea virus, bovine rotavirus, enterotoxin-producing Escherichia coli, bovine infectious rhinotracheitis virus Or a kit of small ruminant virus;
  • test sample contains foot-and-mouth disease virus and/or bluetongue virus and/or vesicular stomatitis virus and/or bovine viral diarrhea virus and/or bovine rotavirus and/or enterotoxin-producing Escherichia coli And/or bovine infectious rhinotracheitis virus and/or small ruminant virus;
  • test sample contains foot-and-mouth disease virus and/or bluetongue virus and/or vesicular stomatitis virus and/or bovine viral diarrhea virus and/or bovine rotavirus and/or enteral Kit for toxin E. coli and/or bovine infectious rhinotracheitis virus and/or Peste des Petits Ruminants virus.
  • the invention also protects the use of the primer combination as follows: (b1) to (b6):
  • (b3) Check whether the pathogen to be tested is foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhea virus, bovine rotavirus, enterotoxin-producing Escherichia coli, bovine infectious rhinotracheitis virus or small ruminant Animal disease virus
  • (b4) Preparation for detecting whether the pathogen to be tested is foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhea virus, bovine rotavirus, enterotoxin-producing Escherichia coli, bovine infectious rhinotracheitis virus Or a kit of small ruminant virus;
  • test sample contains foot-and-mouth disease virus and/or bluetongue virus and/or vesicular stomatitis virus and/or bovine viral diarrhea virus and/or bovine rotavirus and/or enterotoxin-producing Escherichia coli And/or bovine infectious rhinotracheitis virus and/or small ruminant virus;
  • test sample contains foot-and-mouth disease virus and/or bluetongue virus and/or vesicular stomatitis virus and/or bovine viral diarrhea virus and/or bovine rotavirus and/or enteral Kit for toxin E. coli and/or bovine infectious rhinotracheitis virus and/or Peste des Petits Ruminants virus.
  • the invention also protects a kit comprising the primer combination; the use of the kit is as follows (c1) or (c2) or (c3):
  • (c2) Detecting whether the pathogen to be tested is foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhea virus, bovine rotavirus, enterotoxin-producing Escherichia coli, bovine infectious rhinotracheitis virus or small ruminant Animal disease virus
  • test sample contains foot-and-mouth disease virus and/or bluetongue virus and/or vesicular stomatitis virus and/or bovine viral diarrhea virus and/or bovine rotavirus and/or enterotoxin-producing Escherichia coli And/or bovine infectious rhinotracheitis virus and/or Peste des Petits Ruminants virus.
  • the invention also protects the method of making the kit comprising the steps of individually packaging the individual primers.
  • the invention also protects a method for identifying eight bovine pathogens, comprising the following steps (d1) or (d2):
  • d1 performing genomic DNA extraction operation and/or RNA extraction operation on the pathogen to be tested (DNA extraction operation and RNA extraction operation may be performed in the same system or post-mixing separately) to obtain a nucleic acid; the content of the pathogen to be tested RNA or nucleic acid containing RNA may be reverse transcribed; using the obtained DNA and/or cDNA as a template, PCR amplification may be performed using the primer combination (specifically, GeXP multiplex PCR amplification may be performed; amplification products may be subjected to capillary electrophoresis detection) If the amplified product contains a 165-167 bp DNA fragment, the pathogen to be tested is or is a foot-and-mouth disease virus, if the amplified product contains a 135-137 bp DNA fragment, the pathogen to be tested is or the candidate is a bluetongue virus, if The product has a DNA fragment of 278-281 bp, a pathogen to be tested or a candidate for
  • the pathogen to be tested is or the candidate is enterotoxin-producing Escherichia coli, if the amplified product contains a DNA fragment of 187-189 bp.
  • the pathogen to be tested is or candidate for bovine infectious rhinotracheitis virus;
  • the pathogen to be tested is or is a foot-and-mouth disease virus, if the cDNA contains the target sequence of the primer pair II, the pathogen to be tested is or is a candidate for bluetongue virus, if the cDNA contains the primer pair III
  • the target sequence, the pathogen to be tested is or is a vesicular stomatitis virus, if the cDNA contains the target sequence of the primer pair IV, the pathogen to be tested is or is a candidate for bovine viral diarrhea virus, if the cDNA contains The target sequence of
  • the invention also protects whether the pathogen to be tested is foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhea virus, bovine rotavirus, enterotoxin-producing Escherichia coli, bovine infectious rhinotracheitis virus Or a small anti-veterinary virus method, including the following steps (e1) or (e2):
  • RNA extraction operation performing genomic DNA extraction operation and/or RNA extraction operation on the pathogen to be tested (the DNA extraction operation and the RNA extraction operation may be performed in the same system or may be separately mixed) to obtain a nucleic acid; the content of the pathogen to be tested is RNA or nucleic acid containing RNA may be reverse transcribed; using the obtained DNA and/or cDNA as a template, PCR amplification may be performed using the primer combination (specifically, GeXP multiplex PCR amplification may be performed; amplification products may be subjected to capillary electrophoresis detection) If the amplified product contains a 165-167 bp DNA fragment, the pathogen to be tested is or is a foot-and-mouth disease virus, if the amplified product contains a 135-137 bp DNA fragment, the pathogen to be tested is or the candidate is a bluetongue virus, if The product has a DNA fragment of 278-281 bp, a pathogen to be tested or a
  • the pathogen to be tested is or is candidate for the small ruminant virus, if the amplified product contains a 252-254 bp DNA fragment, the pathogen to be tested Or candidate for enterotoxin-producing E. coli, if the amplification product contains a DNA fragment of 187-189 bp, the pathogen to be tested is or is candidate for bovine infectious rhinotracheitis virus;
  • the pathogen to be tested is or is a foot-and-mouth disease virus, if the cDNA contains the target sequence of the primer pair II, the pathogen to be tested is or is a candidate for bluetongue virus, if the cDNA contains the primer pair III
  • the target sequence, the pathogen to be tested is or is a vesicular stomatitis virus, if the cDNA contains the target sequence of the primer pair IV, the pathogen to be tested is or is a candidate for bovine viral diarrhea virus, if the cDNA contains The target sequence of the primer pair V
  • the invention also protects a test sample for the presence or absence of foot-and-mouth disease virus and/or bluetongue virus and/or vesicular stomatitis virus and/or bovine viral diarrhea virus and/or bovine rotavirus and/or enteral
  • a method of toxin E. coli and/or bovine infectious rhinotracheitis virus and/or Peste des Petits Ruminants virus comprising the following steps (f1) or (f2):
  • (f1) performing genomic DNA extraction operation and/or RNA extraction operation on the sample to be tested (the DNA extraction operation and the RNA extraction operation may be performed in the same system or may be separately mixed) to obtain a nucleic acid; the sample to be tested is contained RNA or nucleic acid containing RNA may be reverse transcribed, and the obtained DNA and/or cDNA may be used as a template, and the primer combination may be used for PCR amplification (specifically, GeXP multiplex PCR amplification may be performed; amplification products may be subjected to capillary electrophoresis detection).
  • the sample to be tested contains or suspects to contain foot-and-mouth disease virus, if the amplified product contains a 135-137 bp DNA fragment, the sample to be tested contains or suspects to contain bluetongue virus, if expanded The product has a DNA fragment of 278-281 bp, and the sample to be tested contains or is suspected of containing vesicular Oral inflammation virus, if the amplified product contains a 308-310 bp DNA fragment, the sample to be tested contains or suspects to contain bovine viral diarrhea virus, if the amplified product contains a 211-214 bp DNA fragment, the sample to be tested contains or is suspected of containing a cattle wheel Virus, if the amplified product contains 342-345 bp DNA fragment, the sample to be tested contains or suspects to contain P.
  • vaccae virus if the amplified product contains 252-254 bp DNA fragment, the sample to be tested contains or suspects to contain enterotoxin-producing large intestine Bacillus, if the amplified product contains a DNA fragment of 187-189 bp, the sample to be tested contains or suspects to contain bovine infectious rhinotracheitis virus;
  • the invention also protects the primer combinations as follows (g1) or (g2):
  • primer pair I the primer pair I, the primer pair II, the primer pair III, the primer pair IV, the primer pair V, the primer pair VI, the primer pair VII, and the primer pair VIII
  • a combination of any two primer pairs a combination of any three primer pairs, a combination of any four primer pairs, a combination of any five primer pairs, a combination of any six primer pairs, or a combination of any seven primer pairs.
  • the use of the primer combination is to identify foot-and-mouth disease virus and/or bluetongue virus and/or vesicular stomatitis virus and/or bovine viral diarrhea virus and/or bovine rotavirus and/or enterotoxin-producing Escherichia coli and/or Or Bovine infectious rhinotracheitis virus and / or small ruminant disease virus.
  • the invention also protects the use of the primer combination for the identification of foot-and-mouth disease virus and/or bluetongue virus and/or vesicular stomatitis virus and/or bovine viral diarrhea virus and/or bovine rotavirus and/or enteral Toxin E. coli and/or bovine infectious rhinotracheitis virus and/or Peste des Petits Ruminants virus.
  • the invention also protects a kit comprising the primer combination; the kit is for the purpose of identifying foot-and-mouth disease virus and/or bluetongue virus and/or vesicular stomatitis virus and/or bovine viral diarrhea virus and/or cattle Rotavirus and/or enterotoxin-producing Escherichia coli and/or bovine infectious rhinotracheitis virus and/or Peste des Petits Ruminants virus.
  • bovine pathogens mentioned above are foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhea virus, bovine rotavirus, enterotoxin-producing Escherichia coli, bovine infectious rhinotracheitis virus and small Anti-veterinary virus.
  • the pathogen to be tested according to any of the above may specifically be FMDV O-type inactivated virus, FMDV type A inactivated virus, FMDV Asia type I inactivated virus, VSV NJ type inactivated virus, VSV IND type inactivated virus, BTV type 4 Inactivated virus, BTV type 8 inactivated virus, BTV type 9 inactivated virus, BTV type 15 inactivated virus, BTV type 17 inactivated virus, BTV type 18 inactivated virus, PPRV vaccine strain, BVDV reference strain Oregon CV24 strain (BVDV-1 type), BVDV reference strain NADL strain (BVDV-1 type), BVDV reference strain yak strain (BVDV-1 type), BRV reference strain NCDV, BRV reference strain BRV014, IBRV virus, ETEC reference Strain 1676, ETEC reference strain 1751, ETEC reference strain B41, BVDV strain GX-BVDV1, BVDV strain GX-BVDV2, BVDV strain GX-BVDV3, BVDV strain GX-BVDV4, BVDV
  • target sequence of the primer pair I may specifically be a DNA molecule represented by the sequence 27 of the following (h1) or (h2) or (h3): (h1) sequence listing; (h1) sequence listing Sequence 27 is a DNA molecule from nucleotides 19 to 146 at the 5' end; (h3) and (h1) or (h2) A DNA molecule having more than 98% homology.
  • target sequence of the primer pair II may specifically be a DNA molecule represented by the sequence of the following (h4) or (h5) or (h6): (h4) sequence listing; (h5) sequence listing The sequence 28 is a DNA molecule represented by nucleotides 19 to 117 at the 5' end; (h6) has a DNA molecule having 98% or more homology with (h4) or (h5).
  • target sequence of the primer pair III may specifically be a DNA molecule represented by the sequence of the following (h7) or (h8) or (h9): (h7) sequence listing; (h8) sequence listing The sequence 29 is a DNA molecule represented by nucleotides 19 to 259 at the 5' end; (h9) has a DNA molecule having 98% or more homology with (h7) or (h8).
  • target sequences of the primer pair IV may specifically be the following (h10) or (h11) or (h12): (h10) DNA molecules represented by the sequence 30 of the sequence listing; (h11) sequence listing The sequence 30 is a DNA molecule represented by nucleotides 19 to 289 at the 5' end; (h12) a DNA molecule having 98% or more homology with (h10) or (h11).
  • target sequences of the primer pair V may specifically be a DNA molecule represented by the sequence of the following (h13) or (h14) or (h15): (h13) sequence listing; (h14) sequence listing The sequence 31 is a DNA molecule represented by nucleotides 19 to 192 at the 5' end; (h15) a DNA molecule having 98% or more homology with (h13) or (h14).
  • target sequence of the primer pair VIII may specifically be a DNA molecule represented by the sequence 32 of the sequence of the following (h16) or (h17) or (h18): (h16); (h17) sequence listing
  • the sequence 32 is a DNA molecule represented by nucleotides 19 to 325 at the 5' end; (h18) a DNA molecule having 98% or more homology with (h16) or (h17).
  • target sequence of the primer pair VI may specifically be a DNA molecule represented by the sequence of the following (h19) or (h20) or (h21): (h19) sequence listing; (h20) sequence listing
  • the sequence 33 is a DNA molecule represented by nucleotides 19 to 234 at the 5' end; (h21) has a DNA molecule having 98% or more homology with (h19) or (h20).
  • target sequence of the primer pair VII may specifically be a DNA molecule represented by the sequence 34 of the sequence of the following (h22) or (h23) or (h24): (h22); (h23) sequence listing Sequence 34 is a DNA molecule from nucleotides 19 to 169 at the 5' end; (h24) and (h22) or (h23) A DNA molecule having 98% or more homology.
  • the sample to be tested according to any of the above may be a stool swab, an eye test, a nasal mucus swab, an anticoagulant, an OP solution (esophage-pharyngeal secretion), a vesicular fluid, a rectal mucosal tissue sample, and a blister tissue.
  • Sample or lymph node tissue sample may be a stool swab, an eye test, a nasal mucus swab, an anticoagulant, an OP solution (esophage-pharyngeal secretion), a vesicular fluid, a rectal mucosal tissue sample, and a blister tissue.
  • Sample or lymph node tissue sample may be a stool swab, an eye test, a nasal mucus swab, an anticoagulant, an OP solution (esophage-pharyngeal secretion), a vesicular fluid, a rectal mucosal tissue sample,
  • the concentration of each primer in the primer combination is as follows: the concentrations of FMDV-F and FMDV-R are both 0.2 ⁇ mol/ ⁇ L, and BTV-F and BTV-R The concentration was 0.2 ⁇ mol/ ⁇ L, the concentrations of VSV-F and VSV-R were both 0.2 ⁇ mol/ ⁇ L, the concentrations of BVDV-F and BVDV-R were both 2 ⁇ mol/ ⁇ L, and the concentrations of BRV-F and BRV-R were both.
  • ETEC-F and ETEC-R were both 0.2 ⁇ mol/ ⁇ L
  • concentrations of IBRV-F and IBRV-R were both 0.2 ⁇ mol/ ⁇ L
  • concentrations of PPRV-F and PPRV-R were both 2 ⁇ mol/ ⁇ L.
  • the reaction system (20 ⁇ L) of any of the above GeXP multiplex PCR amplifications may specifically be: template 1 ⁇ L (10-100 ng), Genome Lab GeXP Starter Kit 5 ⁇ buffer 4 ⁇ L (buffer contains universal primers, universal primers by sequence table Primer A shown in SEQ ID NO: 25 and primer B shown in SEQ ID NO: 26 in the Sequence Listing, wherein the primer A has a CY5 fluorophore label at the 5' end, and the primer A and the primer B have a working concentration of 0.25 ⁇ M, MgCl. 2 (25 ⁇ M) 4 ⁇ L, 1 ⁇ L of a primer mixture containing all the primers in the primer combination, DNA polymerase 10 U, supplemented to 20 ⁇ L with ultrapure water.
  • the reaction procedure of any of the above GeXP multiplex PCR amplifications may specifically be: 95 ° C for 5 minutes pre-denaturation; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 94 ° C for 30 seconds, 68 ° C 30 seconds, 72 ° C for 30 seconds, 10 cycles; 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 72 ° C extension for 5 min, the reaction was terminated.
  • the electrophoresis conditions of any of the above capillary electrophoresis were: 90 ° C for 120 seconds, denaturation; 2.0 KV for 30 seconds, inhalation of the sample; 6.0 KV for 35 minutes, and the sample was separated.
  • the GeXP detection method established by the invention can simultaneously identify foot-and-mouth disease virus, bluetongue virus, vesicular stomatitis virus, bovine viral diarrhea virus, bovine rotavirus, enterotoxin-producing Escherichia coli, bovine infectious rhinotracheitis virus and small
  • the pathogen of eight kinds of bovine infectious diseases of anti-veterinary virus has the characteristics of high throughput, specificity and high sensitivity, and can be used for monitoring and epidemic disease of bovine epidemiology
  • the differential diagnosis of love ensures the healthy development of the cattle industry.
  • Example 1 is a graph showing the results of multiplex PCR amplification of each sample to be tested in Example 2.
  • Figure 2 is a graph showing the results of multiplex PCR amplification of eight mixed bovine pathogen samples in Example 2.
  • Fig. 3 is a graph showing the results of amplification of multiplex PCR using the reaction system 1-5 in Example 5.
  • Fig. 4 is a graph showing the results of amplification of multiplex PCR using the mixture A and the mixture B as templates in Example 6.
  • the following examples are provided to facilitate a better understanding of the invention but are not intended to limit the invention.
  • the experimental methods in the following examples are conventional methods unless otherwise specified.
  • the test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. For the quantitative tests in the following examples, three replicate experiments were set, and the results were averaged. Due to the error of the GeXP system itself, the deviation of the amplified fragment size from the theoretical value of ⁇ 2bp is a correct result.
  • FMDV O-type inactivated virus FMDVA type inactivated virus, FMDVAsia type I inactivated virus, VSV NJ type inactivated virus, VSV IND type inactivated virus, BTV type 4 inactivated virus, BTV type 8 inactivated virus, BTV 9 Inactivated virus, BTV type 15 inactivated virus, BTV type 17 inactivated virus, BTV type 18 inactivated virus, PPRV vaccine strain: References: Qin Min, Zou Fengcai, Yang Yunqing, et al.
  • BVDV reference strain NADL strain (BVDV-1 type): China Veterinary Drug Inspection Institute, article number: AV67.
  • BVDV reference strain yak strain (BVDV-1 type): China Veterinary Drug Inspection Institute, article number: AV68.
  • BRV reference strain NCDV China Veterinary Drug Inspection Institute, article number: AV51.
  • BRV reference strain BRV014 China Veterinary Drug Inspection Institute, article number: AV52.
  • IBRV virus China Veterinary Microbial Culture Collection Management Center, article number: AV21.
  • ETEC reference strain 1676 China Veterinary Drug Inspection Institute, article number: 212.
  • ETEC reference strain 1751 China Veterinary Drug Inspection Institute, article number: 214.
  • ETEC reference strain B41 China Veterinary Drug Inspection Institute, article number: 215.
  • Genome Lab GeXP Starter Kit 5 ⁇ buffer which contains the primer A shown in SEQ ID NO: 25 of the sequence listing and the primer B shown in SEQ ID NO: 26 of the Sequence Listing, wherein the 5' end of the primer A has a label of a CY5 fluorophore; Mankurt company.
  • Sample buffer Beckman Coulter, USA, article number: M409196.
  • DNA size standard kit-400Base Pairs Beckman Coulter, USA, article number: 608098.
  • DNApolymerase SIGMA, USA, article number: D4184-1.5KU.
  • a number of primers for identifying eight bovine pathogens of FMDV, BTV, VSV, BVDV, BRV, ETEC, IBRV and PPRV were obtained by extensive sequence analysis and alignment. Each primer was pre-experimented, and the sensitivity, specificity and the like were compared, and finally eight pairs of specific primers for identifying eight bovine pathogens were obtained.
  • Each specific primer pair, the forward primer and the reverse primer are composed of a targeting segment and a universal primer segment, and the universal primer segment is located at the 5' end of the targeting segment.
  • the primer pair used to identify FMDV consists of the following two primers (5' ⁇ 3'):
  • FMDV-F (sequence 1 of the sequence listing ): AGGTGACACTATAGAATA GCCGTGGGACCATACAGG;
  • FMDV-R sequence 2 of the Sequence Listing: GTACGACTCACTATAGGGA AAGTGATCTGTAGCTTGGAATCTC.
  • the underlined portion is a universal primer segment.
  • the primer pair used to identify BTV consists of the following two primers (5' ⁇ 3'):
  • BTV-F sequence 3 of the sequence listing : AGGTGACACTATAGAATA AGGGTAACTCACAGCAAACTCAA;
  • the underlined portion is a universal primer segment.
  • the primer pair used to identify VSV consists of the following two primers (5' ⁇ 3'):
  • VSV-F sequence 5 of the sequence listing ): AGGTGACACTATAGAATA AAACTACTGGACGGGCTTGA;
  • VSV-R (sequence 6 of the Sequence Listing): GTACGACTCACTATAGGGA TGAGATGCCCAAATGTTGC.
  • the underlined portion is a universal primer segment.
  • the primer pair used to identify BVDV consists of two primers (5' ⁇ 3'):
  • BVDV-F (sequence 7 of the sequence listing ): AGGTGACACTATAGAATA GTGAGTTCGTTGGATGGC;
  • BVDV-R (sequence 8 of the sequence listing ): GTACGACTCACTATAGGGA TATGTTTTGTATAAGAGTTCATTTG.
  • the underlined portion is a universal primer segment.
  • the primer pair used to identify the BRV consists of the following two primers (5' ⁇ 3'):
  • BRV-F sequence 9 of the sequence listing ): AGGTGACACTATAGAATA CAGTGGCTTCCATTAGAAGCAT;
  • BRV-R (sequence 10 of the Sequence Listing): GTACGACTCACTATAGGGA GGTCACATCCTCTCACTA.
  • the underlined portion is a universal primer segment.
  • the primer pair used to identify ETEC consists of the following two primers (5' ⁇ 3'):
  • ETEC-F sequence 11 of the sequence listing : AGGTGACACTATAGAATA CTCAGGTGCGAAAGCGTG;
  • ETEC-R (sequence 12 of the Sequence Listing): GTACGACTCACTATAGGGA CGTTGCATCGAATTAAACCAC.
  • the underlined portion is a universal primer segment.
  • the primer pair used to identify ETEC consists of the following two primers (5' ⁇ 3'):
  • IBRV-F sequence 13 of the Sequence Listing: AGGTGACACTATAGAATA GCGTCATTTACAAGGAGAACATC;
  • IBRV-R (sequence 14 of the Sequence Listing): GTACGACTCACTATAGGGA ATCTCGCCCATGCCCAC.
  • the underlined portion is a universal primer segment.
  • the primer pair used to identify PPRV consists of the following two primers (5' ⁇ 3'):
  • PPRV-F sequence 15 of the sequence listing : AGGTGACACTATAGAATA TGGTTTGAGAACAGAGAAATAATAGA;
  • PPRV-R (sequence 16 of the Sequence Listing): GTACGACTCACTATAGGGA GCTTGTTGCCGGGGGTC.
  • the underlined portion is a universal primer segment.
  • primer pair I The primer pair used to identify FMDV was named primer pair I.
  • primer pair II The primer pair used to identify BTV was named primer pair II.
  • primer pair III The primer pair used to identify VSV was named primer pair III.
  • primer pair IV The primer pair used to identify BVDV was named primer pair IV.
  • primer pair V The primer pair used to identify BRV was named primer pair V.
  • primer pair VI The primer pair used to identify ETEC was named primer pair VI.
  • primer pair VII The primer pair used to identify IBRV was named primer pair VII.
  • primer pair VIII The primer pair used to identify PPRV was named primer pair VIII.
  • Each of the above primer pairs constitutes a primer combination.
  • RNA of the sample to be tested extracts the total RNA of the sample to be tested and reverse-transcribe into cDNA.
  • the samples to be tested were: FMDV O-type inactivated virus, BTV type 4 inactivated virus, VSV NJ type inactivated virus, BVDV reference strain Oregon CV24 strain (BVDV-1 type), BRV reference strain NCDV, PPRV vaccine strain .
  • the samples to be tested were ETEC reference strain 1676 and IBRV virus, respectively.
  • the GeXP multiplex PCR was carried out using the primer obtained in Example 1 using the cDNA obtained in the step 1 and the genomic DNA obtained in the step 2 as a template.
  • Multiplex PCR reaction system (20 ⁇ L): template 1 ⁇ L, Genome Lab GeXP Starter Kit 5 ⁇ buffer 4 ⁇ L (buffer contains universal primers, universal primers are shown by primer A shown in sequence 25 of the sequence listing and primers shown in sequence 26 of the sequence listing)
  • Composition B in which the 5' end of primer A has a label of CY5 fluorophore, primer A and primer B each have a working concentration of 0.25 ⁇ M), MgCl 2 (25 ⁇ M) 4 ⁇ L, and a primer mixture containing all primers in the primer combination 1 ⁇ L , DNA polymerase 10U, supplemented to 20 ⁇ L with ultrapure water.
  • the concentrations of FMDV-F and FMDV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of BTV-F and BTV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of VSV-F and VSV-R were both.
  • the concentration of BVDV-F and BVDV-R were both 2 ⁇ mol/ ⁇ L
  • the concentrations of BRV-F and BRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of ETEC-F and ETEC-R were both 0.2 ⁇ mol/
  • the concentrations of ⁇ L, IBRV-F and IBRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of PPRV-F and PPRV-R were both 2 ⁇ mol/ ⁇ L.
  • a negative control with equal volume of water as a template was set.
  • the DNA content in the 1 ⁇ L template is about 100 ng;
  • the DNA content in the 1 ⁇ L template is about 100 ng;
  • Multiplex PCR reaction procedure pre-denaturation at 95 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C 30 seconds, 10 cycles; 94 ° C 30 seconds, 68 ° C 30 seconds, 72 ° C 30 seconds, 10 cycles; 94 ° C 30 seconds, 50 ° C 30 seconds, 72 ° C 30 seconds, 10 cycles; 72 ° C extension At 5 min, the reaction was terminated.
  • Capillary electrophoresis of the multiplex PCR amplification product of step 3 the specific steps are: mixing 3 ⁇ L of multiplex PCR amplification product, 38.75 ⁇ L sample buffer and 0.25 ⁇ L DNA size standard kit-400 Base Pairs, and adding the sample plate. In the middle, add 1 drop of paraffin to each well to block the liquid surface to avoid the oxidation of formamide and the evaporation of the sample. 180 ⁇ L of sample buffer was added to each well of the buffer plate for capillary electrophoresis. Capillary electrophoresis conditions: 90 ° C for 120 seconds, denaturation; 2.0 KV for 30 seconds, inhalation of the sample; 6.0 KV for 35 minutes, separation of the sample.
  • the PCR products of different size fragments are separated by electrophoresis, and the instrument recognizes the fragment size and signal intensity by detecting the fluorescent group carried by the PCR product. After the electrophoresis is completed, the results are analyzed using the instrument's own software, Express Profiler software.
  • the amplified fragments of the eight bovine pathogens are: FMDV: 165-167 bp, BTV: 135-137 bp, VSV: 278-281 bp, BVDV: 308-310 bp, BRV: 211-214 bp, PPRV: 342-345 bp, ETEC: 252-254 bp, IBRV: 187-189 bp. Due to the error of the GeXP system itself, the deviation of the amplified fragment size from the theoretical value of ⁇ 2bp is a correct result.
  • FIG. 1 The electrophoresis results are shown in Figure 1.
  • the abscissa indicates the fragment size (unit: bp), and the ordinate indicates the signal intensity, that is, the content of the PCR amplification product.
  • Fig. 1A shows the results of multiplex PCR amplification of FMDV O-type inactivated virus cDNA, and a DNA fragment of 165.03 bp was amplified.
  • Figure 1B shows the results of multiplex PCR amplification of the BTV4 type inactivated virus cDNA, and a 137.62 bp DNA fragment was amplified.
  • Figure 1C shows the results of multiplex PCR amplification of VSV NJ-type inactivated virus cDNA, and a 278.04 bp DNA fragment was amplified.
  • Fig. 1D shows the multiplex PCR amplification results of the BVDV reference strain Oregon CV24 strain (BVDV-1 type) cDNA, and a 309.58 bp DNA fragment was amplified.
  • Figure 1E shows the multiplex PCR amplification of the BRV reference strain NCDV cDNA, and a 211.71 bp DNA fragment was amplified.
  • Figure 1F shows the multiplex PCR amplification of the PPRV vaccine strain cDNA, and a 344.20 bp DNA fragment was amplified.
  • Fig. 1D shows the multiplex PCR amplification results of the BVDV reference strain Oregon CV24 strain (BVDV-1 type) cDNA, and a 309.58 bp DNA fragment was amplified.
  • Figure 1E shows the multiplex PCR amplification of the
  • FIG. 1G shows the multiplex PCR amplification results of the ECX strain GX-ETEC1 genomic DNA, and a 252.24 bp DNA fragment was amplified.
  • Figure 1H shows the results of multiplex PCR amplification of IBRV virus genomic DNA, and a 188.21 bp DNA fragment was amplified. Only one specific single peak appeared in each reaction, and there were no other signal peaks, and the fragment size was consistent with the criterion. None of the negative controls were amplified and there was no peak of the target signal.
  • RNA of the sample to be tested extracts the total RNA of the sample to be tested and reverse-transcribe into cDNA.
  • the samples to be tested were: FMDV O-type inactivated virus, BTV type 4 inactivated virus, VSV NJ type inactivated virus, BVDV reference strain Oregon CV24 strain (BVDV-1 type), BRV reference strain NCDV, PPRV vaccine strain .
  • the samples to be tested were ETEC reference strain 1676 and IBRV virus, respectively.
  • Multiplex PCR reaction system (20 ⁇ L): template 1 ⁇ L, Genome Lab GeXP Starter Kit 5 ⁇ buffer 4 ⁇ L (buffer contains universal primers, universal primers are shown by primer A shown in sequence 25 of the sequence listing and primers shown in sequence 26 of the sequence listing) a B composition in which the primer A has a label of a CY5 fluorophore at the 5' end, a primer A and a primer B have a working concentration of 0.25 ⁇ M, a MgC12 (25 ⁇ M) 4 ⁇ L, and a primer mixture containing all the primers in the primer combination, 1 ⁇ L. DNA polymerase 10U, supplemented to 20 ⁇ L with ultrapure water.
  • the concentrations of FMDV-F and FMDV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of BTV-F and BTV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of VSV-F and VSV-R were both.
  • the concentration of BVDV-F and BVDV-R were both 2 ⁇ mol/ ⁇ L
  • the concentrations of BRV-F and BRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of ETEC-F and ETEC-R were both 0.2 ⁇ mol/
  • the concentrations of ⁇ L, IBRV-F and IBRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of PPRV-F and PPRV-R were both 2 ⁇ mol/ ⁇ L.
  • a negative control with equal volume of water as a template was set.
  • the cDNA of the FMDV O-type inactivated virus in the 1 ⁇ L template is about 100 ng
  • the cDNA of the inactivated virus of the BTV type 4 is about 100 ng
  • the cDNA of the inactivated virus of the VSVNJ type is about 100 ng
  • the reference strain of the BVDV is the Oregon CV24 strain (BVDV-1 type).
  • the cDNA is about 100 ng
  • the genomic DNA of the BRV reference strain NCDV is about 100 ng
  • the genomic DNA of the PPRV vaccine strain is about 100 ng
  • the genomic DNA of the ECTC strain GX-ETEC1 is about 100 ng
  • the genomic DNA of the IBRV virus is about 100 ng.
  • Multiplex PCR reaction procedure pre-denaturation at 95 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 94 ° C for 30 seconds, 68 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles ; 94 ° C 30 seconds, 50 ° C 30 seconds, 72 ° C 30 seconds, 10 cycles; 72 ° C extension 5 minutes, end the opposite should.
  • Capillary electrophoresis of the multiplex PCR amplification product of step 4 the specific steps are: mixing 3 ⁇ L of multiplex PCR amplification product, 38.75 ⁇ L sample buffer and 0.25 ⁇ L DNA size standard kit-400 Base Pairs, and adding the sample plate. In the middle, add 1 drop of paraffin to each well to block the liquid surface to avoid the oxidation of formamide and the evaporation of the sample. 180 ⁇ L of sample buffer was added to each well of the buffer plate for capillary electrophoresis. Capillary electrophoresis conditions: 90 ° C for 120 seconds, denaturation; 2.0 KV for 30 seconds, inhalation of the sample; 6.0 KV for 35 minutes, separation of the sample.
  • the PCR products of different size fragments are separated by electrophoresis, and the instrument recognizes the fragment size and signal intensity by detecting the fluorescent group carried by the PCR product. After the electrophoresis is completed, the results are analyzed using the instrument's own software, Express Profiler software.
  • the judgment criteria are: the size of the amplified fragments of the eight bovine pathogens are FMDV: 165-167 bp, BTV: 135-137 bp, VSV: 278-281 bp, BVDV: 308-310 bp, BRV: 211 -214 bp, PPRV: 342-345 bp, ETEC: 252-254 bp, IBRV: 187-189 bp.
  • the test results are shown in Figure 2.
  • the abscissa indicates the fragment size (unit: bp), and the ordinate indicates the signal intensity, that is, the content of the PCR amplification product.
  • the results showed that 8 signal peaks corresponding to 8 pathogens could be detected simultaneously using GeXP multiplex PCR, FMDV: 166.39 bp, BTV: 136.17 bp, VSV: 280.40 bp, BVDV: 309.59 bp, BRV: 213.71 bp, PPRV: 342.16 bp. , ETEC: 253.23 bp, IBRV: 187.25 bp, no other peaks. None of the negative controls were amplified and there was no peak of the target signal.
  • the samples to be tested are: FMDV O-type inactivated virus, FMDV type A inactivated virus, FMDV Asia type I inactivated virus, BTV type 4 inactivated virus, BTV type 8 inactivated virus, BTV type 9 inactivated virus, BTV Type 15 inactivated virus, BTV17 inactivated virus, BTV type 18 inactivated virus, VSVNJ type inactivated virus, VSV IND type inactivated virus, BVDV reference strain Oregon CV24 strain (BVDV-1 type), BVDV reference strain NADL strain (BVDV-1 type), BVDV reference strain yak strain (BVDV-1 type), BVDV strain GX-BVDV1, BVDV strain GX-BVDV2, BVDV strain GX-BVDV3, BVDV strain GX-BVDV4, BVDV strain GX-BVDV5, BVDV strain GX-BVDV6, BVDV strain GX-BVDV7, BVDV strain GX-BVDV8,
  • the samples to be tested were: ETEC reference strain 1676, ETEC reference strain 1751, ETEC reference strain B41, and IBRV virus.
  • the GeXP multiplex PCR was carried out using the primer obtained in Example 1 using the cDNA obtained in the step 1 and the genomic DNA obtained in the step 2 as a template.
  • Multiplex PCR reaction system (20 ⁇ L): template 1 ⁇ L, Genome Lab GeXP Starter Kit 5 ⁇ buffer 4 ⁇ L (buffer contains universal primers, universal primers are shown by primer A shown in sequence 25 of the sequence listing and primers shown in sequence 26 of the sequence listing)
  • Composition B in which the 5' end of primer A has a label of CY5 fluorophore, primer A and primer B each have a working concentration of 0.25 ⁇ M), MgCl 2 (25 ⁇ M) 4 ⁇ L, and a primer mixture containing all primers in the primer combination 1 ⁇ L , DNA polymerase 10U, supplemented to 20 ⁇ L with ultrapure water.
  • the concentrations of FMDV-F and FMDV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of BTV-F and BTV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of VSV-F and VSV-R were both.
  • the concentration of BVDV-F and BVDV-R were both 2 ⁇ mol/ ⁇ L
  • the concentrations of BRV-F and BRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of ETEC-F and ETEC-R were both 0.2 ⁇ mol/
  • the concentrations of ⁇ L, IBRV-F and IBRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of PPRV-F and PPRV-R were both 2 ⁇ mol/ ⁇ L.
  • a negative control with equal volume of water as a template was set.
  • the DNA content in the 1 ⁇ L template is about 100 ng;
  • the DNA content in the 1 ⁇ L template is about 100 ng;
  • Multiplex PCR reaction procedure pre-denaturation at 95 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 94 ° C for 30 seconds, 68 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 72 ° C extension for 5 min, the reaction was terminated.
  • Capillary electrophoresis of the multiplex PCR amplification product of step 3 the specific steps are: 3 ⁇ L multiplex PCR amplification product, 38.75 ⁇ L sample buffer and 0.25 ⁇ L DNA size standard kit-400Base Pairs vortex and mix, add to the sample plate, add 1 drop of paraffin to each surface to avoid the formamide oxidation and sample evaporation. . 180 ⁇ L of sample buffer was added to each well of the buffer plate for capillary electrophoresis. Capillary electrophoresis conditions: 90 ° C for 120 seconds, denaturation; 2.0 KV for 30 seconds, inhalation of the sample; 6.0 KV for 35 minutes, separation of the sample.
  • the PCR products of different size fragments are separated by electrophoresis, and the instrument recognizes the fragment size and signal intensity by detecting the fluorescent group carried by the PCR product. After the electrophoresis is completed, the results are analyzed using the instrument's own software, Express Profiler software.
  • the judgment criteria are: the size of the amplified fragments of the eight bovine pathogens are FMDV: 165-167 bp, BTV: 135-137 bp, VSV: 278-281 bp, BVDV: 308-310 bp, BRV: 211 -214 bp, PPRV: 342-345 bp, ETEC: 252-254 bp, IBRV: 187-189 bp. Due to the error of the GeXP system itself, the deviation of the amplified fragment size from the theoretical value of ⁇ 2bp is a correct result.
  • Example 1 The primers of Example 1 were used for multiplex PCR amplification, and each sample showed only a specific single peak corresponding to the pathogen, no other signal peaks, and the fragment size was consistent with the judgment standard, and the results showed that the design of Example 1 was Primer combinations are generally applicable to 8 bovine pathogens.
  • the specific fragment designed by the present invention was used to amplify the target fragment of each strain, and the obtained target fragment was ligated with the pEASY-T1 vector to construct a recombinant plasmid of each target fragment of GeXP (double-stranded DNA molecule corresponding to sequence 17 of the sequence listing)
  • the recombinant plasmid pEASY-T1-FMDV was obtained by ligation with the pEASY-T1 vector; the double-stranded DNA molecule corresponding to the sequence 18 of the sequence listing was ligated to the pEASY-T1 vector to obtain the recombinant plasmid pEASY-T1-BTV;
  • the corresponding double-stranded DNA molecule is ligated to the pEASY-T1 vector to obtain the recombinant plasmid pEASY-T1-VSV; the double-stranded DNA molecule corresponding to the sequence 20 of the sequence listing is
  • pEASY-T1-PPRV the double-stranded DNA molecule corresponding to the sequence 23 of the sequence listing was ligated to the pEASY-T1 vector to obtain the recombinant plasmid pEASY-T1-ETEC; the double-stranded DNA molecule corresponding to the sequence 24 of the sequence listing and pEASY-T1 Vector ligation, resulting in recombinant plasmid pEASY-T1-IBRV), positive clone plasmid was extracted.
  • the pEASY-T1-IBRV and pEASY-T1-ETEC plasmid concentrations were determined by a NanoDrop UV spectrophotometer.
  • the positive clone plasmids extracted from the T7 in vitro transcription kit instructions (pEASY-T1-BTV, pEASY-T1-FMDV, pEASY-T1-BRV, pEASY-T1-VSV, pEASY-T1-BVDV and pEASY-T1--PPRV)
  • In vitro transcription into RNA determination of RNA concentration by NanoDrop, calculation of copy number of each plasmid as a copy number according to molecular weight and concentration, and dilution of each plasmid to 10 8 copies / ⁇ l ⁇ 1copies / ⁇ l, to obtain FMDV standard, BTV standard, VSV standard, BVDV standard, BRV standard, PPRV standard, ETEC standard and IBRV standard, kept at -70 °C.
  • the mixture obtained in the step 2 was diluted 10 times with ddH 2 O to obtain each dilution.
  • the GeXP multiplex PCR was carried out using the primer combination prepared in Example 1.
  • Multiplex PCR reaction system (20 ⁇ L): template 1 ⁇ L, Genome Lab GeXP Starter Kit 5 ⁇ buffer 4 ⁇ L (buffer contains universal primers, universal primers are shown by primer A shown in sequence 25 of the sequence listing and primers shown in sequence 26 of the sequence listing) a B composition in which the 5' end of the primer A has a label of a CY5 fluorescent group, the working concentrations of the primer A and the primer B are both 0.25 ⁇ M), MgCl 2 (25 ⁇ M) 4 ⁇ L, and a primer mixture containing all the primers in the primer combination, 1 ⁇ L, DNA polymerase 10U, supplemented to 20 ⁇ L with ultrapure water.
  • the concentrations of FMDV-F and FMDV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of BTV-F and BTV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of VSV-F and VSV-R were both.
  • the concentration of BVDV-F and BVDV-R were both 2 ⁇ mol/ ⁇ L
  • the concentrations of BRV-F and BRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of ETEC-F and ETEC-R were both 0.2 ⁇ mol/
  • the concentrations of ⁇ L, IBRV-F and IBRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of PPRV-F and PPRV-R were both 2 ⁇ mol/ ⁇ L.
  • a negative control with equal volume of water as a template was set.
  • the initial concentrations of FMDV standard, BTV standard, VSV standard, BVDV standard, BRV standard, ETEC standard, IBRV standard and PPRV standard are all 10 6 copies/ ⁇ L;
  • the initial concentrations of FMDV standard, BTV standard, VSV standard, BVDV standard, BRV standard, ETEC standard, IBRV standard and PPRV standard are all 10 5 copies/ ⁇ L;
  • the initial concentrations of FMDV standard, BTV standard, VSV standard, BVDV standard, BRV standard, ETEC standard, IBRV standard and PPRV standard are all 10 4 copies/ ⁇ L;
  • the initial concentrations of FMDV standards, BTV standards, VSV standards, BVDV standards, BRV standards, ETEC standards, IBRV standards, and PPRV standards are all 10 3 copies/ ⁇ L;
  • the initial concentrations of the FMDV standard, the BTV standard, the VSV standard, the BVDV standard, the BRV standard, the ETEC standard, the IBRV standard, and the PPRV standard are all 10 2 copies/ ⁇ L;
  • reaction system 6 the initial concentrations of FMDV standard, BTV standard, VSV standard, BVDV standard, BRV standard, ETEC standard, IBRV standard and PPRV standard are all 10 copies/ ⁇ L;
  • the initial concentrations of the FMDV standard, the BTV standard, the VSV standard, the BVDV standard, the BRV standard, the ETEC standard, the IBRV standard, and the PPRV standard were all 1 copy/ ⁇ L.
  • Multiplex PCR reaction procedure pre-denaturation at 95 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 94 ° C for 30 seconds, 68 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 72 ° C extension for 5 min, the reaction was terminated.
  • Capillary electrophoresis of the multiplex PCR amplification product of step 3 the specific steps are: mixing 3 ⁇ L of multiplex PCR amplification product, 38.75 ⁇ L sample buffer and 0.25 ⁇ L DNA size standard kit-400 Base Pairs, and adding the sample plate. In the middle, add 1 drop of paraffin to each well to block the liquid surface to avoid the oxidation of formamide and the evaporation of the sample. 180 ⁇ L of sample buffer was added to each well of the buffer plate for capillary electrophoresis. Capillary electrophoresis conditions: 90 ° C for 120 seconds, denaturation; 2.0 KV for 30 seconds, inhalation of the sample; 6.0 KV for 35 minutes, separation of the sample.
  • the instrument recognizes fragment size and signal intensity by detecting the fluorescent group carried by the PCR product. After the electrophoresis is completed, the results are analyzed using the instrument's own software, Express Profiler software.
  • the judgment criteria are: the size of the amplified fragments of the eight bovine pathogens are FMDV: 165-167 bp, BTV: 135-137 bp, VSV: 278-281 bp, BVDV: 308-310 bp, BRV: 211 -214 bp, PPRV: 342-345 bp, ETEC: 252-254 bp, IBRV: 187-189 bp. Due to the error of the GeXP system itself, the deviation of the amplified fragment size from the theoretical value of ⁇ 2bp is a correct result.
  • test results are shown in Figure 3.
  • 3A to 3E sequentially correspond to amplification results of multiplex PCR using the reaction system 1-5.
  • the abscissa indicates the fragment size (unit: bp), and the ordinate indicates the signal intensity, that is, the content of the PCR amplification product.
  • the results showed that when the concentration of the DNA to be tested in the detection system was as low as 100 copies/ ⁇ L, eight pathogens could also be detected.
  • the IBRV standard, the BRV standard, the ETEC standard, the BVDV standard, and the PPRV standard prepared in Example 4 were mixed to obtain a mixed solution A.
  • the FMDV standard, the BTV standard, the IBRV standard, the BRV standard, and the ETEC standard prepared in Example 4 were mixed to obtain a mixed solution B.
  • GeXP multiplex PCR was carried out using the primer combination prepared in Example 1 using the mixture A obtained in the step 1 and the mixture B obtained in the step 2 as a template.
  • Multiplex PCR reaction system (20 ⁇ L): template 1 ⁇ L, Genome Lab GeXP Starter Kit 5 ⁇ buffer 4 ⁇ L (buffer contains universal primers, universal primers are shown by primer A shown in sequence 25 of the sequence listing and primers shown in sequence 26 of the sequence listing) a B composition in which the 5' end of the primer A has a label of a CY5 fluorescent group, the working concentrations of the primer A and the primer B are both 0.25 ⁇ M), MgCl 2 (25 ⁇ M) 4 ⁇ L, and a primer mixture containing all the primers in the primer combination, 1 ⁇ L, DNA polymerase 10U, supplemented to 20 ⁇ L with ultrapure water.
  • the concentrations of FMDV-F and FMDV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of BTV-F and BTV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of VSV-F and VSV-R were both.
  • the concentration of BVDV-F and BVDV-R were both 2 ⁇ mol/ ⁇ L
  • the concentrations of BRV-F and BRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of ETEC-F and ETEC-R were both 0.2 ⁇ mol/
  • the concentrations of ⁇ L, IBRV-F and IBRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of PPRV-F and PPRV-R were both 2 ⁇ mol/ ⁇ L.
  • the concentration of the IBRV standard is 10 3 copies/ ⁇ L in the reaction system
  • the concentration of the BRV standard is 10 5 copies/ ⁇ L
  • the concentration of the ETEC standard is 10 5 copies/ ⁇ L
  • the BVDV standard concentration of the product was 105 copies / ⁇ L
  • a concentration of the standard PPRV 107 copies / ⁇ L is 10 3 copies/ ⁇ L in the reaction system
  • the concentration of the FMDV standard is 10 4 copies/ ⁇ L in the reaction system
  • the concentration of the BTV standard is 10 8 copies/ ⁇ L
  • the concentration of the IBRV standard is 10 4 copies/ ⁇ L
  • the BRV standard concentration of the product was 105 copies / ⁇ L
  • a concentration of the standard ETEC 105 copies / ⁇ L is 10 4 copies/ ⁇ L in the reaction system
  • Multiplex PCR reaction procedure pre-denaturation at 95 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 94 ° C for 30 seconds, 68 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 72 ° C extension for 5 min, the reaction was terminated.
  • Capillary electrophoresis of the multiplex PCR amplification product of step 3 the specific steps are: mixing 3 ⁇ L of multiplex PCR amplification product, 38.75 ⁇ L sample buffer and 0.25 ⁇ L DNA size standard kit-400 Base Pairs, and adding the sample plate. In the middle, add 1 drop of paraffin to each well to block the liquid surface to avoid the oxidation of formamide and the evaporation of the sample. 180 ⁇ L of sample buffer was added to each well of the buffer plate for capillary electrophoresis. Capillary electrophoresis conditions: 90 ° C for 120 seconds, denaturation; 2.0 KV for 30 seconds, inhalation of the sample; 6.0 KV for 35 minutes, separation of the sample.
  • the PCR products of different size fragments are separated by electrophoresis, and the instrument recognizes the fragment size and signal intensity by detecting the fluorescent group carried by the PCR product. After the electrophoresis is completed, the results are analyzed using the instrument's own software, Express Profiler software.
  • the judgment criteria are: the size of the amplified fragments of the eight bovine pathogens are FMDV: 165-167 bp, BTV: 135-137 bp, VSV: 278-281 bp, BVDV: 308-310 bp, BRV: 211 -214 bp, PPRV: 342-345 bp, ETEC: 252-254 bp, IBRV: 187-189 bp. Due to the error of the GeXP system itself, the deviation of the amplified fragment size from the theoretical value of ⁇ 2bp is a correct result.
  • FIG. 4 shows the results of amplification of multiplex PCR using the mixture A as a template
  • FIG. 4B shows the amplification results of multiplex PCR using the mixture B as a template. The results show that when the concentration of the starting template in the reaction system is very large, the template with low concentration can be detected accurately and sensitively, and the interference is small.
  • the samples to be tested were: 305 clinical samples, including 156 stool swabs, 30 eye tests, 30 nasal mucus swabs, 70 anticoagulation, and 2 OP fluids (esophage-pharyngeal secretions). 2 parts of vesicular fluid, 15 tissue samples (10 parts of rectal mucosa, 2 parts of blister, 3 parts of lymph nodes). Clinical samples were collected from Guangxi in 2012-2014. About 1/2 of the samples were from asymptomatic dairy cows in dairy farms. About one-fourth of the samples were from cattle with clinical symptoms such as diarrhea, weight loss and runny nose. The samples were derived from cows with mental discomfort, difficulty swallowing, fever, blisters in the mouth, and white nose and nose.
  • the DNA/cDNA mixed solution obtained in the step 2 was used as a template, and the GeXP multiplex PCR was carried out using the primer combination prepared in Example 1.
  • Multiplex PCR reaction system (20 ⁇ L): template 1 ⁇ L (DNA content 10-100 ng), Genome Lab GeXP Starter Kit 5 ⁇ buffer 4 ⁇ L (buffer contains universal primers, universal primers are shown by sequence 25 of sequence listing primer A and Primer B consisting of sequence 26 of the Sequence Listing, wherein primer 5 has a CY5 fluorophore label at the 5' end, primer A and primer B each have a working concentration of 0.25 ⁇ M, and MgCl 2 (25 ⁇ M) 4 ⁇ L contains primers. 1 ⁇ L of the primer mixture of all the primers in the combination, DNA polymerase 10 U, was made up to 20 ⁇ L with ultrapure water.
  • the concentrations of FMDV-F and FMDV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of BTV-F and BTV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of VSV-F and VSV-R were both.
  • the concentration of BVDV-F and BVDV-R were both 2 ⁇ mol/ ⁇ L
  • the concentrations of BRV-F and BRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of ETEC-F and ETEC-R were both 0.2 ⁇ mol/
  • the concentrations of ⁇ L, IBRV-F and IBRV-R were both 0.2 ⁇ mol/ ⁇ L
  • the concentrations of PPRV-F and PPRV-R were both 2 ⁇ mol/ ⁇ L.
  • a negative control with equal volume of water as a template was set.
  • Multiplex PCR reaction procedure pre-denaturation at 95 ° C for 5 minutes; 94 ° C for 30 seconds, 55 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 94 ° C for 30 seconds, 68 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles 94 ° C for 30 seconds, 50 ° C for 30 seconds, 72 ° C for 30 seconds, 10 cycles; 72 ° C extension for 5 min, the reaction was terminated.
  • Capillary electrophoresis of the multiplex PCR amplification product of step 3 the specific steps are: 3 ⁇ L multiplex PCR amplification product, 38.75 ⁇ L sample buffer and 0.25 ⁇ L DNA size standard kit-400Base Pairs vortex and mix, add to the sample plate, add 1 drop of paraffin to each surface to avoid the formamide oxidation and sample evaporation. . 180 ⁇ L of sample buffer was added to each well of the buffer plate for capillary electrophoresis. Capillary electrophoresis conditions: 90 ° C for 120 seconds, denaturation; 2.0 KV for 30 seconds, inhalation of the sample; 6.0 KV for 35 minutes, separation of the sample.
  • the PCR products of different size fragments are separated by electrophoresis, and the instrument recognizes the fragment size and signal intensity by detecting the fluorescent group carried by the PCR product. After the electrophoresis is completed, the results are analyzed using the instrument's own software, Express Profiler software.
  • the judgment criteria are: the size of the amplified fragments of the eight bovine pathogens are FMDV: 165-167 bp, BTV: 135-137 bp, VSV: 278-281 bp, BVDV: 308-310 bp, BRV: 211 -214 bp, PPRV: 342-345 bp, ETEC: 252-254 bp, IBRV: 187-189 bp. Due to the error of the GeXP system itself, the deviation of the amplified fragment size from the theoretical value of ⁇ 2bp is a correct result.
  • step 4 The positive amplification product obtained in step 4 was sequenced to verify the correctness of the results.

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Abstract

本发明公开了一种同时鉴别8种牛病原体的引物组合及GeXP检测方法。本发明的引物组合由引物对Ⅰ、引物对Ⅱ、引物对Ⅲ、引物对Ⅳ、引物对Ⅴ、引物对Ⅵ、引物对Ⅶ和引物对Ⅷ组成。本发明还提供了同时鉴别口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒和小反刍兽疫病毒的GeXP检测方法。

Description

一种同时鉴别8种牛病原体的引物组合及GeXP检测方法
交叉引用
本发明要求广西壮族自治区兽医研究所向中国专利局提交的、申请号为CN201610569423.4、发明名称为“一种同时鉴别8种牛病原体的引物组合及GeXP检测方法”的中国专利申请的优先权，该申请的全部内容通过引用结合在本发明中。
技术领域
本发明涉及一种同时鉴别8种牛病原体的引物组合及GeXP检测方法。
背景技术
目前我国现有牛存栏头数1.38亿头，牛肉产量达到675.9万吨，是世界第四大牛肉生产国。近年来广西养牛业发展也十分迅速，拥有全国第5位的牛存栏量，其中水牛存栏数达到450万头，占全国水牛总数的1/5，居全国之首，世界第二。随着养牛业的发展，牛传染病的发病率也在逐年升高，已经成为制约养牛业发展的一大重要因素，具体表现为：老病仍在流行，并且病原出现了新的血清型或变异株，新疾病陆续出现，给牛病的防控带来困难。每年大量牛因疾病死亡，造成了严重的经济损失，据统计2015年全年国内肉牛的因传染病全程死亡率高达5％，导致的直接经济损失高达90-150亿元，牛传染病严重地影响了我国肉类及其制品进入国际市场。口蹄疫病毒(Foot and Mouth Disease Virus，FMDV)、蓝舌病病毒(Bluetongue Virus，BTV)、水泡性口炎病毒(Vesicular Stomatitis Virus，VSV)、牛病毒性腹泻病毒(Bovine Viral Diarrheal Virus，BVDV)、牛轮状病毒(Bovine Rotavirus，BRV)、产肠毒大肠杆菌(Enterotoxigenic E.coli，ETEC)、牛传染性鼻气管炎病毒(Infectious Bovine Rhinotracheitis Virus，IBRV)和小反刍兽疫病毒(Peste des Petits Ruminants Virus，PPRV)是严重危害养牛业的8种主要传染性疾病的病原体，这些病原体的存在，时时刻刻危害着养牛业的发展。由FMDV引发 的牛口蹄疫、由VSV引发的牛水泡性口炎和由BTV引发的牛蓝舌病是牛的高度急性传染病，一般呈暴发性流行，临床上在口腔，蹄部及乳房出现病变，症状十分相似难以区分，死亡率高，被世界动物卫生组织(OIE)列为A类传染病。BVDV，BRV和ETEC也是引起牛腹泻的主要病原体，且BVDV以持续性感染无症状形式存在，牛群中相当一部分牛是这些病原体的携带者，并且常随着其它病暴发，患病牛的症状为急性水样腹泻，急速消瘦。IBRV属于免疫抑制性疾病病毒，感染机体后还可继发细菌感染，据调查不仅各品种的牛均有感染的报道，而且感染地区遍布全国各省市，感染率居高。由PPRV引发的小反刍兽疫病也是近年来出现在的外来新病，OIE将其列为必须报告的动物疫病，在我国列为一类动物疫病。这些疾病是养牛业的巨大潜患，一旦暴发，将会造成巨大的经济损失，因此开展牛传染病的快速检测技术的研究势在必行。
快速准确地检测诊断传染病是进行有效防控的前提和基础。目前用于鉴别诊断这些牛传染病的主要方法有病原分离鉴定和血清学试验等，但这些方法常受临床病料新鲜度、污染程度或血清效价的限制，导致结果有误，且耗时耗力，在实际应用中有一定的局限性。随着分子生物学的进步，以PCR技术为基础发展起来的分子生物学诊断方法已广泛应用于传染病的检测诊断，包括PCR、荧光PCR和LAMP等，但这些方法只能对单一或2至4种病原体进行检测，无法实现真正意义上的同时对多种病原体实行高通量检测。GeXP多基因表达分析系统是一种新型的高通量的基因检测技术，将多重PCR技术和毛细管电泳技术有效的结合起来，采用荧光标记通用引物和特异性嵌合引物(即基因特异性引物5’端连接通用引物)相结合从而引发多重PCR体系的扩增，可同时对多达30个目的基因进行有效检测分析，实现真正意义上的高通量检测鉴别多种病原体的目的。
发明内容
本发明的目的是提供一种同时鉴别8种牛病原体的引物组合及GeXP检测方法。
本发明提供了一种引物组合，由引物对I、引物对II、引物对III、引物 对IV、引物对V、引物对VI、引物对VII和引物对VIII组成；
所述引物对I由引物FMDV-F和引物FMDV-R组成；
所述引物FMDV-F为如下(a1)或(a2)或(a3)：
(a1)序列表的序列1所示的单链DNA分子；
(a2)序列表的序列1自5′末端第19至36位核苷酸所示的单链DNA分子；
(a3)将(a1)或(a2)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a1)或(a2)相同功能的DNA分子；
所述引物FMDV-R为如下(a4)或(a5)或(a6)：
(a4)序列表的序列2所示的单链DNA分子；
(a5)序列表的序列2自5′末端第20至43位核苷酸所示的单链DNA分子；
(a6)将(a4)或(a5)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a4)或(a5)相同功能的DNA分子；
所述引物对II由引物BTV-F和引物BTV-R组成；
所述引物BTV-F为如下(a7)或(a8)或(a9)：
(a7)序列表的序列3所示的单链DNA分子；
(a8)序列表的序列3自5′末端第19至41位核苷酸所示的单链DNA分子；
(a9)将(a7)或(a8)经过一个或几个核苷酸的取代和/或缺失和/或添加且与(a7)或(a8)具有相同功能的DNA分子；
所述引物BTV-R为如下(a10)或(a11)或(a12)：
(a10)序列表的序列4所示的单链DNA分子；
(a11)序列表的序列4自5′末端第20至37位核苷酸所示的单链DNA分子；
(a12)将(a10)或(a11)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a10)或(a11)相同功能的DNA分子；
所述引物对III由引物VSV-F和引物VSV-R组成；
所述引物VSV-F为如下(a13)或(a14)或(a15)：
(a13)序列表的序列5所示的单链DNA分子；
(a14)序列表的序列5自5′末端第19至38位核苷酸所示的单链DNA分子；
(a15)将(a13)或(a14)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a13)或(a14)相同功能的DNA分子；
所述引物VSV-R为如下(a16)或(a17)或(a18)：
(a16)序列表的序列6所示的单链DNA分子；
(a17)序列表的序列6自5′末端第20至38位核苷酸所示的单链DNA分子；
(a18)将(a16)或(a17)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a16)或(a17)相同功能的DNA分子；
所述引物对IV由引物BVDV-F和引物BVDV-R组成；
所述引物BVDV-F为如下(a19)或(a20)或(a21)：
(a19)序列表的序列7所示的单链DNA分子；
(a20)序列表的序列7自5′末端第19至36位核苷酸所示的单链DNA分子；
(a21)将(a19)或(a20)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a19)或(a20)相同功能的DNA分子；
所述引物BVDV-R为如下(a22)或(a23)或(a24)：
(a22)序列表的序列8所示的单链DNA分子；
(a23)序列表的序列8自5′末端第20至44位核苷酸所示的单链DNA分子；
(a24)将(a22)或(a23)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a22)或(a23)相同功能的DNA分子；
所述引物对V由引物BRV-F和引物BRV-R组成；
所述引物BRV-F为如下(a25)或(a26)或(a27)：
(a25)序列表的序列9所示的单链DNA分子；
(a26)序列表的序列9自5′末端第19至40位核苷酸所示的单链DNA分子；
(a27)将(a25)或(a26)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a25)或(a26)相同功能的DNA分子；
所述引物BRV-R为如下(a28)或(a29)或(a30)：
(a28)序列表的序列10所示的单链DNA分子；
(a29)序列表的序列10自5′末端第20至37位核苷酸所示的单链DNA分子；
(a30)将(a28)或(a29)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a28)或(a29)相同功能的DNA分子；
所述引物对VI由引物ETEC-F和引物ETEC-R组成：
所述引物ETEC-F为如下(a31)或(a32)或(a33)：
(a31)序列表的序列11所示的单链DNA分子；
(a32)序列表的序列11自5′末端第19至36位核苷酸所示的单链DNA分子；
(a33)将(a31)或(a32)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a31)或(a32)相同功能的DNA分子；
所述引物ETEC-R为如下(a34)或(a35)或(a36)：
(a34)序列表的序列12所示的单链DNA分子；
(a35)序列表的序列12自5′末端第20至40位核苷酸所示的单链DNA分子；
(a36)将(a34)或(a35)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a34)或(a35)相同功能的DNA分子；
所述引物对VII由引物IBRV-F和引物IBRV-R组成；
所述引物IBRV-F为如下(a37)或(a38)或(a39)：
(a37)序列表的序列13所示的单链DNA分子；
(a38)序列表的序列13自5′末端第19至41位核苷酸所示的单链DNA分子；
(a39)将(a37)或(a38)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a37)或(a38)相同功能的DNA分子；
所述引物IBRV-R为如下(a40)或(a41)或(a42)：
(a40)序列表的序列14所示的单链DNA分子；
(a41)序列表的序列14自5′末端第20至36位核苷酸所示的单链DNA分子；
(a42)将(a40)或(a41)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a40)或(a41)相同功能的DNA分子；
所述引物对VIII由引物PPRV-F和引物PPRV-R组成：
所述引物PPRV-F为如下(a43)或(a44)或(a45)：
(a43)序列表的序列15所示的单链DNA分子；
(a44)序列表的序列15自5′末端第19至44位核苷酸所示的单链DNA分子；
(a45)将(a43)或(a44)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a43)或(a44)相同功能的DNA分子；
所述引物PPRV-R为如下(a46)或(a47)或(a48)：
(a46)序列表的序列16所示的单链DNA分子；
(a47)序列表的序列16自5′末端第20至36位核苷酸所示的单链DNA分子；
(a48)将(a46)或(a47)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a46)或(a47)相同功能的DNA分子。
所述引物组合的用途为如下(b1)至(b6)中的任意一种：
(b1)鉴别8种牛病原体；
(b2)制备用于鉴别8种牛病原体的试剂盒；
(b3)检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒；
(b4)制备用于检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒的试剂盒；
(b5)检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒；
(b6)制备用于检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒的试剂盒。
本发明还保护所述引物组合的应用，为如下(b1)至(b6)中的任意一种：
(b1)鉴别8种牛病原体；
(b2)制备用于鉴别8种牛病原体的试剂盒；
(b3)检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒；
(b4)制备用于检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒的试剂盒；
(b5)检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒；
(b6)制备用于检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒的试剂盒。
本发明还保护含有所述引物组合的试剂盒；所述试剂盒的用途为如下(c1)或(c2)或(c3)：
(c1)鉴别8种牛病原体；
(c2)检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒；
(c3)检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒。
本发明还保护所述试剂盒的制备方法，包括将各条引物单独包装的步骤。
本发明还保护一种鉴别8种牛病原体的方法，包括如下步骤(d1)或(d2)：
(d1)将待测病原体进行基因组DNA提取操作和/或RNA提取操作(DNA提取操作和RNA提取操作可在同一体系中进行，也可分别进行后混合)，得到核酸；将待测病原体的含有RNA或可能含有RNA的核酸进行反转录；以得到的DNA和/或cDNA作为模板，采用所述引物组合进行PCR扩增(具体可进行GeXP多重PCR扩增；扩增产物可进行毛细管电泳检测)，如果扩增产物含有165-167bp的DNA片段、待测病原体为或候选为口蹄疫病毒，如果扩增产物含有135-137bp的DNA片段、待测病原体为或候选为蓝舌病病毒，如果扩增产物含有278-281bp的DNA片段、待测病原体为或候选为水泡性口炎病毒，如果扩增产物含有308-310bp的DNA片段、待测病原体为或候选为牛病毒性腹泻病毒，如果扩增产物含有211-214bp的DNA片段、待测病原体为或候选为牛轮状病毒，如果扩增产物含有342-345bp的DNA片 段、待测病原体为或候选为小反刍兽疫病毒，如果扩增产物含有252-254bp的DNA片段、待测病原体为或候选为产肠毒素大肠杆菌，如果扩增产物含有187-189bp的DNA片段、待测病原体为或候选为牛传染性鼻气管炎病毒；
(d2)检测待病原体的基因组DNA或cDNA中是否含有所述引物对I的靶序列、所述引物对II的靶序列、所述引物对III的靶序列、所述引物对IV的靶序列、所述引物对V的靶序列、所述引物对VI的靶序列、所述引物对VII的靶序列或所述引物对VIII的靶序列，如果所述cDNA中含有所述引物对I的靶序列、待测病原体为或候选为口蹄疫病毒，如果所述cDNA中含有所述引物对II的靶序列、待测病原体为或候选为蓝舌病病毒，如果所述cDNA中含有所述引物对III的靶序列、待测病原体为或候选为水泡性口炎病毒，如果所述cDNA中含有所述引物对IV的靶序列、待测病原体为或候选为牛病毒性腹泻病毒，如果所述cDNA中含有所述引物对V的靶序列、待测病原体为或候选为牛轮状病毒，如果所述基因组DNA中含有所述引物对VI的靶序列、待测病原体为或候选为产肠毒素大肠杆菌，如果所述cDNA中含有所述引物对VII的靶序列、待测病原体为或候选为牛传染性鼻气管炎病毒，如果所述cDNA中含有所述引物对VIII的靶序列、待测病原体为或候选为小反刍兽疫病毒。
本发明还保护一种检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒的方法，包括如下步骤(e1)或(e2)：
(e1)将待测病原体进行基因组DNA提取操作和/或RNA提取操作(DNA提取操作和RNA提取操作可在同一体系中进行，也可分别进行后混合)，得到核酸；将待测病原体的含有RNA或可能含有RNA的核酸进行反转录；以得到的DNA和/或cDNA作为模板，采用所述引物组合进行PCR扩增(具体可进行GeXP多重PCR扩增；扩增产物可进行毛细管电泳检测)，如果扩增产物含有165-167bp的DNA片段、待测病原体为或候选为口蹄疫病毒，如果扩增产物含有135-137bp的DNA片段、待测病原体为或候选为蓝舌病病毒，如果扩增产物含有278-281bp的DNA片段、待测病原体为或候选为水泡性口炎病毒，如果扩增产物含有308-310bp的DNA片段、待测病原体为或候选为牛病毒性腹泻病毒，如果扩增产物含有211-214bp的DNA片段、待测病 原体为或候选为牛轮状病毒，如果扩增产物含有342-345bp的DNA片段、待测病原体为或候选为小反刍兽疫病毒，如果扩增产物含有252-254bp的DNA片段、待测病原体为或候选为产肠毒素大肠杆菌，如果扩增产物含有187-189bp的DNA片段、待测病原体为或候选为牛传染性鼻气管炎病毒；
(e2)检测待病原体的基因组DNA或cDNA中是否含有所述引物对I的靶序列、所述引物对II的靶序列、所述引物对III的靶序列、所述引物对IV的靶序列、所述引物对V的靶序列、所述引物对VI的靶序列、所述引物对VII的靶序列或所述引物对VIII的靶序列，如果所述cDNA中含有所述引物对I的靶序列、待测病原体为或候选为口蹄疫病毒，如果所述cDNA中含有所述引物对II的靶序列、待测病原体为或候选为蓝舌病病毒，如果所述cDNA中含有所述引物对III的靶序列、待测病原体为或候选为水泡性口炎病毒，如果所述cDNA中含有所述引物对IV的靶序列、待测病原体为或候选为牛病毒性腹泻病毒，如果所述cDNA中含有所述引物对V的靶序列、待测病原体为或候选为牛轮状病毒，如果所述基因组DNA中含有所述引物对VI的靶序列、待测病原体为或候选为产肠毒素大肠杆菌，如果所述cDNA中含有所述引物对VII的靶序列、待测病原体为或候选为牛传染性鼻气管炎病毒，如果所述cDNA中含有所述引物对VIII的靶序列、待测病原体为或候选为小反刍兽疫病毒。
本发明还保护一种检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒的方法，包括如下步骤(f1)或(f2)：
(f1)将待测样本进行基因组DNA提取操作和/或RNA提取操作(DNA提取操作和RNA提取操作可在同一体系中进行，也可分别进行后混合)，得到核酸；将待测样本的含有RNA或可能含有RNA的核酸进行反转录，以得到的DNA和/或cDNA作为模板，采用所述引物组合进行PCR扩增(具体可进行GeXP多重PCR扩增；扩增产物可进行毛细管电泳检测)，如果扩增产物含有165-167bp的DNA片段、待测样本含有或疑似含有口蹄疫病毒，如果扩增产物含有135-137bp的DNA片段、待测样本含有或疑似含有蓝舌病病毒，如果扩增产物含有278-281bp的DNA片段、待测样本含有或疑似含有水泡性 口炎病毒，如果扩增产物含有308-310bp的DNA片段、待测样本含有或疑似含有牛病毒性腹泻病毒，如果扩增产物含有211-214bp的DNA片段、待测样本含有或疑似含有牛轮状病毒，如果扩增产物含有342-345bp的DNA片段、待测样本含有或疑似含有小反刍兽疫病毒，如果扩增产物含有252-254bp的DNA片段、待测样本含有或疑似含有产肠毒素大肠杆菌，如果扩增产物含有187-189bp的DNA片段、待测样本含有或疑似含有牛传染性鼻气管炎病毒；
(f2)检测待测样本的基因组DNA或cDNA中是否含有所述引物对I的靶序列、所述引物对II的靶序列、所述引物对III的靶序列、所述引物对IV的靶序列、所述引物对V的靶序列、所述引物对VI的靶序列、所述引物对VII的靶序列或所述引物对VIII的靶序列，如果所述cDNA中含有所述引物对I的靶序列、待测样本含有或疑似含有口蹄疫病毒，如果所述cDNA中含有所述所述引物对II的靶序列、待测样本含有或疑似含有蓝舌病病毒，如果所述cDNA中含有所述所述引物对III的靶序列、待测样本含有或疑似含有水泡性口炎病毒，如果所述cDNA中含有所述所述引物对IV的靶序列、待测样本含有或疑似含有牛病毒性腹泻病毒，如果所述cDNA中含有所述所述引物对V的靶序列、待测样本含有或疑似含有牛轮状病毒，如果基因组DNA含有所述引物对VI的靶序列、待测样本含有或疑似含有产肠毒素大肠杆菌，如果所述cDNA中含有所述引物对VII的靶序列、待测样本含有或疑似含有牛传染性鼻气管炎病毒，如果所述cDNA中含有所述引物对VIII的靶序列、待测样本含有或疑似含有小反刍兽疫病毒。
本发明还保护引物组合，为如下(g1)或(g2)：
(g1)所述引物对I或所述引物对II或所述引物对III或所述引物对IV或所述引物对V或所述引物对VI或所述引物对VII或所述引物对VIII；
(g2)所述引物对I、所述引物对II、所述引物对III、所述引物对IV、所述引物对V、所述引物对VI、所述引物对VII和所述引物对VIII中的任意两个引物对的组合、任意三个引物对的组合、任意四个引物对的组合、任意五个引物对的组合、任意六个引物对的组合或任意七个引物对的组合。
所述引物组合的用途为鉴别口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或 牛传染性鼻气管炎病毒和/或小反刍兽疫病毒。
本发明还保护所述引物组合的应用，为鉴别口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒。
本发明还保护含有所述引物组合的试剂盒；所述试剂盒的用途为鉴别口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒。
以上任一所述8种牛病原体为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒和小反刍兽疫病毒。
以上任一所述待测病原体具体可为FMDV O型灭活病毒、FMDV A型灭活病毒、FMDV Asia I型灭活病毒、VSV NJ型灭活病毒、VSV IND型灭活病毒、BTV 4型灭活病毒、BTV 8型灭活病毒、BTV 9型灭活病毒、BTV 15型灭活病毒、BTV 17型灭活病毒、BTV 18型灭活病毒、PPRV疫苗株、BVDV参考毒株Oregon CV24株(BVDV-1型)、BVDV参考毒株NADL株(BVDV-1型)、BVDV参考毒株牦牛株(BVDV-1型)、BRV参考毒株NCDV、BRV参考毒株BRV014、IBRV病毒、ETEC参考株1676、ETEC参考株1751、ETEC参考株B41、BVDV毒株GX-BVDV1、BVDV毒株GX-BVDV2、BVDV毒株GX-BVDV3、BVDV毒株GX-BVDV4、BVDV毒株GX-BVDV5、BVDV毒株GX-BVDV6、BVDV毒株GX-BVDV7、BVDV毒株GX-BVDV8、BVDV毒株GX-BVDV9、BVDV毒株GX-BVDV10、BVDV毒株GX-BVDV11、BVDV毒株GX-BVDV12、BVDV毒株GX-BVDV13、BVDV毒株GX-041、BRV毒株GX-BRV-1、BRV毒株GX-BRV-2、BRV毒株GX-BRV-3、BRV毒株GX-BRV-4、BRV毒株GX-BRV-5、BRV毒株GX-BRV-6、BRV毒株GX-BRV-7或BRV毒株GX-BRV-8。
以上任一所述“所述引物对I的靶序列”具体可为如下(h1)或(h2)或(h3)：(h1)序列表的序列27所示的DNA分子；(h1)序列表的序列27自5′末端第19至146位核苷酸所示的DNA分子；(h3)与(h1)或(h2) 具有98％以上同源性的DNA分子。
以上任一所述“所述引物对II的靶序列”具体可为如下(h4)或(h5)或(h6)：(h4)序列表的序列28所示的DNA分子；(h5)序列表的序列28自5′末端第19至117位核苷酸所示的DNA分子；(h6)与(h4)或(h5)具有98％以上同源性的DNA分子。
以上任一所述“所述引物对III的靶序列”具体可为如下(h7)或(h8)或(h9)：(h7)序列表的序列29所示的DNA分子；(h8)序列表的序列29自5′末端第19至259位核苷酸所示的DNA分子；(h9)与(h7)或(h8)具有98％以上同源性的DNA分子。
以上任一所述“所述引物对IV的靶序列”具体可为如下(h10)或(h11)或(h12)：(h10)序列表的序列30所示的DNA分子；(h11)序列表的序列30自5′末端第19至289位核苷酸所示的DNA分子；(h12)与(h10)或(h11)具有98％以上同源性的DNA分子。
以上任一所述“所述引物对V的靶序列”具体可为如下(h13)或(h14)或(h15)：(h13)序列表的序列31所示的DNA分子；(h14)序列表的序列31自5′末端第19至192位核苷酸所示的DNA分子；(h15)与(h13)或(h14)具有98％以上同源性的DNA分子。
以上任一所述“所述引物对VIII的靶序列”具体可为如下(h16)或(h17)或(h18)：(h16)序列表的序列32所示的DNA分子；(h17)序列表的序列32自5′末端第19至325位核苷酸所示的DNA分子；(h18)与(h16)或(h17)具有98％以上同源性的DNA分子。
以上任一所述“所述引物对VI的靶序列”具体可为如下(h19)或(h20)或(h21)：(h19)序列表的序列33所示的DNA分子；(h20)序列表的序列33自5′末端第19至234位核苷酸所示的DNA分子；(h21)与(h19)或(h20)具有98％以上同源性的DNA分子。
以上任一所述“所述引物对VII的靶序列”具体可为如下(h22)或(h23)或(h24)：(h22)序列表的序列34所示的DNA分子；(h23)序列表的序列34自5′末端第19至169位核苷酸所示的DNA分子；(h24)与(h22)或 (h23)具有98％以上同源性的DNA分子。
以上任一所述待测样本具体可为粪便拭子、眼试子、鼻粘液拭子、抗凝血、OP液(食道-咽部分泌物)、水泡液、直肠黏膜组织样本、水泡皮组织样本或淋巴结组织样本。
以上任一所述GeXP多重PCR扩增的反应体系中，引物组合中的各条引物的浓度如下：FMDV-F和FMDV-R的浓度均为0.2μmol/μL，BTV-F和BTV-R的浓度均为0.2μmol/μL，VSV-F和VSV-R的浓度均为0.2μmol/μL，BVDV-F和BVDV-R的浓度均为2μmol/μL，BRV-F和BRV-R的浓度均为0.2μmol/μL，ETEC-F和ETEC-R的浓度均为0.2μmol/μL，IBRV-F和IBRV-R的浓度均为0.2μmol/μL，PPRV-F和PPRV-R的浓度均为2μmol/μL。
以上任一所述GeXP多重PCR扩增的反应体系(20μL)具体可为：模板1μL(10-100ng)，Genome Lab GeXP Starter Kit 5×buffer 4μL(buffer内含有通用引物，通用引物由序列表的序列25所示的引物A和序列表的序列26所示的引物B组成，其中引物A的5’末端具有CY5荧光基团的标记，引物A和引物B的工作浓度均为0.25μM)，MgCl2(25μM)4μL，含有引物组合中的所有引物的引物混合物1μL，DNA polymerase 10U，用超纯水补足至20μL。
以上任一所述GeXP多重PCR扩增的反应程序具体可为：95℃5分钟预变性；94℃30秒，55℃30秒，72℃30秒，10个循环；94℃30秒，68℃30秒，72℃30秒，10个循环；94℃30秒，50℃30秒，72℃30秒，10个循环；72℃延伸5min，结束反应。
以上任一所述毛细管电泳的电泳条件为：90℃120秒，变性；2.0KV 30秒，吸入样品；6.0KV 35分钟，分离样品。
本发明建立的GeXP检测方法可以同时鉴别口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒和小反刍兽疫病毒8种牛传染病的病原体。本方法具有高通量、特异性和灵敏度较高的特点，可用于牛病流行病学的监测和突发疫 情的鉴别诊断，保障养牛业的健康发展。
附图说明
图1为实施例2中各待测样本的多重PCR扩增结果图。
图2为实施例2中8种牛病原体混合样本的多重PCR扩增结果图。
图3为实施例5中采用反应体系1-5时多重PCR的扩增结果图。
图4为实施例6中采用混合液A和混合液B为模板进行多重PCR的扩增结果图。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂商店购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。由于GeXP本身系统的误差，扩增片断大小与理论值存在±2bp偏差均属于正确结果。
FMDV O型灭活病毒、FMDVA型灭活病毒、FMDVAsia I型灭活病毒、VSV NJ型灭活病毒、VSV IND型灭活病毒、BTV 4型灭活病毒、BTV 8型灭活病毒、BTV 9型灭活病毒、BTV 15型灭活病毒、BTV 17型灭活病毒、BTV 18型灭活病毒、PPRV疫苗株：参考文献：秦敏，邹丰才，杨云庆，等.蓝舌病、口蹄疫、小反刍兽疫和水泡性口炎多重PCR检测方法的建立[J].动物医学进展，2015，36(9)：18-22.；由云南出入境检验检疫局惠赠，公众可从广西壮族自治区兽医研究所获得。
BVDV参考毒株Oregon CV24株(BVDV-1型)：中国兽医药品监察所，货号：AV69。
BVDV参考毒株NADL株(BVDV-1型)：中国兽医药品监察所，货号：AV67。
BVDV参考毒株牦牛株(BVDV-1型)：中国兽医药品监察所，货号：AV68。
BRV参考毒株NCDV：中国兽医药品监察所，货号：AV51。
BRV参考毒株BRV014：中国兽医药品监察所，货号：AV52。
IBRV病毒：中国兽医微生物菌种保藏管理中心，货号：AV21。
ETEC参考株1676：中国兽医药品监察所，货号：212。
ETEC参考株1751：中国兽医药品监察所，货号：214。
ETEC参考株B41：中国兽医药品监察所，货号：215。
BVDV毒株GX-BVDV1、BVDV毒株GX-BVDV2、BVDV毒株GX-BVDV3、BVDV毒株GX-BVDV4、BVDV毒株GX-BVDV5、BVDV毒株GX-BVDV6、BVDV毒株GX-BVDV7、BVDV毒株GX-BVDV8、BVDV毒株GX-BVDV9、BVDV毒株GX-BVDV10、BVDV毒株GX-BVDV11、BVDV毒株GX-BVDV12、BVDV毒株GX-BVDV13、BVDV毒株GX-041：参考文献：Fan Q，Xie Z，Xie L，et al.A reverse transcription loop-mediated isothermal amplification method for rapid detection of bovine viral diarrhea virus[J].Journal of Virological Methods，2012，186(1-2)：43-48.；公众可以从广西壮族自治区兽医研究所获得。
BRV毒株GX-BRV-1、BRV毒株GX-BRV-2、BRV毒株GX-BRV-3、BRV毒株GX-BRV-4、BRV毒株GX-BRV-5、BRV毒株GX-BRV-6、BRV毒株GX-BRV-7、BRV毒株GX-BRV-8：参考文献：Xie Z，Fan Q，Liu J，et al.Reverse transcription loop-mediated isothermal amplification assay for rapid detection of Bovine Rotavirus[J].Bmc Veterinary Research，2012，8(1)：451-452.；公众可以从广西壮族自治区兽医研究所获得。
Genome Lab GeXP Starter Kit 5×buffer：其中含有序列表的序列25所示的引物A和序列表的序列26所示的引物B，其中引物A的5’末端具有CY5荧光基团的标记；美国贝克曼库尔特公司。
样品缓冲液：美国贝克曼库尔特公司，货号：M409196。
DNA size standard kit-400Base Pairs：美国贝克曼库尔特公司产品，货号：608098。
DNApolymerase：美国SIGMA公司，货号：D4184-1.5KU。
MgCl2(25μM)：美国SIGMA公司，货号：M8787-1.5ML。
EasyPure Viral DNA/RNA Kit：北京全式金生物技术有限公司，货号：ER201-01。
实施例1、引物组合的设计和制备
进行大量序列分析、比对获得了用于鉴别FMDV、BTV、VSV、BVDV、BRV、ETEC、IBRV和PPRV 8种牛病原体的若干引物。将各个引物进行预实验，比较灵敏度、特异性等性能，最终得到用于鉴别8种牛病原体的8对特异性引物。每个特异性引物对，正向引物和反向引物均由靶向区段和通用引物区段组成，通用引物区段位于靶向区段的5’端。
用于鉴定FMDV的引物对由如下两条引物组成(5’→3’)：
FMDV-F(序列表的序列1)：AGGTGACACTATAGAATAGCCGTGGGACCATACAGG；
FMDV-R(序列表的序列2)：GTACGACTCACTATAGGGAAAGTGATCTGTAGCTTGGAATCTC。
下划线部分为通用引物区段。
用于鉴定BTV的引物对由如下两条引物组成(5’→3’)：
BTV-F(序列表的序列3)：AGGTGACACTATAGAATAAGGGTAACTCACAGCAAACTCAA；
BTV-R(序列表的序列4)：GTACGACTCACTATAGGGAGAGCAGCCTGTCCATCCC。
下划线部分为通用引物区段。
用于鉴定VSV的引物对由如下两条引物组成(5’→3’)：
VSV-F(序列表的序列5)：AGGTGACACTATAGAATAAAACTACTGGACGGGCTTGA；
VSV-R(序列表的序列6)：GTACGACTCACTATAGGGATGAGATGCCCAAATGTTGC。
下划线部分为通用引物区段。
用于鉴定BVDV的引物对由如下两条引物组成(5’→3’)：
BVDV-F(序列表的序列7)：AGGTGACACTATAGAATAGTGAGTTCGTTGGATGGC；
BVDV-R(序列表的序列8)：GTACGACTCACTATAGGGATATGTTTTGTATAAGAGTTCATTTG。
下划线部分为通用引物区段。
用于鉴定BRV的引物对由如下两条引物组成(5’→3’)：
BRV-F(序列表的序列9)：AGGTGACACTATAGAATACAGTGGCTTCCATTAGAAGCAT；
BRV-R(序列表的序列10)：GTACGACTCACTATAGGGAGGTCACATCCTCTCACTA。
下划线部分为通用引物区段。
用于鉴定ETEC的引物对由如下两条引物组成(5’→3’)：
ETEC-F(序列表的序列11)：AGGTGACACTATAGAATACTCAGGTGCGAAAGCGTG；
ETEC-R(序列表的序列12)：GTACGACTCACTATAGGGACGTTGCATCGAATTAAACCAC。
下划线部分为通用引物区段。
用于鉴定ETEC的引物对由如下两条引物组成(5’→3’)：
IBRV-F(序列表的序列13)：AGGTGACACTATAGAATAGCGTCATTTACAAGGAGAACATC；
IBRV-R(序列表的序列14)：GTACGACTCACTATAGGGAATCTCGCCCATGCCCAC。
下划线部分为通用引物区段。
用于鉴定PPRV的引物对由如下两条引物组成(5’→3’)：
PPRV-F(序列表的序列15)：AGGTGACACTATAGAATATGGTTTGAGAACAGAGAAATAATAGA；
PPRV-R(序列表的序列16)：GTACGACTCACTATAGGGAGCTTGTTGCCGGGGGTC。
下划线部分为通用引物区段。
用于鉴定FMDV的引物对命名为引物对I。
用于鉴定BTV的引物对命名为引物对II。
用于鉴定VSV的引物对命名为引物对III。
用于鉴定BVDV的引物对命名为引物对IV。
用于鉴定BRV的引物对命名为引物对V。
用于鉴定ETEC的引物对命名为引物对VI。
用于鉴定IBRV的引物对命名为引物对VII。
用于鉴定PPRV的引物对命名为引物对VIII。
上述各引物对组成引物组合。
实施例2、特异性
一、单一模板实验
1、提取待测样本的总RNA，并反转录为cDNA。待测样本分别为：FMDV O型灭活病毒、BTV 4型灭活病毒、VSV NJ型灭活病毒、BVDV参考毒株Oregon CV24株(BVDV-1型)、BRV参考毒株NCDV、PPRV疫苗株。
2、提取待测样本的基因组DNA。待测样本分别为ETEC参考株1676和IBRV病毒。
3、分别以步骤1得到的cDNA和步骤2得到的基因组DNA为模板，采用实施例1的引物组合进行GeXP多重PCR。
多重PCR的反应体系(20μL)：模板1μL，Genome Lab GeXP Starter Kit5×buffer 4μL(buffer内含有通用引物，通用引物由序列表的序列25所示的引物A和序列表的序列26所示的引物B组成，其中引物A的5’末端具有CY5荧光基团的标记，引物A和引物B的工作浓度均为0.25μM)，MgCl2(25μM)4μL，含有引物组合中的所有引物的引物混合物1μL，DNA polymerase 10U，用超纯水补足至20μL。多重PCR的反应体系中，FMDV-F和FMDV-R的浓度均为0.2μmol/μL，BTV-F和BTV-R的浓度均为0.2μmol/μL，VSV-F和VSV-R的浓度均为0.2μmol/μL，BVDV-F和BVDV-R的浓度均为2μmol/μL，BRV-F和BRV-R的浓度均为0.2μmol/μL，ETEC-F和ETEC-R的浓度均为0.2μmol/μL，IBRV-F和IBRV-R的浓度均为0.2μmol/μL，PPRV-F和PPRV-R的浓度均为2μmol/μL。设置用等体积水作为模板的阴性对照。
模板为步骤1得到的各个cDNA样本时，1μL模板中的DNA含量约100ng；
模板为步骤2得到的基因组DNA样本时，1μL模板中的DNA含量约100ng；
多重PCR的反应程序：95℃5分钟预变性；94℃30秒，55℃30秒， 72℃30秒，10个循环；94℃30秒，68℃30秒，72℃30秒，10个循环；94℃30秒，50℃30秒，72℃30秒，10个循环；72℃延伸5min，结束反应。
4、将步骤3的多重PCR扩增产物进行毛细管电泳，具体步骤为：将3μL多重PCR扩增产物、38.75μL样品缓冲液和0.25μL DNA size standard kit-400Base Pairs漩涡震荡混匀，加入上样板中，每孔滴加1滴石蜡封闭液面，避免甲酰胺氧化和样品蒸发。在缓冲液板上每孔加入180μL的样品缓冲液，进行毛细管电泳。毛细管电泳条件：90℃120秒，变性；2.0KV 30秒，吸入样品；6.0KV 35分钟，分离样品。不同大小片段的PCR产物在电泳中分离，仪器通过检测PCR产物携带的荧光基团辨认其片段大小和信号强度。电泳完成后，使用仪器自带软件Express Profiler software分析结果。
根据电泳结果进行判断，判断标准为：8种牛病原体目的基因的扩增片断大小分别为：FMDV：165-167bp，BTV：135-137bp，VSV：278-281bp，BVDV：308-310bp，BRV：211-214bp，PPRV：342-345bp，ETEC：252-254bp，IBRV：187-189bp。由于GeXP本身系统的误差，扩增片断大小与理论值存在±2bp偏差均属于正确结果。
电泳结果如图1所示。图1中，横坐标表示片段大小(单位为bp)，纵坐标表示信号强度，即PCR扩增产物的含量。图1A为FMDV O型灭活病毒cDNA的多重PCR扩增结果，扩增得到165.03bp的DNA片段。图1B为BTV4型灭活病毒cDNA的多重PCR扩增结果，扩增得到136.72bp的DNA片段。图1C为VSV NJ型灭活病毒cDNA的多重PCR扩增结果，扩增得到278.04bp的DNA片段。图1D为BVDV参考毒株Oregon CV24株(BVDV-1型)cDNA的多重PCR扩增结果，扩增得到309.58bp的DNA片段。图1E为BRV参考毒株NCDV cDNA的多重PCR扩增结果，扩增得到211.71bp的DNA片段。图1F为PPRV疫苗株cDNA的多重PCR扩增结果，扩增得到344.20bp的DNA片段。图1G为ECTC菌株GX-ETEC1基因组DNA的多重PCR扩增结果，扩增得到252.24bp的DNA片段。图1H为IBRV病毒基因组DNA的多重PCR扩增结果，扩增得到188.21bp的DNA片段。每个反应只出现特异性单峰，无其它信号峰，且片段大小与判断标准相符。阴性对照均无扩增，无目的信号峰值。
二、混合模板实验
1、提取待测样本的总RNA，并反转录为cDNA。待测样本分别为：FMDV O型灭活病毒、BTV 4型灭活病毒、VSV NJ型灭活病毒、BVDV参考毒株Oregon CV24株(BVDV-1型)、BRV参考毒株NCDV、PPRV疫苗株。
2、提取待测样本的基因组DNA。待测样本分别为ETEC参考株1676和IBRV病毒。
3、将步骤1得到的6种cDNA、步骤2得到的2种基因组DNA混合。
4、以步骤3得到的混合液为模板，采用实施例1的引物组合进行GeXP多重PCR。
多重PCR的反应体系(20μL)：模板1μL，Genome Lab GeXP Starter Kit5×buffer 4μL(buffer内含有通用引物，通用引物由序列表的序列25所示的引物A和序列表的序列26所示的引物B组成，其中引物A的5’末端具有CY5荧光基团的标记，引物A和引物B的工作浓度均为0.25μM)，MgC12(25μM)4μL，含有引物组合中的所有引物的引物混合物1μL，DNA polymerase 10U，用超纯水补足至20μL。多重PCR的反应体系中，FMDV-F和FMDV-R的浓度均为0.2μmol/μL，BTV-F和BTV-R的浓度均为0.2μmol/μL，VSV-F和VSV-R的浓度均为0.2μmol/μL，BVDV-F和BVDV-R的浓度均为2μmol/μL，BRV-F和BRV-R的浓度均为0.2μmol/μL，ETEC-F和ETEC-R的浓度均为0.2μmol/μL，IBRV-F和IBRV-R的浓度均为0.2μmol/μL，PPRV-F和PPRV-R的浓度均为2μmol/μL。设置用等体积水作为模板的阴性对照。
1μL模板中FMDV O型灭活病毒的cDNA为约100ng，BTV 4型灭活病毒的cDNA为约100ng，VSVNJ型灭活病毒的cDNA为约100ng，BVDV参考毒株Oregon CV24株(BVDV-1型)的cDNA为约100ng，BRV参考毒株NCDV的基因组DNA为约100ng，PPRV疫苗株的基因组DNA为约100ng，ECTC菌株GX-ETEC1的基因组DNA为约100ng，IBRV病毒的基因组DNA为约100ng。
多重PCR的反应程序：95℃5分钟预变性；94℃30秒，55℃30秒，72℃30秒，10个循环；94℃30秒，68℃30秒，72℃30秒，10个循环；94℃30秒，50℃30秒，72℃30秒，10个循环；72℃延伸5min，结束反 应。
5、将步骤4的多重PCR扩增产物进行毛细管电泳，具体步骤为：将3μL多重PCR扩增产物、38.75μL样品缓冲液和0.25μL DNA size standard kit-400Base Pairs漩涡震荡混匀，加入上样板中，每孔滴加1滴石蜡封闭液面，避免甲酰胺氧化和样品蒸发。在缓冲液板上每孔加入180μL的样品缓冲液，进行毛细管电泳。毛细管电泳条件：90℃120秒，变性；2.0KV 30秒，吸入样品；6.0KV 35分钟，分离样品。不同大小片段的PCR产物在电泳中分离，仪器通过检测PCR产物携带的荧光基团辨认其片段大小和信号强度。电泳完成后，使用仪器自带软件Express Profiler software分析结果。
根据电泳结果进行判断，判断标准为：8种牛病原体目的基因的扩增片断大小分别为FMDV：165-167bp，BTV：135-137bp，VSV：278-281bp，BVDV：308-310bp，BRV：211-214bp，PPRV：342-345bp，ETEC：252-254bp，IBRV：187-189bp。
检测结果如图2所示。图2中，横坐标表示片段大小(单位为bp)，纵坐标表示信号强度，即PCR扩增产物的含量。结果表明，使用GeXP多重PCR可以同时检测出8种病原体对应的8种信号峰，FMDV：166.39bp，BTV：136.17bp，VSV：280.40bp，BVDV：309.59bp，BRV：213.71bp，PPRV：342.16bp，ETEC：253.23bp，IBRV：187.25bp，无其它杂峰。阴性对照均无扩增，无目的信号峰值。
实施例3、普适性
1、提取待测样本的总RNA，并反转录为cDNA。待测样本分别为：FMDV O型灭活病毒、FMDV A型灭活病毒、FMDV Asia I型灭活病毒、BTV 4型灭活病毒、BTV 8型灭活病毒、BTV 9型灭活病毒、BTV 15型灭活病毒、BTV17型灭活病毒、BTV 18型灭活病毒、VSVNJ型灭活病毒、VSV IND型灭活病毒、BVDV参考毒株Oregon CV24株(BVDV-1型)、BVDV参考毒株NADL株(BVDV-1型)、BVDV参考毒株牦牛株(BVDV-1型)、BVDV毒株GX-BVDV1、BVDV毒株GX-BVDV2、BVDV毒株GX-BVDV3、BVDV毒株GX-BVDV4、BVDV毒株GX-BVDV5、BVDV毒株GX-BVDV6、BVDV毒株GX-BVDV7、BVDV毒株GX-BVDV8、BVDV毒株GX-BVDV9、BVDV 毒株GX-BVDV10、BVDV毒株GX-BVDV11、BVDV毒株GX-BVDV12、BVDV毒株GX-BVDV13、BVDV毒株GX-041、BRV参考毒株NCDV、BRV参考毒株BRV014、BRV毒株GX-BRV-1、BRV毒株GX-BRV-2、BRV毒株GX-BRV-3、BRV毒株GX-BRV-4、BRV毒株GX-BRV-5、BRV毒株GX-BRV-6、BRV毒株GX-BRV-7、BRV毒株GX-BRV-8、PPRV疫苗株。
2、提取待测样本的基因组DNA。待测样本分别为：ETEC参考株1676、ETEC参考株1751、ETEC参考株B41和IBRV病毒。
3、分别以步骤1得到的cDNA和步骤2得到的基因组DNA为模板，采用实施例1的引物组合进行GeXP多重PCR。
多重PCR的反应体系(20μL)：模板1μL，Genome Lab GeXP Starter Kit5×buffer 4μL(buffer内含有通用引物，通用引物由序列表的序列25所示的引物A和序列表的序列26所示的引物B组成，其中引物A的5’末端具有CY5荧光基团的标记，引物A和引物B的工作浓度均为0.25μM)，MgCl2(25μM)4μL，含有引物组合中的所有引物的引物混合物1μL，DNA polymerase 10U，用超纯水补足至20μL。多重PCR的反应体系中，FMDV-F和FMDV-R的浓度均为0.2μmol/μL，BTV-F和BTV-R的浓度均为0.2μmol/μL，VSV-F和VSV-R的浓度均为0.2μmol/μL，BVDV-F和BVDV-R的浓度均为2μmol/μL，BRV-F和BRV-R的浓度均为0.2μmol/μL，ETEC-F和ETEC-R的浓度均为0.2μmol/μL，IBRV-F和IBRV-R的浓度均为0.2μmol/μL，PPRV-F和PPRV-R的浓度均为2μmol/μL。设置用等体积水作为模板的阴性对照。
模板为步骤1得到的各个cDNA样本时，1μL模板中的DNA含量约100ng；
模板为步骤2得到的各个基因组DNA样本时，1μL模板中的DNA含量约100ng；
多重PCR的反应程序：95℃5分钟预变性；94℃30秒，55℃30秒，72℃30秒，10个循环；94℃30秒，68℃30秒，72℃30秒，10个循环；94℃30秒，50℃30秒，72℃30秒，10个循环；72℃延伸5min，结束反应。
4、将步骤3的多重PCR扩增产物进行毛细管电泳，具体步骤为：将3 μL多重PCR扩增产物、38.75μL样品缓冲液和0.25μL DNA size standard kit-400Base Pairs漩涡震荡混匀，加入上样板中，每孔滴加1滴石蜡封闭液面，避免甲酰胺氧化和样品蒸发。在缓冲液板上每孔加入180μL的样品缓冲液，进行毛细管电泳。毛细管电泳条件：90℃ 120秒，变性；2.0KV 30秒，吸入样品；6.0KV 35分钟，分离样品。不同大小片段的PCR产物在电泳中分离，仪器通过检测PCR产物携带的荧光基团辨认其片段大小和信号强度。电泳完成后，使用仪器自带软件Express Profiler software分析结果。
根据电泳结果进行判断，判断标准为：8种牛病原体目的基因的扩增片断大小分别为FMDV：165-167bp，BTV：135-137bp，VSV：278-281bp，BVDV：308-310bp，BRV：211-214bp，PPRV：342-345bp，ETEC：252-254bp，IBRV：187-189bp。由于GeXP本身系统的误差，扩增片断大小与理论值存在±2bp偏差均属于正确结果。
采用实施例1的引物组合对待测样本进行多重PCR扩增，每个样本只出现对应病原体的特异性单峰，无其他信号峰，且片段大小与判断标准相符，结果表明，实施例1设计的引物组合普遍适用于8种牛病原体。
实施例4、质粒标准品制备
使用本发明中设计的特异性引物扩增各毒株的目的片断，将所得目的片段与pEASY-T1载体连接后构建GeXP各目的片断的重组质粒(将序列表的序列17对应的双链DNA分子与pEASY-T1载体连接，得到重组质粒pEASY-T1-FMDV；将序列表的序列18对应的双链DNA分子与pEASY-T1载体连接，得到重组质粒pEASY-T1-BTV；将序列表的序列19对应的双链DNA分子与pEASY-T1载体连接，得到重组质粒pEASY-T1-VSV；将序列表的序列20对应的双链DNA分子与pEASY-T1载体连接，得到重组质粒pEASY-T1-BVDV；将序列表的序列21对应的双链DNA分子与pEASY-T1载体连接，得到重组质粒pEASY-T1-BRV；将序列表的序列22对应的双链DNA分子与pEASY-T1载体连接，得到重组质粒pEASY-T1-PPRV；将序列表的序列23对应的双链DNA分子与pEASY-T1载体连接，得到重组质粒pEASY-T1-ETEC；将序列表的序列24对应的双链DNA分子与pEASY-T1载体连接，得到重组质粒pEASY-T1-IBRV)，提取阳性克隆质粒。通过NanoDrop 紫外分光光度计测定pEASY-T1-IBRV和pEASY-T1-ETEC质粒浓度。参照T7体外转录试剂盒说明书将提取的阳性克隆质粒(pEASY-T1-BTV，pEASY-T1-FMDV，pEASY-T1-BRV，pEASY-T1-VSV，pEASY-T1-BVDV和pEASY-T1--PPRV)体外转录为RNA，用NanoDrop测定RNA浓度，根据分子量及浓度计算各质粒拷贝数为拷贝数制备标准品，将各质粒浓度稀释到108copies/μl～1copies/μl，即得到FMDV标准品、BTV标准品、VSV标准品、BVDV标准品、BRV标准品、PPRV标准品、ETEC标准品和IBRV标准品，-70℃保存备用。
实施例5、敏感性
1、将实施例4制备的FMDV标准品、BTV标准品、VSV标准品、BVDV标准品、BRV标准品、ETEC标准品、IBRV标准品和PPRV标准品等拷贝数混合，得到混合液。
2、用ddH2O 10倍梯度稀释步骤2得到的混合液，得到各个稀释液。
3、将步骤2得到的稀释液作为模板，采用实施例1制备的引物组合进行GeXP多重PCR。
多重PCR的反应体系(20μL)：模板1μL，Genome Lab GeXP Starter Kit5×buffer 4μL(buffer内含有通用引物，通用引物由序列表的序列25所示的引物A和序列表的序列26所示的引物B组成，其中引物A的5’末端具有CY5荧光基团的标记，引物A和引物B的工作浓度均为0.25μM)，MgCl2(25μM)4μL，含有引物组合中的所有引物的引物混合物1μL，DNA polymerase 10U，用超纯水补足至20μL。多重PCR的反应体系中，FMDV-F和FMDV-R的浓度均为0.2μmol/μL，BTV-F和BTV-R的浓度均为0.2μmol/μL，VSV-F和VSV-R的浓度均为0.2μmol/μL，BVDV-F和BVDV-R的浓度均为2μmol/μL，BRV-F和BRV-R的浓度均为0.2μmol/μL，ETEC-F和ETEC-R的浓度均为0.2μmol/μL，IBRV-F和IBRV-R的浓度均为0.2μmol/μL，PPRV-F和PPRV-R的浓度均为2μmol/μL。设置用等体积水作为模板的阴性对照。
由于采用的稀释液的稀释度不同，形成如下不同的反应体系：
反应体系1中，FMDV标准品、BTV标准品、VSV标准品、BVDV标 准品、BRV标准品、ETEC标准品、IBRV标准品和PPRV标准品的初始浓度均为106拷贝/μL；
反应体系2中，FMDV标准品、BTV标准品、VSV标准品、BVDV标准品、BRV标准品、ETEC标准品、IBRV标准品和PPRV标准品的初始浓度均为105拷贝/μL；
反应体系3中，FMDV标准品、BTV标准品、VSV标准品、BVDV标准品、BRV标准品、ETEC标准品、IBRV标准品和PPRV标准品的初始浓度均为104拷贝/μL；
反应体系4中，FMDV标准品、BTV标准品、VSV标准品、BVDV标准品、BRV标准品、ETEC标准品、IBRV标准品和PPRV标准品的初始浓度均为103拷贝/μL；
反应体系5中，FMDV标准品、BTV标准品、VSV标准品、BVDV标准品、BRV标准品、ETEC标准品、IBRV标准品和PPRV标准品的初始浓度均为102拷贝/μL；
反应体系6中，FMDV标准品、BTV标准品、VSV标准品、BVDV标准品、BRV标准品、ETEC标准品、IBRV标准品和PPRV标准品的初始浓度均为10拷贝/μL；
反应体系7中，FMDV标准品、BTV标准品、VSV标准品、BVDV标准品、BRV标准品、ETEC标准品、IBRV标准品和PPRV标准品的初始浓度均为1拷贝/μL。
多重PCR的反应程序：95℃5分钟预变性；94℃30秒，55℃30秒，72℃30秒，10个循环；94℃30秒，68℃30秒，72℃30秒，10个循环；94℃30秒，50℃30秒，72℃30秒，10个循环；72℃延伸5min，结束反应。
4、将步骤3的多重PCR扩增产物进行毛细管电泳，具体步骤为：将3μL多重PCR扩增产物、38.75μL样品缓冲液和0.25μL DNA size standard kit-400Base Pairs漩涡震荡混匀，加入上样板中，每孔滴加1滴石蜡封闭液面，避免甲酰胺氧化和样品蒸发。在缓冲液板上每孔加入180μL的样品缓冲液，进行毛细管电泳。毛细管电泳条件：90℃120秒，变性；2.0KV 30秒，吸入样品；6.0KV 35分钟，分离样品。不同大小片段的PCR产物在电泳中 分离，仪器通过检测PCR产物携带的荧光基团辨认其片段大小和信号强度。电泳完成后，使用仪器自带软件Express Profiler software分析结果。
根据电泳结果进行判断，判断标准为：8种牛病原体目的基因的扩增片断大小分别为FMDV：165-167bp，BTV：135-137bp，VSV：278-281bp，BVDV：308-310bp，BRV：211-214bp，PPRV：342-345bp，ETEC：252-254bp，IBRV：187-189bp。由于GeXP本身系统的误差，扩增片断大小与理论值存在±2bp偏差均属于正确结果。
检测结果如图3所示。图3A-图3E依次对应采用反应体系1-5时多重PCR的扩增结果，图3中，横坐标表示片段大小(单位为bp)，纵坐标表示信号强度，即PCR扩增产物的含量。结果表明，当检测体系中待测DNA的浓度低至100拷贝/μL时，也可以检测出8种病原体。
实施例6、干扰性
1、将实施例4制备的IBRV标准品、BRV标准品、ETEC标准品、BVDV标准品和PPRV标准品混合，得到混合液A。
2、将实施例4制备的FMDV标准品、BTV标准品、IBRV标准品、BRV标准品和ETEC标准品混合，得到混合液B。
3、分别以步骤1得到的混合液A和步骤2得到的混合液B作为模板，采用实施例1制备的引物组合进行GeXP多重PCR。
多重PCR的反应体系(20μL)：模板1μL，Genome Lab GeXP Starter Kit5×buffer 4μL(buffer内含有通用引物，通用引物由序列表的序列25所示的引物A和序列表的序列26所示的引物B组成，其中引物A的5’末端具有CY5荧光基团的标记，引物A和引物B的工作浓度均为0.25μM)，MgCl2(25μM)4μL，含有引物组合中的所有引物的引物混合物1μL，DNA polymerase 10U，用超纯水补足至20μL。多重PCR的反应体系中，FMDV-F和FMDV-R的浓度均为0.2μmol/μL，BTV-F和BTV-R的浓度均为0.2μmol/μL，VSV-F和VSV-R的浓度均为0.2μmol/μL，BVDV-F和BVDV-R的浓度均为2μmol/μL，BRV-F和BRV-R的浓度均为0.2μmol/μL，ETEC-F和ETEC-R的浓度均为0.2μmol/μL，IBRV-F和IBRV-R的浓度均为0.2μmol/μL，PPRV-F和PPRV-R的浓度均为2μmol/μL。设置用等体积水作为模板 的阴性对照。
以混合液A为模板时，反应体系中，IBRV标准品的浓度为103拷贝/μL，BRV标准品的浓度为105拷贝/μL，ETEC标准品的浓度为105拷贝/μL，BVDV标准品的浓度为105拷贝/μL，PPRV标准品的浓度为107拷贝/μL。
以混合液B为模板时，反应体系中，FMDV标准品的浓度为104拷贝/μL，BTV标准品的浓度为108拷贝/μL，IBRV标准品的浓度为104拷贝/μL，BRV标准品的浓度为105拷贝/μL，ETEC标准品的浓度为105拷贝/μL。
多重PCR的反应程序：95℃5分钟预变性；94℃30秒，55℃30秒，72℃30秒，10个循环；94℃30秒，68℃30秒，72℃30秒，10个循环；94℃30秒，50℃30秒，72℃30秒，10个循环；72℃延伸5min，结束反应。
4、将步骤3的多重PCR扩增产物进行毛细管电泳，具体步骤为：将3μL多重PCR扩增产物、38.75μL样品缓冲液和0.25μL DNA size standard kit-400Base Pairs漩涡震荡混匀，加入上样板中，每孔滴加1滴石蜡封闭液面，避免甲酰胺氧化和样品蒸发。在缓冲液板上每孔加入180μL的样品缓冲液，进行毛细管电泳。毛细管电泳条件：90℃ 120秒，变性；2.0KV 30秒，吸入样品；6.0KV 35分钟，分离样品。不同大小片段的PCR产物在电泳中分离，仪器通过检测PCR产物携带的荧光基团辨认其片段大小和信号强度。电泳完成后，使用仪器自带软件Express Profiler software分析结果。
根据电泳结果进行判断，判断标准为：8种牛病原体目的基因的扩增片断大小分别为FMDV：165-167bp，BTV：135-137bp，VSV：278-281bp，BVDV：308-310bp，BRV：211-214bp，PPRV：342-345bp，ETEC：252-254bp，IBRV：187-189bp。由于GeXP本身系统的误差，扩增片断大小与理论值存在±2bp偏差均属于正确结果。
检测结果如图4所示。图4中，横坐标表示片段大小(单位为bp)，纵坐标表示信号强度，即PCR扩增产物的含量。图4A为采用混合液A为模板进行多重PCR的扩增结果，图4B为采用混合液B为模板进行多重PCR的扩增结果。结果表明，当反应体系中起始模板的浓度悬殊较大时，仍能准确敏感的检测出低浓度的模板，干扰性小。
实施例7、临床样本检测
待测样本为：305份临床样本，其中包括156份粪便拭子，30份眼试子，30份鼻粘液拭子，70份抗凝血，2份OP液(食道-咽部分泌物)，2份水泡液，15份组织样品(10份直肠黏膜，2份水泡皮，3份淋巴结)。临床样品采集于2012-2014年广西各地，约1/2的样品来源于各地奶牛场无症状奶牛，约1/4的样品来源于具有腹泻，消瘦，流鼻涕等临床症状的牛，1/4样品来源于具有精神不撅，吞咽困难，发烧，口腔糜烂出现水泡，口鼻白沫的牛。
1、使用EasyPure Viral DNA/RNA Kit提取待测样本的DNA/RNA，得到DNA/RNA混合溶液。
2、将步骤1得到的DNA/RNA混合溶液反转录得到DNA/cDNA混合溶液。
3、将步骤2得到的DNA/cDNA混合溶液作为模板，采用实施例1制备的引物组合进行进行GeXP多重PCR。
多重PCR的反应体系(20μL)：模板1μL(DNA含量为10-100ng)，Genome Lab GeXP Starter Kit 5×buffer 4μL(buffer内含有通用引物，通用引物由序列表的序列25所示的引物A和序列表的序列26所示的引物B组成，其中引物A的5’末端具有CY5荧光基团的标记，引物A和引物B的工作浓度均为0.25μM)，MgCl2(25μM)4μL，含有引物组合中的所有引物的引物混合物1μL，DNA polymerase 10U，用超纯水补足至20μL。多重PCR的反应体系中，FMDV-F和FMDV-R的浓度均为0.2μmol/μL，BTV-F和BTV-R的浓度均为0.2μmol/μL，VSV-F和VSV-R的浓度均为0.2μmol/μL，BVDV-F和BVDV-R的浓度均为2μmol/μL，BRV-F和BRV-R的浓度均为0.2μmol/μL，ETEC-F和ETEC-R的浓度均为0.2μmol/μL，IBRV-F和IBRV-R的浓度均为0.2μmol/μL，PPRV-F和PPRV-R的浓度均为2μmol/μL。设置用等体积水作为模板的阴性对照。
多重PCR的反应程序：95℃5分钟预变性；94℃30秒，55℃30秒，72℃30秒，10个循环；94℃30秒，68℃30秒，72℃30秒，10个循环；94℃30秒，50℃30秒，72℃30秒，10个循环；72℃延伸5min，结束反应。
4、将步骤3的多重PCR扩增产物进行毛细管电泳，具体步骤为：将3 μL多重PCR扩增产物、38.75μL样品缓冲液和0.25μL DNA size standard kit-400Base Pairs漩涡震荡混匀，加入上样板中，每孔滴加1滴石蜡封闭液面，避免甲酰胺氧化和样品蒸发。在缓冲液板上每孔加入180μL的样品缓冲液，进行毛细管电泳。毛细管电泳条件：90℃ 120秒，变性；2.0KV 30秒，吸入样品；6.0KV 35分钟，分离样品。不同大小片段的PCR产物在电泳中分离，仪器通过检测PCR产物携带的荧光基团辨认其片段大小和信号强度。电泳完成后，使用仪器自带软件Express Profiler software分析结果。
根据电泳结果进行判断，判断标准为：8种牛病原体目的基因的扩增片断大小分别为FMDV：165-167bp，BTV：135-137bp，VSV：278-281bp，BVDV：308-310bp，BRV：211-214bp，PPRV：342-345bp，ETEC：252-254bp，IBRV：187-189bp。由于GeXP本身系统的误差，扩增片断大小与理论值存在±2bp偏差均属于正确结果。
5、将步骤4得到的阳性扩增产物进行测序以验证结果的正确性。
检测结果如表1所示。
表1 临床样本检测结果统计
Figure PCTCN2017081056-appb-000001
305份样品中，146份检测结果为阳性，其中单重感染为90份(单冲感染样本占检测样本的29.5％)，混合感染28份(混合感染样本占检测样本的9.2％)。测序结果显示阳性结果均为对应的病毒，无非特异性扩增的假阳性。

Claims (10)

  1. 引物组合，由引物对Ⅰ、引物对Ⅱ、引物对Ⅲ、引物对Ⅳ、引物对Ⅴ、引物对Ⅵ、引物对Ⅶ和引物对Ⅷ组成；
    所述引物对Ⅰ由引物FMDV-F和引物FMDV-R组成；
    所述引物FMDV-F为如下(a1)或(a2)或(a3)：
    (a1)序列表的序列1所示的单链DNA分子；
    (a2)序列表的序列1自5′末端第19至36位核苷酸所示的单链DNA分子；
    (a3)将(a1)或(a2)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a1)或(a2)相同功能的DNA分子；
    所述引物FMDV-R为如下(a4)或(a5)或(a6)：
    (a4)序列表的序列2所示的单链DNA分子；
    (a5)序列表的序列2自5′末端第20至43位核苷酸所示的单链DNA分子；
    (a6)将(a4)或(a5)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a4)或(a5)相同功能的DNA分子；
    所述引物对Ⅱ由引物BTV-F和引物BTV-R组成；
    所述引物BTV-F为如下(a7)或(a8)或(a9)：
    (a7)序列表的序列3所示的单链DNA分子；
    (a8)序列表的序列3自5′末端第19至41位核苷酸所示的单链DNA分子；
    (a9)将(a7)或(a8)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a7)或(a8)相同功能的DNA分子；
    所述引物BTV-R为如下(a10)或(a11)或(a12)：
    (a10)序列表的序列4所示的单链DNA分子；
    (a11)序列表的序列4自5′末端第20至37位核苷酸所示的单链DNA分子；
    (a12)将(a10)或(a11)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a10)或(a11)相同功能的DNA分子；
    所述引物对Ⅲ由引物VSV-F和引物VSV-R组成；
    所述引物VSV-F为如下(a13)或(a14)或(a15)：
    (a13)序列表的序列5所示的单链DNA分子；
    (a14)序列表的序列5自5′末端第19至38位核苷酸所示的单链DNA分子；
    (a15)将(a13)或(a14)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a13)或(a14)相同功能的DNA分子；
    所述引物VSV-R为如下(a16)或(a17)或(a18)：
    (a16)序列表的序列6所示的单链DNA分子；
    (a17)序列表的序列6自5′末端第20至38位核苷酸所示的单链DNA分子；
    (a18)将(a16)或(a17)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a16)或(a17)相同功能的DNA分子；
    所述引物对Ⅳ由引物BVDV-F和引物BVDV-R组成；
    所述引物BVDV-F为如下(a19)或(a20)或(a21)：
    (a19)序列表的序列7所示的单链DNA分子；
    (a20)序列表的序列7自5′末端第19至36位核苷酸所示的单链DNA分子；
    (a21)将(a19)或(a20)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a19)或(a20)相同功能的DNA分子；
    所述引物BVDV-R为如下(a22)或(a23)或(a24)：
    (a22)序列表的序列8所示的单链DNA分子；
    (a23)序列表的序列8自5′末端第20至44位核苷酸所示的单链DNA分子；
    (a24)将(a22)或(a23)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a22)或(a23)相同功能的DNA分子；
    所述引物对Ⅴ由引物BRV-F和引物BRV-R组成；
    所述引物BRV-F为如下(a25)或(a26)或(a27)：
    (a25)序列表的序列9所示的单链DNA分子；
    (a26)序列表的序列9自5′末端第19至40位核苷酸所示的单链DNA 分子；
    (a27)将(a25)或(a26)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a25)或(a26)相同功能的DNA分子；
    所述引物BRV-R为如下(a28)或(a29)或(a30)：
    (a28)序列表的序列10所示的单链DNA分子；
    (a29)序列表的序列10自5′末端第20至37位核苷酸所示的单链DNA分子；
    (a30)将(a28)或(a29)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a28)或(a29)相同功能的DNA分子；
    所述引物对Ⅵ由引物ETEC-F和引物ETEC-R组成：
    所述引物ETEC-F为如下(a31)或(a32)或(a33)：
    (a31)序列表的序列11所示的单链DNA分子；
    (a32)序列表的序列11自5′末端第19至36位核苷酸所示的单链DNA分子；
    (a33)将(a31)或(a32)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a31)或(a32)相同功能的DNA分子；
    所述引物ETEC-R为如下(a34)或(a35)或(a36)：
    (a34)序列表的序列12所示的单链DNA分子；
    (a35)序列表的序列12自5′末端第20至40位核苷酸所示的单链DNA分子；
    (a36)将(a34)或(a35)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a34)或(a35)相同功能的DNA分子；
    所述引物对Ⅶ由引物IBRV-F和引物IBRV-R组成；
    所述引物IBRV-F为如下(a37)或(a38)或(a39)：
    (a37)序列表的序列13所示的单链DNA分子；
    (a38)序列表的序列13自5′末端第19至41位核苷酸所示的单链DNA分子；
    (a39)将(a37)或(a38)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a37)或(a38)相同功能的DNA分子；
    所述引物IBRV-R为如下(a40)或(a41)或(a42)：
    (a40)序列表的序列14所示的单链DNA分子；
    (a41)序列表的序列14自5′末端第20至36位核苷酸所示的单链DNA分子；
    (a42)将(a40)或(a41)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a40)或(a41)相同功能的DNA分子；
    所述引物对Ⅷ由引物PPRV-F和引物PPRV-R组成：
    所述引物PPRV-F为如下(a43)或(a44)或(a45)：
    (a43)序列表的序列15所示的单链DNA分子；
    (a44)序列表的序列15自5′末端第19至44位核苷酸所示的单链DNA分子；
    (a45)将(a43)或(a44)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a43)或(a44)相同功能的DNA分子；
    所述引物PPRV-R为如下(a46)或(a47)或(a48)：
    (a46)序列表的序列16所示的单链DNA分子；
    (a47)序列表的序列16自5′末端第20至36位核苷酸所示的单链DNA分子；
    (a48)将(a46)或(a47)经过一个或几个核苷酸的取代和/或缺失和/或添加且具有与(a46)或(a47)相同功能的DNA分子。
  2. 权利要求1所述引物组合的应用，为如下(b1)至(b6)中的任意一种：
    (b1)鉴别8种牛病原体；
    (b2)制备用于鉴别8种牛病原体的试剂盒；
    (b3)检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒；
    (b4)制备用于检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒的试剂盒；
    (b5)检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/ 或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒；
    (b6)制备用于检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒的试剂盒；
    所述8种牛病原体为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒和小反刍兽疫病毒。
  3. 含有权利要求1所述引物组合的试剂盒；所述试剂盒的用途为如下(c1)或(c2)或(c3)：
    (c1)鉴别8种牛病原体；
    (c2)检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒；
    (c3)检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒；
    所述8种牛病原体为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒和小反刍兽疫病毒。
  4. 权利要求3所述试剂盒的制备方法，包括将各条引物单独包装的步骤。
  5. 一种鉴别8种牛病原体的方法，包括如下步骤(d1)或(d2)：
    (d1)将待测病原体进行基因组DNA提取操作和/或RNA提取操作，得到核酸；将待测病原体的含有RNA或可能含有RNA的核酸进行反转录，以得到的DNA和/或cDNA作为模板，采用所述引物组合进行PCR扩增，如果扩增产物含有165-167bp的DNA片段、待测病原体为或候选为口蹄疫病毒，如果扩增产物含有135-137bp的DNA片段、待测病原体为或候选为蓝舌病病毒，如果扩增产物含有278-281bp的DNA片段、待测病原体为或候选为水泡性口炎病毒，如果扩增产物含有308-310bp的DNA片段、待测病原体为或候选为牛病毒性腹泻病毒，如果扩增产物含有211-214bp的DNA片段、待测病原体为或候选为牛轮状病毒，如果扩增产物含有342-345bp的DNA片段、 待测病原体为或候选为小反刍兽疫病毒，如果扩增产物含有252-254bp的DNA片段、待测病原体为或候选为产肠毒素大肠杆菌，如果扩增产物含有187-189bp的DNA片段、待测病原体为或候选为牛传染性鼻气管炎病毒；
    (d2)检测待病原体的基因组DNA或cDNA中是否含有权利要求1所述引物对Ⅰ的靶序列、所述引物对Ⅱ的靶序列、所述引物对Ⅲ的靶序列、所述引物对Ⅳ的靶序列、所述引物对Ⅴ的靶序列、所述引物对Ⅵ的靶序列、所述引物对Ⅶ的靶序列或所述引物对Ⅷ的靶序列，如果所述cDNA中含有所述引物对Ⅰ的靶序列、待测病原体为或候选为口蹄疫病毒，如果所述cDNA中含有所述引物对Ⅱ的靶序列、待测病原体为或候选为蓝舌病病毒，如果所述cDNA中含有所述引物对Ⅲ的靶序列、待测病原体为或候选为水泡性口炎病毒，如果所述cDNA中含有所述引物对Ⅳ的靶序列、待测病原体为或候选为牛病毒性腹泻病毒，如果所述cDNA中含有所述引物对Ⅴ的靶序列、待测病原体为或候选为牛轮状病毒，如果所述基因组DNA中含有所述引物对Ⅵ的靶序列、待测病原体为或候选为产肠毒素大肠杆菌，如果所述cDNA中含有所述引物对Ⅶ的靶序列、待测病原体为或候选为牛传染性鼻气管炎病毒，如果所述cDNA中含有所述引物对Ⅷ的靶序列、待测病原体为或候选为小反刍兽疫病毒；
    所述8种牛病原体为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒和小反刍兽疫病毒。
  6. 一种检测待测病原体是否为口蹄疫病毒、蓝舌病病毒、水泡性口炎病毒、牛病毒性腹泻病毒、牛轮状病毒、产肠毒素大肠杆菌、牛传染性鼻气管炎病毒或小反刍兽疫病毒的方法，包括如下步骤(e1)或(e2)：
    (e1)将待测病原体进行基因组DNA提取操作和/或RNA提取操作，得到核酸；将待测病原体的含有RNA或可能含有RNA的核酸进行反转录，以得到的DNA和/或cDNA作为模板，采用所述引物组合进行PCR扩增，如果扩增产物含有165-167bp的DNA片段、待测病原体为或候选为口蹄疫病毒，如果扩增产物含有135-137bp的DNA片段、待测病原体为或候选为蓝舌病病毒，如果扩增产物含有278-281bp的DNA片段、待测病原体为或候选为水泡性口炎病毒，如果扩增产物含有308-310bp的DNA片段、待测病原体为 或候选为牛病毒性腹泻病毒，如果扩增产物含有211-214bp的DNA片段、待测病原体为或候选为牛轮状病毒，如果扩增产物含有342-345bp的DNA片段、待测病原体为或候选为小反刍兽疫病毒，如果扩增产物含有252-254bp的DNA片段、待测病原体为或候选为产肠毒素大肠杆菌，如果扩增产物含有187-189bp的DNA片段、待测病原体为或候选为牛传染性鼻气管炎病毒；
    (e2)检测待病原体的基因组DNA或cDNA中是否含有权利要求1所述引物对Ⅰ的靶序列、所述引物对Ⅱ的靶序列、所述引物对Ⅲ的靶序列、所述引物对Ⅳ的靶序列、所述引物对Ⅴ的靶序列、所述引物对Ⅵ的靶序列、所述引物对Ⅶ的靶序列或所述引物对Ⅷ的靶序列，如果所述cDNA中含有所述引物对Ⅰ的靶序列、待测病原体为或候选为口蹄疫病毒，如果所述cDNA中含有所述引物对Ⅱ的靶序列、待测病原体为或候选为蓝舌病病毒，如果所述cDNA中含有所述引物对Ⅲ的靶序列、待测病原体为或候选为水泡性口炎病毒，如果所述cDNA中含有所述引物对Ⅳ的靶序列、待测病原体为或候选为牛病毒性腹泻病毒，如果所述cDNA中含有所述引物对Ⅴ的靶序列、待测病原体为或候选为牛轮状病毒，如果所述基因组DNA中含有所述引物对Ⅵ的靶序列、待测病原体为或候选为产肠毒素大肠杆菌，如果所述cDNA中含有所述引物对Ⅶ的靶序列、待测病原体为或候选为牛传染性鼻气管炎病毒，如果所述cDNA中含有所述引物对Ⅷ的靶序列、待测病原体为或候选为小反刍兽疫病毒。
  7. 一种检测待测样本中是否含有口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒的方法，包括如下步骤(f1)或(f2)：
    (f1)将待测样本进行基因组DNA提取操作和/或RNA提取操作，得到核酸溶液；将待测样本的含有RNA或可能含有RNA的核酸进行反转录，以得到的DNA和/或cDNA作为模板，采用所述引物组合进行PCR扩增，如果扩增产物含有165-167bp的DNA片段、待测样本含有或疑似含有口蹄疫病毒，如果扩增产物含有135-137bp的DNA片段、待测样本含有或疑似含有蓝舌病病毒，如果扩增产物含有278-281bp的DNA片段、待测样本含有或疑似含有水泡性口炎病毒，如果扩增产物含有308-310bp的DNA片段、待测样本含有 或疑似含有牛病毒性腹泻病毒，如果扩增产物含有211-214bp的DNA片段、待测样本含有或疑似含有牛轮状病毒，如果扩增产物含有342-345bp的DNA片段、待测样本含有或疑似含有小反刍兽疫病毒，如果扩增产物含有252-254bp的DNA片段、待测样本含有或疑似含有产肠毒素大肠杆菌，如果扩增产物含有187-189bp的DNA片段、待测样本含有或疑似含有牛传染性鼻气管炎病毒；
    (f2)检测待测样本的基因组DNA或cDNA中是否含有权利要求1所述引物对Ⅰ的靶序列、所述引物对Ⅱ的靶序列、所述引物对Ⅲ的靶序列、所述引物对Ⅳ的靶序列、所述引物对Ⅴ的靶序列、所述引物对Ⅵ的靶序列、所述引物对Ⅶ的靶序列或所述引物对Ⅷ的靶序列，如果所述cDNA中含有所述引物对Ⅰ的靶序列、待测样本含有或疑似含有口蹄疫病毒，如果所述cDNA中含有所述所述引物对Ⅱ的靶序列、待测样本含有或疑似含有蓝舌病病毒，如果所述cDNA中含有所述所述引物对Ⅲ的靶序列、待测样本含有或疑似含有水泡性口炎病毒，如果所述cDNA中含有所述所述引物对Ⅳ的靶序列、待测样本含有或疑似含有牛病毒性腹泻病毒，如果所述cDNA中含有所述所述引物对Ⅴ的靶序列、待测样本含有或疑似含有牛轮状病毒，如果基因组DNA含有所述引物对Ⅵ的靶序列、待测样本含有或疑似含有产肠毒素大肠杆菌，如果所述cDNA中含有所述引物对Ⅶ的靶序列、待测样本含有或疑似含有牛传染性鼻气管炎病毒，如果所述cDNA中含有所述引物对Ⅷ的靶序列、待测样本含有或疑似含有小反刍兽疫病毒。
  8. 引物组合，为如下(g1)或(g2)：
    (g1)权利要求1中的所述引物对Ⅰ或所述引物对Ⅱ或所述引物对Ⅲ或所述引物对Ⅳ或所述引物对Ⅴ或所述引物对Ⅵ或所述引物对Ⅶ或所述引物对Ⅷ；
    (g2)权利要求1中的所述引物对Ⅰ、所述引物对Ⅱ、所述引物对Ⅲ、所述引物对Ⅳ、所述引物对Ⅴ、所述引物对Ⅵ、所述引物对Ⅶ和所述引物对Ⅷ中的任意两个引物对的组合、任意三个引物对的组合、任意四个引物对的组合、任意五个引物对的组合、任意六个引物对的组合或任意七个引物对的组合。
  9. 权利要求8所述引物组合在制备试剂盒中的应用，所述试剂盒的用途 为鉴别口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒。
  10. 含有权利要求8所述引物组合的试剂盒，所述试剂盒的用途为鉴别口蹄疫病毒和/或蓝舌病病毒和/或水泡性口炎病毒和/或牛病毒性腹泻病毒和/或牛轮状病毒和/或产肠毒素大肠杆菌和/或牛传染性鼻气管炎病毒和/或小反刍兽疫病毒。
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