CN113755565B - Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying wild strain and vaccine strain of African swine fever - Google Patents
Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying wild strain and vaccine strain of African swine fever Download PDFInfo
- Publication number
- CN113755565B CN113755565B CN202110951918.4A CN202110951918A CN113755565B CN 113755565 B CN113755565 B CN 113755565B CN 202110951918 A CN202110951918 A CN 202110951918A CN 113755565 B CN113755565 B CN 113755565B
- Authority
- CN
- China
- Prior art keywords
- swine fever
- african swine
- seq
- primer
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 229960005486 vaccine Drugs 0.000 title claims abstract description 43
- 208000007407 African swine fever Diseases 0.000 title claims abstract description 31
- 239000007850 fluorescent dye Substances 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 55
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 30
- 241000700605 Viruses Species 0.000 claims abstract description 14
- 241000701386 African swine fever virus Species 0.000 claims description 55
- 101100166610 African swine fever virus (strain Badajoz 1971 Vero-adapted) Ba71V-058 gene Proteins 0.000 claims description 17
- 239000013612 plasmid Substances 0.000 claims description 16
- 238000011144 upstream manufacturing Methods 0.000 claims description 15
- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims description 10
- 238000003753 real-time PCR Methods 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 239000013642 negative control Substances 0.000 claims description 7
- 239000013641 positive control Substances 0.000 claims description 7
- UDGUGZTYGWUUSG-UHFFFAOYSA-N 4-[4-[[2,5-dimethoxy-4-[(4-nitrophenyl)diazenyl]phenyl]diazenyl]-n-methylanilino]butanoic acid Chemical compound COC=1C=C(N=NC=2C=CC(=CC=2)N(C)CCCC(O)=O)C(OC)=CC=1N=NC1=CC=C([N+]([O-])=O)C=C1 UDGUGZTYGWUUSG-UHFFFAOYSA-N 0.000 claims description 6
- 108700026220 vif Genes Proteins 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 230000035945 sensitivity Effects 0.000 abstract description 5
- 108020004414 DNA Proteins 0.000 description 25
- 230000003321 amplification Effects 0.000 description 21
- 238000003199 nucleic acid amplification method Methods 0.000 description 21
- 238000012224 gene deletion Methods 0.000 description 11
- 238000001514 detection method Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 125000006853 reporter group Chemical group 0.000 description 8
- 238000012408 PCR amplification Methods 0.000 description 7
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 230000001018 virulence Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 108700005084 Multigene Family Proteins 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 241000282898 Sus scrofa Species 0.000 description 3
- 238000012217 deletion Methods 0.000 description 3
- 230000037430 deletion Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000010791 quenching Methods 0.000 description 3
- 230000000171 quenching effect Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- ABZLKHKQJHEPAX-UHFFFAOYSA-N tetramethylrhodamine Chemical compound C=12C=CC(N(C)C)=CC2=[O+]C2=CC(N(C)C)=CC=C2C=1C1=CC=CC=C1C([O-])=O ABZLKHKQJHEPAX-UHFFFAOYSA-N 0.000 description 3
- 241000710777 Classical swine fever virus Species 0.000 description 2
- 241000710842 Japanese encephalitis virus Species 0.000 description 2
- 241000202347 Porcine circovirus Species 0.000 description 2
- 241000702619 Porcine parvovirus Species 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 108091036078 conserved sequence Proteins 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 241000238888 Argasidae Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 206010020741 Hyperpyrexia Diseases 0.000 description 1
- 101150003837 MGF gene Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000002238 attenuated effect Effects 0.000 description 1
- 229940031567 attenuated vaccine Drugs 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 208000027744 congestion Diseases 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001351 cycling effect Effects 0.000 description 1
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000013615 primer Substances 0.000 description 1
- 239000002987 primer (paints) Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000010025 steaming Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 229940125575 vaccine candidate Drugs 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000009278 visceral effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12Q1/70—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
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a quadruple quantitative fluorescent probe primer combination, a kit and an identification method for identifying African swine fever wild virus and vaccine strains, and belongs to the technical field of molecular biology. The invention discloses a four-fold quantitative fluorescent probe primer combination for identifying African swine fever wild virus and vaccine strain, which comprises the following steps: (a) A first primer probe combination shown in SEQ ID NO. 1-SEQ ID NO. 3; (b) A second primer probe combination shown in SEQ ID NO. 4-SEQ ID NO. 6; (c) A third primer probe combination shown in SEQ ID NO. 7-SEQ ID NO. 9; and (d) a fourth primer probe combination shown as SEQ ID NO. 10-SEQ ID NO. 12. The kit and the identification method can simultaneously identify four genes of the African swine fever wild strain and the vaccine strain, primers of different genes have no interference with each other, and the kit and the identification method have the characteristics of high sensitivity, strong specificity, good repeatability and good stability.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a quadruple quantitative fluorescent probe primer combination, a kit and an identification method for identifying african swine fever wild virus and vaccine strain.
Background
African swine fever (African swine fever, ASF) is an acute, febrile, highly contagious disease in pigs caused by African swine fever virus (African swine fever virus, ASFV). Pigs and wild boars are generally susceptible, and soft ticks are the storage hosts and mediums of ASFV. The incubation period of ASFV is 5-15 days, and once the outbreak disease course is short, the death rate is high and can reach 100%. The symptoms in situ are complex and indistinguishable from other diseases, and are often manifested as hyperpyrexia, skin congestion, abortion, edema, and visceral hemorrhage. However, there is no effective therapeutic measure after the onset of African swine fever, and the virus has a complex immune evasion mechanism, lacks typical neutralizing antibodies, and is not effective for vaccine immunization.
ASFV is a positive 20-face virus with a diameter of about 170-200nm, consisting of double-stranded DNA inside and capsid and envelope around the periphery, and is about 170-190 kb in size. The two ends of the ASFV genome form hairpin loops through partial base pairing, the middle region is relatively conservative, the parts of the two ends close to the hairpin loops are provided with terminal repeated sequences and variable regions, and the size change of the ASFV genome is determined by the variable regions.
The current domestic mainstream vaccine takes the separated African swine fever epidemic strain (Pig/CN/HLJ/2018) of Heilongjiang as a parent by the high-grade of Harbin veterinary research institute of China academy of agricultural sciences to construct attenuated live vaccines with MGF360-505R gene deletion and CD2V (EP 402R) and MGF360-505R double gene combined deletion, which are named rASFV delta 360-eGFP and rASFV delta CD2V/360-eGFP-mCherry respectively. In addition, the strain (SY 18) separated from the first African swine fever epidemic situation in China is taken as a parent strain, an African swine fever virus gene deletion vaccine candidate strain with MGF gene and CD2V (EP 402R) gene deletion is constructed, and the main deleted virulence genes are multi-gene family MGF360-505 (MGF 360-9L, MGF360-10L, MGF360-11L, MGF360-12L, MGF360-13L, MGF360-14L, 505-1R, 505-2R, 505-3R) and EP402R, so that the method for rapidly, sensitively and accurately identifying the African swine fever wild strain and the gene deletion vaccine strain is particularly important because the African swine fever wild strain infection and the vaccine infection can be greatly different from epidemic prevention and control measures and tooth extraction methods of a swine farm along with the market of the vaccine in the future.
Disclosure of Invention
The invention aims to provide a four-fold quantitative fluorescent probe primer combination, a kit and an identification method for identifying African swine fever wild virus and vaccine strains.
In order to achieve the above object, the present invention adopts the following technical scheme:
a set of four quantitative fluorescent probe primer combinations for identifying african swine fever wild strains and vaccine strains, comprising: primer probe combinations for detecting special EGFP fluorescent reporter genes of African swine fever virus B646L genes, african swine fever virus EP402R genes, african swine fever virus MGF360-14L genes and African swine fever virus vaccine strains;
the primer probe combination for detecting the African swine fever virus B646L gene has the nucleotide sequence as follows:
upstream primer B646L-F: as shown in SEQ ID NO. 1
Downstream primer B646L-R: as shown in SEQ ID NO. 2
Fluorescent probe B646L-P: as shown in SEQ ID NO. 3;
the primer probe combination for detecting the African swine fever virus EP402R gene has the nucleotide sequence as follows:
the upstream primer EP402R-F: as shown in SEQ ID NO. 4
Downstream primer EP402R-R: as shown in SEQ ID NO. 5
Fluorescent probe EP402R-P: as shown in SEQ ID NO. 6;
the primer probe combination for detecting the African swine fever virus MGF360-14L gene has the nucleotide sequence as follows:
the upstream primer MGF360-F: as shown in SEQ ID NO. 7
Downstream primer MGF360-R: as shown in SEQ ID NO. 8
Fluorescent probe MGF360-P: as shown in SEQ ID NO. 9;
the primer probe combination for detecting the EGFP gene of the African swine fever virus vaccine has the nucleotide sequence as follows:
the upstream primer eGFP-F: as shown in SEQ ID NO. 10
The downstream primer eGFP-R: as shown in SEQ ID NO. 11
Fluorescent probe eGFP-P: as shown in SEQ ID NO. 12.
Further, the probe is a hydrolysis probe, and a fluorescent reporter group is marked at the 5 'end and a quenching group is marked at the 3' end of the probe.
Further, the fluorescent reporter group is selected from any one of FAM, HEX, cy and ROX; the quenching group is selected from any one of BHQ1, TAMRA and BHQ2.
Further, the 5' -end labeled fluorescent reporter groups of different specific probes are different.
A kit for identifying the quadruple fluorescence quantitative PCR of African swine fever wild virus and vaccine strain comprises the primer probe combination.
Further, the kit further comprises a positive control. The positive control comprises a recombinant plasmid containing an African swine fever virus B646L gene, a recombinant plasmid containing an African swine fever virus EP402R gene, a recombinant plasmid containing an African swine fever virus MGF360-14L gene and a plasmid containing a fluorescent protein EGFP gene.
Further, the kit further comprises a negative control, wherein the negative control is water.
A method for identifying African swine fever wild strain and vaccine strain comprises performing real-time fluorescence quantitative PCR (polymerase chain reaction) by using the primer probe combination or the kit, collecting fluorescence signals, and determining whether the sample contains African swine fever virus wild strain and/or vaccine strain.
The reaction conditions of the real-time fluorescent quantitative PCR are as follows: 94-96 ℃ for 2-3min;94-96 deg.c for 25-30s; 58-60 ℃ for 30-35s;40-42 cycles.
Compared with the prior art, the invention has the following characteristics:
1. because virulence genes EP402R and MGF deleted from the African swine fever gene deletion vaccine strain are located in the variable region of ASFV, mutation easily occurs in the genetic evolution process, and if the CT value difference is too large, fluorescence inhibition can be caused, so that the detection rate is influenced in the detection process. In the highly conserved sequence with shorter virulence gene, finding out proper primer probe sequence and approaching the amplification efficiency, and simultaneously, adjusting the proportion of the primer and the fluorescent probes to further ensure that the four pairs of fluorescent probes maintain more consistent amplification efficiency.
2. Aiming at the mainstream African swine fever gene deletion vaccine strain developed in China at present, the vaccine strain comprises MGF360 gene deletion vaccine and MGF360-505 gene deletion vaccine, and EP402R single gene deletion vaccine and MGF360-505 and EP402R combined deletion vaccine; the deleted virulence genes are multi-gene families MGF360-505 (comprising MGF360-9L, MGF360-10L, MGF360-11L, MGF360-12L, MGF360-13L, MGF360-14L, MGF505-1R, MGF505-2R, MGF-3R) and EP402R of the African swine fever virus of the type II epidemic gene in China, wherein most of the deleted virulence genes are found to have low homology and more mutation sites after the deletion genes of the multi-gene families are compared, and only one segment of highly conserved gene sequence in MGF360-14L is selected for primer design.
3. When the African swine fever wild virus and the vaccine virus are co-infected, whether the gene-deleted vaccine virus infection exists or not cannot be determined by identifying the deleted genes.
4. The invention has stronger specificity, has no non-specific amplification curve for swine fever virus, porcine pseudorabies virus, porcine reproductive and respiratory syndrome virus, porcine circovirus, porcine parvovirus and porcine Japanese encephalitis virus, and ensures the detection accuracy.
5. The invention establishes a detection kit for rapidly identifying the African swine fever wild strain and the vaccine strain, and has the characteristics of high sensitivity, strong specificity, good repeatability and good stability.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing fluorescence amplification of the African swine fever virus B646L gene.
FIG. 2 is a standard curve of the African swine fever virus B646L gene.
FIG. 3 is a graph showing fluorescence amplification of the EP402R gene of African swine fever virus.
FIG. 4 is a standard graph of the African swine fever virus EP402R gene.
FIG. 5 is a graph showing fluorescence amplification of the MGF360 gene of African swine fever virus.
FIG. 6 is a standard graph of the MGF360 gene of African swine fever virus.
FIG. 7 is a graph showing fluorescence amplification of EGFP gene of African swine fever vaccine strain.
FIG. 8 is a standard graph of EGFP gene for African swine fever vaccine strain.
FIG. 9 is a graph of fluorescence amplification curves from a specificity experiment.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Aiming at the African swine fever gene deletion strain developed in China at present, the invention selects the gene sequence of B646L, EP, 402, R, MGF and 360-14L of African swine fever and the EGFP fluorescent reporter gene specific to the attenuated vaccine strain of the African swine fever gene deletion, designs and synthesizes specific primers and probes for the four genes, establishes a method for identifying the wild strain of the African swine fever and the vaccine strain, and develops a kit convenient to diagnose. The kit and the identification method can simultaneously identify four genes of the African swine fever wild strain and the vaccine strain, primers of different genes have no interference with each other, and the kit and the identification method have the characteristics of high sensitivity, strong specificity, good repeatability and good stability.
The 5 '-end of the fluorescent probe B646L-P, MGF360-P, EP402R-P, eGFP-P can be marked with any one of fluorescent reporter groups FAM, HEX, cy and ROX, and the 3' -end of the fluorescent probe B646L-P, MGF R-P, EP R-P, eGFP-P can be marked with any one of fluorescent quenching groups BHQ1, TAMRA and BHQ2.
The fluorescent probes B646L-P, MGF360-P, EP402R-P, eGFP-P are labeled with three different fluorescent groups, respectively.
Specifically:
the gene sequence of the African swine fever virus B646L is as follows:
GCAGATGCCGATACCACAAGATCAGCCGTAGTGATAGACCCCACGTAATCCGTGTCCCAACTAATATAAAATTCTCTTGCTCTGGATACGTTAATATGACCACTGGGTTGGTATTCCTCCCGTGG(SEQ ID NO:13)
the primer probe combination for detecting the African swine fever virus B646L gene comprises:
upstream primer B646L-F:5'-CCACGGGAGGAATACCAA-3' (SEQ ID NO: 1)
Downstream primer B646L-R:5'-GCAGATGCCGATACCACA-3' (SEQ ID NO: 2)
Fluorescent probe B646L-P: FAM-TCATATTAACGTATCCAGAGCAAGA-BHQ1 (SEQ ID NO: 3);
the gene sequence of the African swine fever virus EP402R is as follows:
TGAAGAAATAGAAAGTCCACCACCTGAATCTAATGAAGAAGAACAATGTCAGCATGATGACACCACTTCCATACATGAACCATCTCCCAGAGAACCATTACTTCCTAAGCCTTACAGTCGTTATCAGTATAATACACCTATTTACTACATGCGTC(SEQ ID NO:14)
primer probe group for detecting the EP402R gene of African swine fever virus and the method comprise the following steps:
the upstream primer EP402R-F:5'-TGAAGAAATAGAAAGTCCACC-3' (SEQ ID NO: 4)
Downstream primer EP402R-R:5'-GACGCATGTAGTAAATAGGTGTA-3' (SEQ ID NO: 5)
Fluorescent probe EP402R-P: HEX-AGGCTTAGGAAGTAATGGTTCTCTGGG-TRAMRA (SEQ ID NO: 6);
the gene sequence of the African swine fever virus MGF360-14L is as follows:
TATTTGTGGATTGGGGCGCAAATCCTGAATATGGGCTTATACGTGTTCCTGCCGTGTATCTAAAGCGGCTGTGTGCGGAACTGGGAGGCTTAACGCCTGTATCCGAACCC(SEQ ID NO:15)
the primer probe combination for detecting the African swine fever virus MGF360-14L gene comprises:
the upstream primer MGF360-F:5'-TATTTGTGGATTGGGGCG-3' (SEQ ID NO: 7)
Downstream primer MGF360-R:5'-GGGTTCGGATACAGGCGT-3' (SEQ ID NO: 8)
Fluorescent probe MGF360-P: cy5-AGCCTCCCAGTTCCGCACACAGCCGCTT-BHQ2 (SEQ ID NO: 9);
the MGF360-P fluorescence reporter gene has the following sequence:
TATTTGTGGATTGGGGCGCAAATCCTGAATATGGGCTTATACGTGTTCCTGCCGTGTATCTAAAGCGGCTGTGTGCGGAACTGGGAGGCTTAACGCCTGTATCCGAACCC(SEQ ID NO:16)
primer probe combinations for detecting EGFP fluorescence reports include:
the upstream primer eGFP-F:5'-ACCCTCGTGACCACCCTGA-3' (SEQ ID NO: 10)
The downstream primer eGFP-R:5'-TAGTTGCCGTCGTCCTTGA-3' (SEQ ID NO: 11)
Fluorescent probe eGFP-P: ROX-AGTGCTTCAGCCGCTACCCCGA-BHQ2 (SEQ ID NO: 12).
The invention also provides a quadruple fluorescence PCR method for identifying and detecting African swine fever virus wild strain and vaccine strain by using the special primer and the probe set, which comprises the following steps:
1. extracting total DNA of the sample to be detected for standby.
2. Preparing a reaction system: sample DNA 5. Mu.L, primer premix 4. Mu.L (400 nM), probe premix 2. Mu.L (200 nM), PCR amplification solution 12.5. Mu.L, ddH 2 O 1.5μL。
3. Amplification procedure
The cycle is 10s at 95 ℃ and 30s at 58 ℃ for 40 cycles in total, wherein the cycle is 2min at 37 ℃ and 30s at 95 ℃. A total of 4 fluorescent channels, respectively: a reporter group "FAM", a quencher group "BHQ1"; a reporter group "HEX", a quencher group "TAMRA"; reporter group "Cy5", quencher group "BHQ2"; reporter group "ROX", quencher group "BHQ2".
4. Result determination
The Ct value of the positive control four channels is less than 30, a specific S-shaped amplification curve appears, the negative control has no Ct value and no specific amplification curve, and the establishment of the Ct value and the specific amplification curve can judge that the experimental result is established.
Detection result judgment standard:
the Ct value of the detected sample is less than or equal to 35, and a specific S-shaped amplification curve appears, and the detected sample is judged to be positive to the African swine fever virus nucleic acid; no Ct value and no specific amplification curve, and judging that the African swine fever virus nucleic acid is negative; and (3) judging that the African swine fever virus nucleic acid is suspicious by a specific amplification curve with a CT value of 35 < 40, re-sampling and extracting DNA from the suspicious sample, and rechecking, wherein the Ct value is less than 40, and judging positive, otherwise, judging negative.
The invention also provides a kit prepared by using the four groups of primers and probes, and the kit can be used for assisting in identifying the African swine fever virus wild strain and the vaccine strain and/or detecting whether the sample contains the African swine fever virus wild strain and/or the vaccine strain.
The kit also comprises the following reagents: positive control, negative control, primer premix, probe premix, PCR amplification solution;
positive control: recombinant plasmid containing B646L, EP402R and MGF360-14L gene fragment and plasmid pCMV-C-EGFP;
negative control: double steaming;
primer premix: comprises a premix solution with an initial concentration of 10 mu M of MGF360-F, MGF360-R, EP402R-F, EP402R-R, B646L-F, B646L-R, eGFP-F, eGFP-R in a molar ratio of 1:1:1:1:1:1:1:1:1;
probe premix: MGF360-P, EP402R-P, EP402R-P, eGFP-P molar ratio of 1:1:1:1 premix at an initial concentration of 10 μm;
the PCR amplification solution comprises: 2X Animal Detection Probe Master Mix.
The invention also provides a method for detecting whether the sample contains African swine fever virus wild strain and vaccine strain. And carrying out PCR amplification reaction on the nucleic acid extracted from the sample to be tested by using the primer probe combination or the kit, collecting fluorescent signals, and determining whether the sample to be tested contains the African swine fever virus wild strain and/or the vaccine strain.
The technical scheme of the invention is described in detail below. The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.
Example 1
1. Preparation of positive standard
Positive standards of B646L, EP402R and MGF360-14L extract DNA with positive samples of known ASFV, and extract total DNA according to DNA virus extraction kit of radix RhizomatisA25. Mu.L system was used, and the reaction system included: 2 XPCR Mix 10. Mu.L, upstream primer 1. Mu.L, downstream primer 1. Mu. L, DNA 2. Mu.L, TRUEscript Enzyme Mix 0.8.8. Mu.L, RNase free H 2 O (nuclease free water) 10.2. Mu.L. PCR amplification procedure: 94 ℃ for 5min; cycling at 94 ℃ for 30s,55 ℃ for 30s, and 72 ℃ for 30s, for 30 cycles; and then extending at 72 ℃ for 10min. After amplification was completed, all products were identified by 1% agarose gel electrophoresis. And (3) purifying and recovering the PCR product identified as positive by using a root gel recovery kit, connecting the PCR product to a pEASY-T1 vector and transforming the PCR product into DH5 alpha competent cells, picking up positive clones, carrying out shaking amplification by using LB culture solution, and then sending bacterial solution to Shanghai biological engineering Co.
The positive standard for eGFP used the plasmid pCMV-eGFP stored in the laboratory.
2. Quadruple fluorescence quantitative PCR condition optimization
Extracting standard plasmids with plasmid extraction kit of radix et rhizoma Nardostachyos, detecting plasmid template concentration with Nanodrop 2000 nucleic acid concentration meter, respectively performing 10-fold gradient dilution on the four standard plasmids, and collecting the concentration 10 4 The primer and the corresponding probes are mixed respectively at different final probe concentrations and final primer concentrations, 0.5 mu L, 0.25 mu L (200 nM:100 nM), 0.5 mu L (200 nM:200 nM), 1 mu L, 0.5 mu L (400 nM:200 nM), 1 mu L and 1 mu L (400 nM:400 nM), and the applied primer concentrations are 10 mu M, and the amplification procedure provided by the invention is carried out by adding 0.5 mu L, 0.25 mu L (200 nM:100 nM), 0.5 mu L,1 mu L, 0.5 mu L (400 nM:200 nM), 1 mu L, and 1 mu L (400 nM), respectively, to the primers (upstream and downstream) and the corresponding probes, respectively, for 40 cycles, and performing PCR amplification to obtain the lowest CT value and the optimal ratio of the primer probe concentration when the fluorescence intensity value is increased at a higher value of 95 ℃ for 10 mu M, at 37 ℃ for 30 seconds.
Primer concentration and probe concentration grouping experiments:
as a result of experimental results, the best reaction system was found by performing experiments using different combinations of probe and primer concentrations, and it was found that Cy5 and ROX fluorescence intensities were highest and CT values were also lowest in the multiplex qPCR reaction when the final probe concentration was 200nM and the final primer concentration was 400nM (since Cy5 and ROX fluorescence itself was relatively weak in the multiplex detection method, the primer probe combination having the highest Cy5 and ROX fluorescence intensities was selected as the best combination).
Respectively performing 10-fold gradient dilution on four standard plasmids to obtain templates with concentration of 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 Sensitivity determination was performed using a single qPCR reaction system: DNA template 5. Mu.L, upstream and downstream primer 1. Mu.L (400 nM), probe 0.5. Mu.L (200 nM), PCR amplification solution 12.5. Mu.L, ddH 2 O6. Mu.L, all primer probes were applied at a concentration of 10. Mu.M. The amplification procedure was carried out at 37℃for 2min, at 95℃for 30s, at 95℃for 10s, and at 58℃for 30s (fluorescence signal was collected), for a total of 40 cycles, and PCR amplification was carried out and the standard curve thereof was analyzed.
As shown in FIGS. 1-8, in the current concentration range of dilution, the template quantity and the corresponding Ct value have a better linear relation, and the correlation coefficient R 2 The fluorescent quantitative method has higher amplification efficiency (99.13% -101.25%) for different virulence genes, and the minimum detection amount of the fluorescent quantitative PCR is 10 2 The fluorescence quantitative PCR established by the invention has higher sensitivity.
3. Repeatability test
Positive standard substances diluted in 10-time gradient of 8 concentrations are respectively used as templates, and the final concentrations are respectively 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 The fluorescence quantitative PCR was performed with a reaction system and a procedure for providing fluorescence quantification, 3 replicates were set for each gradient, and the reproducibility of the method was verified. The results show that the variation coefficient (CV value) of the repeated experiments of the invention is below 0.5%, which shows that the invention has good repeatability.
4. Specificity test
Taking positive samples of swine fever virus, porcine pseudorabies virus, porcine blue-ear virus, porcine circovirus, porcine parvovirus and porcine Japanese encephalitis virus which are preserved by the inventor as templates, and carrying out fluorescent quantitative PCR amplification by using the primers and the probes. The results are shown in FIG. 9, and the detection results of different signal channels in the system are all negative, which shows that the method has strong specificity and no cross reaction with other main infectious pathogens.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Sequence listing
<110> Fujian Aono Biotechnology group Co., ltd
<120> a four-fold quantitative fluorescent probe primer combination, kit and identification method for identifying African swine fever wild virus and vaccine strain
<130> 20210806
<141> 2021-08-18
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
ccacgggagg aataccaa 18
<210> 2
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 2
gcagatgccg ataccaca 18
<210> 3
<211> 25
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 3
tcatattaac gtatccagag caaga 25
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 4
tgaagaaata gaaagtccac c 21
<210> 5
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 5
gacgcatgta gtaaataggt gta 23
<210> 6
<211> 27
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 6
aggcttagga agtaatggtt ctctggg 27
<210> 7
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
tatttgtgga ttggggcg 18
<210> 8
<211> 18
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gggttcggat acaggcgt 18
<210> 9
<211> 28
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
agcctcccag ttccgcacac agccgctt 28
<210> 10
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
accctcgtga ccaccctga 19
<210> 11
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
tagttgccgt cgtccttga 19
<210> 12
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
agtgcttcag ccgctacccc ga 22
<210> 13
<211> 125
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
gcagatgccg ataccacaag atcagccgta gtgatagacc ccacgtaatc cgtgtcccaa 60
ctaatataaa attctcttgc tctggatacg ttaatatgac cactgggttg gtattcctcc 120
cgtgg 125
<210> 14
<211> 155
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
tgaagaaata gaaagtccac cacctgaatc taatgaagaa gaacaatgtc agcatgatga 60
caccacttcc atacatgaac catctcccag agaaccatta cttcctaagc cttacagtcg 120
ttatcagtat aatacaccta tttactacat gcgtc 155
<210> 15
<211> 110
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
tatttgtgga ttggggcgca aatcctgaat atgggcttat acgtgttcct gccgtgtatc 60
taaagcggct gtgtgcggaa ctgggaggct taacgcctgt atccgaaccc 110
<210> 16
<211> 110
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
tatttgtgga ttggggcgca aatcctgaat atgggcttat acgtgttcct gccgtgtatc 60
taaagcggct gtgtgcggaa ctgggaggct taacgcctgt atccgaaccc 110
Claims (4)
1. A four-fold quantitative fluorescent probe primer combination for identifying african swine fever wild virus and vaccine strains, which is characterized in that: comprising the following steps: primer probe combinations for detecting African swine fever virus B646L gene, african swine fever virus EP402R gene, african swine fever virus MGF360-14L gene and African swine fever virus vaccine EGFP gene;
the primer probe combination for detecting the African swine fever virus B646L gene has the nucleotide sequence as follows:
upstream primer B646L-F: as shown in SEQ ID NO. 1
Downstream primer B646L-R: as shown in SEQ ID NO. 2
Fluorescent probe B646L-P: as shown in SEQ ID NO. 3;
fluorescent probe B646L-P has FAM marked on the 5 '-end and BHQ1 marked on the 3' -end;
the primer probe combination for detecting the African swine fever virus EP402R gene has the nucleotide sequence as follows:
the upstream primer EP402R-F: as shown in SEQ ID NO. 4
Downstream primer EP402R-R: as shown in SEQ ID NO. 5
Fluorescent probe EP402R-P: as shown in SEQ ID NO. 6;
the 5 '-end of the fluorescent probe EP402R-P is marked with HEX, the 3' -end is marked with TRAMRA;
the primer probe combination for detecting the African swine fever virus MGF360-14L gene has the nucleotide sequence as follows:
the upstream primer MGF360-F: as shown in SEQ ID NO. 7
Downstream primer MGF360-R: as shown in SEQ ID NO. 8
Fluorescent probe MGF360-P: as shown in SEQ ID NO. 9;
the 5 '-end of the fluorescent probe MGF360-P is marked with Cy5, and the 3' -end is marked with BHQ2;
the primer probe combination for detecting the EGFP gene of the African swine fever virus vaccine has the nucleotide sequence as follows:
the upstream primer eGFP-F: as shown in SEQ ID NO. 10
The downstream primer eGFP-R: as shown in SEQ ID NO. 11
Fluorescent probe eGFP-P: as shown in SEQ ID NO. 12;
the fluorescent probe eGFP-P was labeled with ROX at the 5 '-end and BHQ2 at the 3' -end.
2. A kit for identifying african swine fever wild virus and vaccine strains by quadruple fluorescent quantitative PCR, characterized in that: the kit comprising the probe primer combination of claim 1.
3. The kit of claim 2, wherein: the kit also comprises a positive control, wherein the positive control comprises a recombinant plasmid containing an African swine fever virus B646L gene, a recombinant plasmid containing an African swine fever virus EP402R gene, a recombinant plasmid containing an African swine fever virus MGF360-14L gene and a plasmid containing a fluorescent protein EGFP gene.
4. The kit of claim 2, wherein: the kit further comprises a negative control, wherein the negative control is water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110951918.4A CN113755565B (en) | 2021-08-18 | 2021-08-18 | Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying wild strain and vaccine strain of African swine fever |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110951918.4A CN113755565B (en) | 2021-08-18 | 2021-08-18 | Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying wild strain and vaccine strain of African swine fever |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113755565A CN113755565A (en) | 2021-12-07 |
| CN113755565B true CN113755565B (en) | 2024-03-26 |
Family
ID=78790426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110951918.4A Active CN113755565B (en) | 2021-08-18 | 2021-08-18 | Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying wild strain and vaccine strain of African swine fever |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113755565B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115094164A (en) * | 2022-06-21 | 2022-09-23 | 广西壮族自治区动物疫病预防控制中心 | Multiple qPCR (quantitative polymerase chain reaction) kit and detection method for ASFV (advanced specific immunodeficiency syndrome) with different gene deletion types |
| CN116004925A (en) * | 2023-02-17 | 2023-04-25 | 江苏农牧科技职业学院 | Triple fluorescence PCR primer probe group, reagent and method for identifying wild strain and gene deletion strain of African swine fever virus |
| CN116656874A (en) * | 2023-05-31 | 2023-08-29 | 中国兽医药品监察所 | Quadruple quantitative fluorescent primer probe composition, kit and identification method for identifying African swine fever virus |
| CN117701779A (en) * | 2024-02-04 | 2024-03-15 | 湖南派智生物科技有限公司 | Method for identifying African swine fever strain, primer probe combination, reagent, kit and application |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110872637A (en) * | 2019-12-04 | 2020-03-10 | 深圳海关动植物检验检疫技术中心 | Reagent for identifying African swine fever gene deletion vaccine, detection method and application |
| CN112646934A (en) * | 2021-01-21 | 2021-04-13 | 华南农业大学 | Triple fluorescent quantitative PCR (polymerase chain reaction) detection primer and kit for identifying African swine fever wild strains and gene deletion strains |
| CN112795706A (en) * | 2021-03-30 | 2021-05-14 | 福建傲农生物科技集团股份有限公司 | Fluorescent probe primer group and kit for African swine fever virus P72 gene and application of fluorescent probe primer group and kit |
| CN112831598A (en) * | 2019-11-22 | 2021-05-25 | 洛阳普泰生物技术有限公司 | Real-time fluorescent PCR amplification primer pair and probe primer for African swine fever virus identification and detection and prepared kit |
-
2021
- 2021-08-18 CN CN202110951918.4A patent/CN113755565B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112831598A (en) * | 2019-11-22 | 2021-05-25 | 洛阳普泰生物技术有限公司 | Real-time fluorescent PCR amplification primer pair and probe primer for African swine fever virus identification and detection and prepared kit |
| CN110872637A (en) * | 2019-12-04 | 2020-03-10 | 深圳海关动植物检验检疫技术中心 | Reagent for identifying African swine fever gene deletion vaccine, detection method and application |
| CN112646934A (en) * | 2021-01-21 | 2021-04-13 | 华南农业大学 | Triple fluorescent quantitative PCR (polymerase chain reaction) detection primer and kit for identifying African swine fever wild strains and gene deletion strains |
| CN112795706A (en) * | 2021-03-30 | 2021-05-14 | 福建傲农生物科技集团股份有限公司 | Fluorescent probe primer group and kit for African swine fever virus P72 gene and application of fluorescent probe primer group and kit |
Non-Patent Citations (1)
| Title |
|---|
| Shimozono,S.等.Accession NO: AB787563.1，Synthetic construct gepra-g gene for GEPRA-G, complete cds.GenBank.2013,1-2. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113755565A (en) | 2021-12-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113755565B (en) | Quadruple quantitative fluorescent probe primer combination, kit and identification method for identifying wild strain and vaccine strain of African swine fever | |
| CN107299155B (en) | Primer and probe for real-time fluorescence quantitative PCR detection of goose astrovirus | |
| CN107385111B (en) | Real-time fluorescent quantitative PCR (polymerase chain reaction) detection primer of goose astrovirus and kit thereof | |
| CN108384899B (en) | Fluorescent quantitative PCR kit for detecting novel goose astrovirus and application thereof | |
| CN112795706A (en) | Fluorescent probe primer group and kit for African swine fever virus P72 gene and application of fluorescent probe primer group and kit | |
| CN110669870A (en) | Real-time fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer, probe and detection kit for serotype of Palima serogroup virus | |
| CN114350848B (en) | Dual fluorescent probe primer combination for identifying African swine fever type I strain and type II strain, kit and application thereof | |
| CN113564280A (en) | RAA primer for detecting 12 serotypes of avian adenovirus group I and detection method thereof | |
| CN115896348A (en) | Primer and probe for dual TaqMan fluorescent quantitative PCR (polymerase chain reaction) of canine distemper virus and canine coronavirus and application of primer and probe | |
| CN105463136A (en) | Kit for RT-PCR typing detection of avian infectious bronchitis virus | |
| CN111118215A (en) | Fluorescent PCR (polymerase chain reaction) primer, probe and kit for detecting capripoxvirus virus | |
| CN113122655A (en) | TaqMan fluorescent quantitative PCR (polymerase chain reaction) detection method for African swine fever virus EP402R gene | |
| CN110643740B (en) | Real-time fluorescent quantitative RT-PCR detection primer, probe and detection kit for Pariemam serogroup virus | |
| CN117025846A (en) | Primer group for detecting novel coronavirus by multiple ddPCR and application thereof | |
| CN113073147B (en) | Primer and kit for B-type fluorescence quantitative detection of pigeon adenovirus | |
| CN113046482B (en) | Pigeon adenovirus B-type loop-mediated isothermal amplification detection primer set and kit | |
| CN112626278B (en) | Primer and probe for identifying canine distemper virus wild strain and vaccine strain and application | |
| CN111893218B (en) | Primer and probe for real-time fluorescent quantitative PCR detection of duck hepatitis C virus | |
| CN115094164A (en) | Multiple qPCR (quantitative polymerase chain reaction) kit and detection method for ASFV (advanced specific immunodeficiency syndrome) with different gene deletion types | |
| CN111500774B (en) | Epidemic hemorrhagic disease virus and serotype identification RT-PCR kit | |
| CN109266786B (en) | E184L gene-based African swine fever virus detection kit and detection method | |
| CN109136409B (en) | Kit and method for detecting African swine fever virus based on K196R gene | |
| CN113046481A (en) | Primer, probe and kit for B-type fluorescence quantitative detection of pigeon adenovirus | |
| CN112795697A (en) | Primer pair, kit and detection method for simultaneously detecting multiple infectious bronchitis viruses of chicken | |
| CN107604102B (en) | Double Real time PCR detection kit for pigeon TTV and novel pigeon adenovirus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |