CN116144836B - Quadruple fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer set for PRRSV (porcine reproductive and respiratory syndrome virus) and detection method - Google Patents
Quadruple fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer set for PRRSV (porcine reproductive and respiratory syndrome virus) and detection method Download PDFInfo
- Publication number
- CN116144836B CN116144836B CN202211143428.2A CN202211143428A CN116144836B CN 116144836 B CN116144836 B CN 116144836B CN 202211143428 A CN202211143428 A CN 202211143428A CN 116144836 B CN116144836 B CN 116144836B
- Authority
- CN
- China
- Prior art keywords
- prrsv2
- prrsv
- quadruple
- pcr
- fluorescent
- 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
- 238000001514 detection method Methods 0.000 title claims abstract description 58
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 title claims abstract description 56
- 238000003757 reverse transcription PCR Methods 0.000 title claims abstract description 29
- 239000000523 sample Substances 0.000 claims abstract description 51
- 239000013612 plasmid Substances 0.000 claims description 34
- 238000000034 method Methods 0.000 claims description 30
- 108020004707 nucleic acids Proteins 0.000 claims description 15
- 150000007523 nucleic acids Chemical class 0.000 claims description 15
- 102000039446 nucleic acids Human genes 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 238000000137 annealing Methods 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
- 238000010839 reverse transcription Methods 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 2
- 238000003745 diagnosis Methods 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 11
- 241001123971 Porcine reproductive and respiratory syndrome virus 1 Species 0.000 abstract description 9
- 208000015181 infectious disease Diseases 0.000 abstract description 3
- 210000001557 animal structure Anatomy 0.000 abstract description 2
- 230000005540 biological transmission Effects 0.000 abstract description 2
- 210000004369 blood Anatomy 0.000 abstract description 2
- 239000008280 blood Substances 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 210000000214 mouth Anatomy 0.000 abstract 1
- 210000001331 nose Anatomy 0.000 abstract 1
- 238000003753 real-time PCR Methods 0.000 description 18
- 241000700605 Viruses Species 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 7
- 239000012634 fragment Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- 101710144128 Non-structural protein 2 Proteins 0.000 description 5
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 description 5
- 102100022648 Reticulon-2 Human genes 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 241000202347 Porcine circovirus Species 0.000 description 4
- 101710110895 Uncharacterized 7.3 kDa protein in cox-rep intergenic region Proteins 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
- 241000710777 Classical swine fever virus Species 0.000 description 3
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 3
- 241000702619 Porcine parvovirus Species 0.000 description 3
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 108091093088 Amplicon Proteins 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010017472 Fumbling Diseases 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101150051014 NSP2 gene Proteins 0.000 description 1
- 101150062176 ORF5 gene Proteins 0.000 description 1
- 101150082475 ORF7 gene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 101150023114 RNA1 gene Proteins 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 206010058874 Viraemia Diseases 0.000 description 1
- 108020005202 Viral DNA Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000001165 lymph node Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000002741 palatine tonsil Anatomy 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 244000052613 viral pathogen Species 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 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
- C12Q1/70—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
- 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
- C12Q1/6851—Quantitative amplification
-
- 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
-
- 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
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a quadruple fluorescent quantitative RT-PCR detection primer set and a detection method of PRRSV, wherein the detection primer set comprises a primer pair (PRRSV-F/R) for identifying PRRSV1 and PRRSV2, 2 probes (PRRSV 1-P/PRRSV 2-P), and a primer pair (CA-NSP 2-F/R, HP-NSP 2-F/R) for identifying PRRSV2 subtype CA-PRRSV, HP-PRRSV and N-PRRSV, and 2 probes (CA-NSP 2-P/HP-NSP 2-P); the quadruple fluorescent quantitative RT-PCR detection method developed based on the detection primer set can determine the type, multiple infection and strain abundance of PRRSV in a short time, has good specificity, sensitivity, repeatability and stability, can be used for detecting low-micro-content samples such as animal organs, blood, mouth and nose swabs, environmental samples and the like, and provides a new tool for preventing PRRSV transmission in pig groups.
Description
Technical Field
The invention belongs to the technical field of pathogenic microorganism detection, and particularly relates to a quadruple fluorescent RT-PCR (reverse transcription-polymerase chain reaction) detection primer group for PRRSV (porcine reproductive and respiratory syndrome virus) and a detection method.
Background
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is an infectious disease of pigs caused by porcine reproductive and respiratory syndrome virus (PRRS virus, PRRSV), which can cause a sow reproductive disorder and a piglet respiratory disorder, and can also cause frequent secondary or concurrent viral or bacterial infections of porcine immunosuppression, resulting in higher mortality; and PRRSV can cause long-time viremia, increasing the difficulty of pig farm clearance. PRRS has rapidly spread to europe, asia, and finally all countries around the world since 1987 after its first outbreak in the united states (Boter et al 1994,Francisco et al 2006,Charpin et al 2012).
PRRSV belongs to a single-stranded RNA virus, is extremely susceptible to mutation and recombination, and is currently in a wide variety of domestic PRRSV and is mainly divided into two major categories: european (PRRSV 1) and american (PRRSV 2); of these, the american type is mainly prevalent with 3 subtypes: classical (CA-PRRSV 2), highly pathogenic (HP-PRRSV 2) and NADC 30-like (N-PRRSV 2), the strains are complex and diverse, and all have obvious virulence. Thus, reliable virus typing detection methods are critical for monitoring and controlling the prevalence of PRRSV.
The current commonly used national standard GB/T35912-2018 (fluorescence RT-PCR detection method for porcine reproductive and respiratory syndrome virus) only has double qPCR (quantitative polymerase chain reaction) capable of distinguishing classical and highly pathogenic two strains, the types of the strains can be further distinguished by sequencing viral genome and comparing and analyzing the sequences, the time is up to 24 hours, and the sequencing cost is high; and when a plurality of strains exist in the sample, only one strain can be sequenced, and the abundance of the PRRSV strain in the sample cannot be further diagnosed.
Disclosure of Invention
In view of the above, the present invention aims to provide a multiplex RT-qPCR detection method for porcine reproductive and respiratory syndrome virus, which can detect and quantify the types of 25 nucleic acids of PRRSV1, PRRSV2, CA-PRRSV2, HP-PRRSV2 and N-PRRSV.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
the invention firstly develops a quadruple fluorescent RT-PCR detection primer group for simultaneously detecting the typing of PRRSV1, PRRSV2, CA-PRRSV2, HP-PRRSV2 and N-PRRSV2 nucleic acid through a large number of gene sequence comparison and test verification, and comprises the following primer pairs and probe sequences:
PRRSV-F：CAGCCAGTCAATCARCTGTGCCA(SEQ ID NO.1)，
PRRSV-R：AGRGGRAAATGKGGCTTCTC(SEQ ID NO.2)，
PRRSV1-P：TCCCAGCGCCAGCARCCTAGRGGA(SEQ ID NO.3)，
PRRSV2-P：CCAGTCCAGAGGCAAGGGACCG(SEQ ID NO.4)；
CA-PRRSV2-F：TCACTGGCTAACTACTACTACCG(SEQ ID NO.5)，
CA-PRRSV2-R：CGTGGACCAGGCTCGGTTGG(SEQ ID NO.6)，
CA-PRRSV2-P：AAGACTAACCGCCGTGCTCTCCAAG(SEQ ID NO.7)；
HP-PRRSV2-F：CTAACGGTTCGGAAGAAACT(SEQ ID NO.8)，
HP-PRRSV2-R：TCATCAGCTTGGGGACACGTC(SEQ ID NO.9)，
HP-PRRSV2-P：GTGTCATCGGCTCGGATGGTGT(SEQ ID NO.10)；
wherein the 5 'and 3' ends of PRRSV1-P, PRRSV2-P, CA-PRRSV2-P and HP-PRRSV2-P are both linked with a fluorescent group.
In the primer set, the primer pair shown as SEQ ID NO. 1-2 can amplify partial fragments in ORF7 genes of PRRSV1 and PRRSV2, and can distinguish PRRSV1 from PRRSV2 by matching with probes PRRSV1-P and PRRSV 2-P; primer pairs shown in SEQ ID No. 5-6 can specifically amplify fragments in NSP2 of CA-PRRSV2, primer pairs shown in SEQ ID No. 8-9 can specifically amplify fragments in NSP2 of HP-PRRSV2, and the primer pairs can be matched with probes CA-PRRSV2-P and HP-PRRSV2-P to distinguish the subtypes CA-PRRSV, HP-PRRSV and N-PRRSV of PRRSV 2.
Preferably, in the above primer set, the fluorescent groups at the 5 'and 3' ends of the probe PRRSV1-P are ROX and BHQ2, respectively, the fluorescent groups at the 5 'and 3' ends of the probe PRRSV2-P are FAM and BHQ1, respectively, the fluorescent groups at the 5 'and 3' ends of the probe HP-PRRSV2-P are HEX and BHQ1, respectively, and the fluorescent groups at the 5 'and 3' ends of the probe CA-PRRSV2-P are CY5 and BHQ2, respectively.
Based on the primer group, the invention further provides a quadruple fluorescent RT-PCR detection kit containing the primer group for porcine reproductive and respiratory syndrome virus. It will be appreciated that reagents for nucleic acid extraction, transcription perpetuation, and PCR amplification may also be included in the detection kit.
The invention also provides a quadruple fluorescence quantitative RT-PCR detection method for the porcine reproductive and respiratory syndrome virus, which specifically comprises the following steps:
s1, preparing standard positive plasmid control solutions with different concentrations, carrying out quadruple real-time fluorescent quantitative RT-PCR detection by using the primer set provided by the invention, and establishing standard curves of different strain types under the quadruple real-time fluorescent quantitative RT-PCR by taking the concentration of the standard positive plasmid control solution as an abscissa and a Ct value as an ordinate;
s2, extracting nucleic acid of a sample to be detected, carrying out reverse transcription, carrying out quadruple real-time fluorescent quantitative RT-PCR detection by using the primer set provided by the invention, and determining the type and content of porcine reproductive and respiratory syndrome virus in the sample to be detected through standard defects according to fluorescent signals and Ct values.
Preferably, in the above technical scheme, the preparation method of the standard positive plasmid control solution with different concentrations in the step S1 comprises the following steps: and (3) amplifying nucleic acid corresponding to the standard strain by using a primer pair with a sequence shown as SEQ ID NO. 11-12 and a primer pair with a sequence shown as SEQ ID NO. 13-14 respectively, recovering amplified products, cloning the amplified products into a vector, converting competent cells, purifying recombinant plasmids, measuring the concentration of the recombinant plasmids, and gradually diluting the recombinant plasmids.
More preferably, the concentration is the copy number of the recombinant plasmid.
More preferably, the competent cell is E.coli DH 5. Alpha.
Preferably, in the above technical scheme, the annealing temperature of the quadruple real-time fluorescence quantitative RT-PCR is 59 ℃; more preferably, the reaction conditions for the quadruple real-time fluorescent quantitative PCR are: 50℃2min,95℃30s, (95℃15s, 59℃30s, and fluorescence signal were collected) 40 cycles.
Preferably, in the technical scheme, in a reaction system of quadruple real-time fluorescence quantitative RT-PCR, the final concentration of the primer PRRSV-F/R is 0.8 mu mol/L, the final concentrations of the primers CA-PRRSV2-F/R and HP-PRRSV2-F/R are 0.4 mu mol/L, and the final concentrations of the probes PRRSV1-P, PRRSV2-P, CA-PRRSV2-P and HP-PRRSV2-P are 0.3 mu mol/L.
The beneficial effects of the invention are as follows:
the detection primer group and the detection method provided by the invention can be used for simultaneously identifying PRRSV5 main epidemic isolates (PRRSV 1, PRRSV2, CA-PRRSV, HP-PRRSV and N-PRRSV), further determining the abundance of each PRRSV strain in a sample to be detected, and simultaneously has good specificity, sensitivity, repeatability and stability. The detection method established by the invention greatly reduces the detection cost through joint detection, realizes one-time multi-flux, can be used for detecting low-micro-content samples such as animal organs, blood, mouth-nose swabs, environmental samples and the like, can determine the type and multiple infection of viruses in a short time, and provides a promising substitution tool for preventing the transmission of PRRSV in pig groups.
Drawings
FIG. 1 is a diagram showing the results of PCR detection of the standard plasmid constructed in example 2;
FIG. 2 is a graph comparing the optimal annealing temperatures explored by the single and multiple systems in example 2;
FIG. 3 is a graph of primer and probe concentrations suitable for quadruple fluorescent quantitative PCR detection in example 2;
FIG. 4 is a standard curve of four strain types under a quadruple fluorescent quantitative PCR system provided by the invention;
FIG. 5 is a diagram showing the result of the detection method constructed by the present invention for detecting the specificity of other viruses;
FIG. 6 is a graph showing the results of the detection of PRRSV plasmid and virus by the detection method constructed in accordance with the present invention;
FIG. 7 is a graph showing the sensitivity detection results of different strain types under a quadruple fluorescent quantitative PCR system provided by the invention;
FIG. 8 is a second graph of the sensitivity detection results of different strain types under the quadruple fluorescent quantitative PCR system provided by the invention.
Detailed Description
The present invention will be described in further detail with reference to examples and experimental data in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
In the following examples, unless otherwise specified, the methods are conventional; the reagents and materials described, unless otherwise specified, are commercially available.
Example 1
The invention acquires 150 PRRSV complete genomes (comprising 50 PRRSV1 and 100 PRRSV 2) from a GenBank database and performs multi-sequence comparison, and discovers that a section of region in the ORF7 gene has larger difference in PRRSV1 and PRRSV2, but has high homology between different types of PRRSV2 and 99.9 percent identity. Based on this, 1 pair of primers (PRRSV-F/R) and 2 probes (PRRSV 1-P, PRRSV 2-P) were designed for the region for the discrimination of PRRSV1 and PRRSV2, and the major class of the PRRSV can be distinguished by this set of primers and probes.
The identification of the PRRSV2 subtypes CA-PRRSV2, HP-PRRSV2 and N-PRRSV2 can be further achieved by designing 2 pairs of primers (CA-PRRSV 2-F/R, HP-PRRSV 2-F/R) and 2 probes (CA-PRRSV 2-P, HP-PRRSV 2-P) in a highly conserved NSP2 gene region, but the three strains of PRRSV2 are different from each other.
The sequences of the primers and probes obtained in this example are shown in Table 1:
TABLE 1
In the above table, the positions correspond to GenBank accession numbers PRRSV1 LV strain (M96262), classical PRRSV2 VR2332 strain (EF 536003), highly pathogenic PRRSV2 TJ strain (EU 860248) and PRRSV2 NADC30 strain (JN 654459), respectively.
The 5 'and 3' ends of the probe sequences in the above table are respectively connected with different fluorophores, and in this embodiment, the specific examples are: the fluorescent groups at the 5 'and 3' ends of the probe PRRSV1-P are ROX and BHQ2, respectively, the fluorescent groups at the 5 'and 3' ends of the probe PRRSV2-P are FAM and BHQ1, respectively, the fluorescent groups at the 5 'and 3' ends of the probe HP-PRRSV2-P are HEX and BHQ1, respectively, and the fluorescent groups at the 5 'and 3' ends of the probe CA-PRRSV2-P are CY5 and BHQ2, respectively.
Example 2
This example constructs a method for typing and quantitatively detecting PRRSV using the primer set provided in example 1, comprising in particular the following:
(1) RNA extraction and reverse transcription.
The sample to be tested to which the method is applicable can be clinical samples (such as serum, viscera and the like), supernatant of virus-infected cell cultures and the like.
The virus nucleic acid in the sample to be tested is extracted using a virus total DNA/RNA extraction Kit FastPure Viral DNA/RNA Mini Kit (available from Nanjinozan Biotechnology Co., ltd.). For total RNA extractedIII All-in-one RT SuperMix Perfect for qPCR (Vazyme) first strand cDNA was synthesized, and the reaction system included 5×all-in-one qRT SuperMix 4. Mu.L, enzyme Mix 1. Mu.L, total RNA1 pg-1. Mu.g, RNase-free ddH 2 O to 20. Mu.L; the reverse transcription procedure was 50℃for 15min at 85℃for 5sec. The eluted DNA, or RNA, synthesizes the first strand cDNA, and is stored at-80 ℃.
(2) Preparation of standard positive control.
The primers shown in Table 2 were used to amplify nucleic acids corresponding to the standard strains, and the amplified products were recovered to obtain full-length fragments PRRSV1-ORF7, PRRSV2-ORF7, CA-PRRSV2-NSP2, HP-PRRSV2-NSP2, N-PRRSV2-NSP2 of the different strains ORF7 and NSP2, respectively.
TABLE 2
The fragments are cloned into a PMD-18T vector and transformed into DH5 alpha competent cells respectively, after overnight culture, plasmids are extracted from various bacterial liquids to serve as templates, and PCR detection is carried out by using standard plasmid primers, and the results are shown in figure 1: european PRRSV1-ORF7389 bp, american PRRSV2-ORF7434 bp, CA-PRRSV2-NSP21041 bp, HP-PRRSV2-NSP2951 bp, NL-PRRSV2-NSP2648 bp are all fragments of the expected size.
Recombinant plasmid was purified using HiSpeed Plasmid Mini Kit (Qiagen) and OD was measured using an ultra-micro accounting analyzer (Nano-200, aosheng) 260 Quantification was performed. The resulting plasmid concentration was converted to copy number by the following formula:
y (copies/. Mu.L) = (6.02X1, 023) × (plasmid concentration ng/. Mu.L) ×10) –9 DNA)/(DNA length x 660).
The detection and calculation results are specifically as follows: the concentration of the American PRRSV2-ORF7 plasmid is 219 ng/. Mu.L, the A260/A280 value is 2.95, and the converted copy number is 4.77E+10 couplings/. Mu.L; the concentration of the European PRRSV1-ORF7 plasmid is 322.21 ng/. Mu.L, the A260/A280 value is 3.02, and the converted copy number is 8.09E+10 couplings/. Mu.L; the concentration of the CA-PRRSV2-NSP2 plasmid is 190.44 ng/. Mu.L, the A260/A280 value is 2.07, and the converted copy number is 4.71E+10 couplings/. Mu.L; the concentration of the HP-PRRSV2-NSP2 plasmid is 224.39 ng/. Mu.L, the A260/A280 value is 2.87, and the converted copy number is 5.54E+10 couplings/. Mu.L; the concentration of N-PRRSV2-NSP2 plasmid was 171.17, the A260/A280 value was 1.95, and the calculated copy number was 4.23E+10 couplings/. Mu.L.
Finally, diluting the standard positive control with DEPC water to 10 gradients (10) 1 -10 10 cobies/. Mu.L) and stored at-80℃until use.
In addition, trans5α Chemically Competent Cell (DH 5. Alpha., CD 201) and E.coli Transetta (DE 3) competent cells used in the above procedure were purchased from Beijing full gold Biotechnology Co., ltd, and Gel/PCR Extraction Kit (Gel/PCR product recovery kit) was purchased from Biomiga, USA.
(3) The primer set provided in example 1 was used to detect DNA of the test object as a template by quadruple real-time fluorescent quantitative PCR.
In this example, the optimal reaction system was searched for from various aspects such as annealing temperature, primer concentration, probe concentration, reaction system, etc., for example, when the annealing temperature was searched for, the difference between different annealing temperatures was small in the process of not adjusting other influencing factors, the optimal annealing temperature for PRRSV1-ORF7, PRRSV2-ORF7 and HP-PRRSV2-NSP2 was 57 ℃ (see fig. 2 a and B) for the single system, 59 ℃ (see fig. 2C) for the CA-PRRSV2-NSP2, and 59 ℃ (see fig. 2D) for the quadruple system.
In addition, by fumbling the primer concentration and the probe concentration, when the primer final concentration of the quadruple system is 0.4 mu mol/L and the probe concentration is 0.3 mu mol/L (see table 2), the time for taking off the amplification curve after each amplification template is early, the Ct value is small, the amplification efficiency is close to 100%, and the fluorescence intensity is high (as shown in figure 3).
TABLE 2
| Primer/probe combinations | Primer concentration | Probe concentration |
| PRRSV-F/R+PRRSV1-P | 0.4μmol/L | 0.3μmol/L |
| PRRSV-F/R+PRRSV2-P | 0.4μmol/L | 0.3μmol/L |
| CA-PRRSV2-F/R/P | 0.4μmol/L | 0.3μmol/L |
| HP-PRRSV2-F/R/P | 0.4μmol/L | 0.3μmol/L |
Based on the above, the reaction system of the quadruple real-time fluorescent quantitative PCR can be: 20. Mu.L total, animal Detection U +Probe Master Mix 10.0. Mu.L, 10. Mu. Mol/L PRRSV-F1.6. Mu.L, 10. Mu. Mol/L PRRSV-R1.6. Mu.L, 10. Mu. Mol/L CA-PRRSV 2-F0.8. Mu.L, 10. Mu. Mol/L CA-PRRSV 2-R0.8. Mu.L, 10. Mu. Mol/L HP-PRRSV 2-F0.8. Mu.L, 10. Mu. Mol/L HP-PRRSV 2-R0.8. Mu.L, 10. Mu. Mol/L each Probe 0.6. Mu.L, template 2.0. Mu.L, DEPC H 2 And the balance of O. And the reaction conditions are as follows: 50 ℃ for 2min;95 ℃ for 30s; fluorescence signals were collected at 95℃for 15s and at 59℃for 30s, 40 cycles.
(4) And (3) establishing a standard curve.
And (3) taking a series of standard positive control solutions with different concentrations prepared in step (2) as an object to be detected, adopting the reaction system and the reaction conditions optimized in step (3), and establishing a standard curve of a quadruple TaqMan real-time fluorescence quantitative PCR standard template by taking the copy number as an abscissa and the Ct value as an ordinate, wherein the standard curve is shown in a specific figure 4.
As can be seen from FIG. 4, at 10 2 COPIES/. Mu.L to 10 9 In the gradient concentration interval of copies/. Mu.L, the slope of the standard curve of the quadruple fluorescence quantitative PCR is-2.70 to-3.39, and the correlation coefficient R 2 0.97 to 0.99, and an amplification efficiency (E) of 97% to 134.2%; shows good linear relation, linear equation of the obtained copy number (x) and Ct value (y) and correlation coefficient R 2 The method comprises the following steps:
for ORF7 of PRRSV 1: y= -3.257x+37.794, r2=0.973, e=102.8%;
ORF7 for PRRSV 2: y= -3.397x+39.341, r2=0.995, e=97%;
NSP2 for HP-PRRSV 2: y= -2.853x+41.821, r2=0.991, e=124.1%;
NSP2 for CA-PRRSV 2: y= -2.706x+37.221, r2=0.98, e=134.2%.
According to the standard curve, the Ct value in the object to be detected is detected by the step (3), and the type and the content of PRRSV strain in the object to be detected can be determined according to the type of fluorescent signal.
Example 3
The specificity, sensitivity, reproducibility and stability of the detection method constructed in example 2 were further evaluated in this example.
(1) Specificity verification
Other viral pathogens including Classical Swine Fever Virus (CSFV), E2 strain, porcine Epidemic Diarrhea Virus (PEDV) YN144 strain, pseudorabies virus (PRV) HENXINX strain, porcine Parvovirus (PPV) PPV1 strain, porcine Circovirus (PCV) type 2 DY strain and type 3 KQ strain were used for the specificity test. The nucleic acids of each virus and PRRSV were extracted, and PCR was performed using cDNA obtained by reverse transcription as a reaction template, and the PRRSV plasmid constructed in example 2 was simultaneously detected, and each sample was repeated 3 times.
As shown in FIGS. 5 to 6, it was confirmed that only PRRSV positive templates and corresponding PRRSV plasmids had fluorescent signals, and no positive fluorescent signals were observed when other viruses (CSFV, PEDV, PRV, PPV, PCV and PCV 3) were measured. In addition, in quadruple real-time fluorescent quantitative PCR, when CA-PRRSV2 (VR 2332), HP-PRRSV2 (TJ) and N-PRRSV2 (NADC 30) isolates were used as templates, only CY5, HEX and FAM fluorescent signals could be specifically detected, respectively (G-I in FIG. 6). Thus, the specificity of the quadruple PRRSV real-time fluorescence quantitative PCR between different viruses and in the same virus is good.
(2) Sensitivity test.
Cultures of virus-infected cells of the standard plasmid constructed in example 2 and CA-PRRSV2 (VR 2332), HP-PRRSV2 (TJ) and N-PRRSV2 (NADC 30) isolates (10) 5 TCID 50/mL) 10-fold serial dilutions 10 4 TCID 50/mL-10 0 TCID 50/mL was used as a nucleic acid template for sensitivity testing.
The detection results of the different gradient standard plasmids are shown in FIG. 7, and show that the copy numbers of PRRSV1-ORF7 (A), PRRSV2-ORF7 (B), CA-PRRSV2-NSP2 (C) and HP-PRRSV2-NSP2 (D) detected by the TaqMan multiplex real-time fluorescence quantitative PCR method are respectively 80 copies/. Mu.L, 47 copies/. Mu.L, 50 copies/. Mu.L and 40 copies/. Mu.L at the minimum. The results of the detection of cultures of infected cells from each strain are shown in FIG. 8, with the lowest detection method being 10TCID 50/mL. The method for detecting the multiplex real-time fluorescent quantitative PCR established by the invention is proved to have quite high sensitivity when the multiplex detection is carried out.
(3) Repeatability and stability tests.
The reproducibility (in-batch) and stability (between batches) of the assay of the method of the invention were determined, and plasmid concentrations were diluted to 3 dilutions of 10 5 、10 4 、10 3 The Ct value was determined in triplicate on the same day for each plasmid dilution for reproducibility within the batch using the method of copies/. Mu.L; for batch-to-batch stability, the Ct value was determined by two different operators on different days according to the instructions. When the measured variation coefficient between the batch and the batch is less than 5%, the method has good repeatability and high stability. The test results are shown in Table 3:
TABLE 3 Table 3
The results show that the variation Coefficient (CV) ranges of Ct values in batches and between batches are respectively 0.16% to 2.52% and 0.35% to 4.97%, and are below 5%, which indicates that the four-fold fluorescence quantitative PCR method established by the invention has good repeatability and stability.
Example 4
The reliability of the quadruple real-time fluorescent quantitative PCR is further verified through 100 clinical samples, and the detection results of the quadruple RT-qPCR method and the GBT 35912-2018 detection method (the reaction system is shown in table 4, the amplification conditions are 50 ℃ 2min and 95 ℃ 30s, (95 ℃ 15s and 60 ℃ 30s and fluorescent signals are collected) are compared, and the detection results are specifically shown in tables 5 and 6.
TABLE 4 Table 4
Note that: the primer and probe sequences in the table are specifically shown in GBT 35912-2018.
TABLE 5
TABLE 6
From the result, the national standard GBT 35912-2018 method detects 26 parts of positive nucleic acid and 74 parts of negative nucleic acid; the quadruple fluorescent quantitative RT-PCR method provided by the invention detects 25 parts of PRRSV positive nucleic acid and 75 parts of PRRSV negative nucleic acid. Of these, 24 positive samples and 73 negative samples gave consistent results. Sensitivity was 92% (24/26), specificity was 98.65% (73/74), and total compliance was 97%. Comparing the Ct values of each channel of the two methods, the method of the invention finds that 20 samples with Ct values lower than the GBT 35912-2018 method and 4 samples with Ct values slightly higher than the GBT 35912-2018 method in the FAM channel; the Ct values of HEX channels are lower than those of the national standard method; the method has 2 samples with positive Ct values in CY5 channel, and the samples are CA-PRRSV2 strains after sequencing. Compared with the national standard method, the sensitivity of the self-building method is higher than that of the national standard method, the coincidence rate is consistent, and the self-building method has the advantage of multiple detection.
Example 5
By adopting the detection method provided by the invention, 321 suspected PRRSV clinical samples are detected in the example.
The detection result is as follows: 20 parts of virus liquid (20/20), 60 parts of oronasal anal swab (24/60), 105 parts of mixed sample of disease (lymph node, tonsil, lung, kidney, spleen) (91/105), 96 parts of environment sample (60/96) and 40 parts of serum (35/40), and the concrete steps are shown in table 7:
TABLE 7
To verify the detection results, all detected PRRSV positive clinical samples were subjected to ORF5 gene amplification and the amplicons were Sanger sequenced, with PRRSV results. The method has wide application range and high sensitivity, and can be used for rapid diagnosis of clinical PRRSV.
In conclusion, compared with the national standard method, the detection primer and the detection method provided by the invention can simultaneously realize quantitative analysis of identification of PRRSV5 main epidemic isolates (PRRSV 1, PRRSV2, CA-PRRSV, HP-PRRSV and N-PRRSV), and have high accuracy, low detection limit and very high application value.
The foregoing description of the preferred embodiments of the present invention should not be taken as limiting the scope of the invention, and it should be noted that any modifications, equivalents, improvements and others within the spirit and principles of the present invention will become apparent to those of ordinary skill in the art.
Claims (10)
1. The quadruple fluorescent RT-PCR detection primer probe group for PRRSV is characterized by comprising the following primer and probe sequences:
PRRSV-F：CAGCCAGTCAATCARCTGTGCCA，
PRRSV-R：AGRGGRAAATGKGGCTTCTC，
PRRSV1-P：TCCCAGCGCCAGCARCCTAGRGGA，
PRRSV2-P：CCAGTCCAGAGGCAAGGGACCG；
CA-PRRSV2-F：TCACTGGCTAACTACTACTACCG，
CA-PRRSV2-R：CGTGGACCAGGCTCGGTTGG，
CA-PRRSV2-P：AAGACTAACCGCCGTGCTCTCCAAG；
HP-PRRSV2-F：CTAACGGTTCGGAAGAAACT，
HP-PRRSV2-R：TCATCAGCTTGGGGACACGTC，
HP-PRRSV2-P：GTGTCATCGGCTCGGATGGTGT；
wherein the 5 'and 3' ends of the probes PRRSV1-P, PRRSV2-P, CA-PRRSV2-P and HP-PRRSV2-P are respectively connected with a fluorescent group.
2. The set of quadruple fluorescent RT-PCR detection primers of claim 1, wherein the fluorescent groups at the 5 'and 3' ends of the probe PRRSV1-P are ROX and BHQ2, respectively, the fluorescent groups at the 5 'and 3' ends of the probe PRRSV2-P are FAM and BHQ1, respectively, the fluorescent groups at the 5 'and 3' ends of the probe HP-PRRSV2-P are HEX and BHQ1, respectively, and the fluorescent groups at the 5 'and 3' ends of the probe CA-PRRSV2-P are CY5 and BHQ2, respectively.
3. A quadruple fluorescent RT-PCR detection kit for PRRSV comprising the primer probe set of claim 1 or 2.
4. A quadruple fluorescent quantitative RT-PCR detection method for PRRSV, characterized in that the detection method is not aimed at diagnosis and treatment of diseases and comprises the steps of:
s1, preparing standard positive plasmid control solutions with different concentrations, carrying out quadruple real-time fluorescent quantitative RT-PCR detection by using the primer probe set of claim 1, and establishing standard curves of different strain types under the quadruple real-time fluorescent quantitative RT-PCR by taking the concentration of the standard positive plasmid control solution as an abscissa and a Ct value as an ordinate;
s2, extracting nucleic acid of a sample to be detected, carrying out quadruple real-time fluorescent quantitative RT-PCR detection by using the primer probe set of claim 1 after reverse transcription, and determining the type and content of the strain of PRRSV in the sample to be detected according to fluorescent signals and Ct values and through a standard curve.
5. The method for detecting the quadruple fluorescent quantitative RT-PCR according to claim 4, wherein the preparation method of the standard positive plasmid control solutions with different concentrations in the step S1 comprises the following steps: and amplifying nucleic acid corresponding to the standard strain by using a primer pair with a sequence shown as SEQ ID NO. 11-12 and a primer pair with a sequence shown as SEQ ID NO. 13-14 respectively, recovering amplified products, cloning the amplified products into a vector, converting competent cells, purifying recombinant plasmids, measuring the concentration of the recombinant plasmids, and diluting the recombinant plasmids step by step to obtain the recombinant plasmid.
6. The method of claim 5, wherein the concentration is the copy number of the recombinant plasmid.
7. The method of claim 5, wherein the competent cell is E.coli DH 5. Alpha.
8. The method according to claim 4, wherein the final concentration of the primer PRRSV-F/R, the final concentrations of the primers CA-PRRSV2-F/R and HP-PRRSV2-F/R and the final concentrations of the probes PRRSV1-P, PRRSV2-P, CA-PRRSV2-P and HP-PRRSV2-P are respectively 0.8. Mu. Mol/L and 0.3. Mu. Mol/L, respectively.
9. The method of claim 4, wherein the annealing temperature of the quadruple real-time fluorescent quantitative RT-PCR is 59 ℃.
10. The method for detecting the quadruple fluorescent quantitative RT-PCR according to claim 9, wherein the reaction conditions of the quadruple real-time fluorescent quantitative RT-PCR are as follows: 50 ℃ for 2min;95 ℃ for 30s;95℃for 15s, 59℃for 30s, and fluorescence signal was collected for 40 cycles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211143428.2A CN116144836B (en) | 2022-09-20 | 2022-09-20 | Quadruple fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer set for PRRSV (porcine reproductive and respiratory syndrome virus) and detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202211143428.2A CN116144836B (en) | 2022-09-20 | 2022-09-20 | Quadruple fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer set for PRRSV (porcine reproductive and respiratory syndrome virus) and detection method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116144836A CN116144836A (en) | 2023-05-23 |
| CN116144836B true CN116144836B (en) | 2023-08-18 |
Family
ID=86355092
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211143428.2A Active CN116144836B (en) | 2022-09-20 | 2022-09-20 | Quadruple fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer set for PRRSV (porcine reproductive and respiratory syndrome virus) and detection method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116144836B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101736094A (en) * | 2010-03-02 | 2010-06-16 | 福建省农业科学院畜牧兽医研究所 | Variant porcine reproductive and respiratory syndrome virus (PRRSV) TaqMan fluorescence quantitative RT-PCR detecting kit and application thereof |
| CN107475459A (en) * | 2017-09-30 | 2017-12-15 | 山东省农业科学院畜牧兽医研究所 | Differentiate the detection method of american type PRRSV classical strainses, variation strain and new virus class NADC30 strains simultaneously |
| KR20200069603A (en) * | 2018-12-07 | 2020-06-17 | 대한민국(농림축산식품부 농림축산검역본부장) | PNA probe for detecting Porcine reproductive and respiratory syndrome virus genetic type and a method for detecting Porcine reproductive and respiratory syndrome virus genetic type using the same |
| CN113913559A (en) * | 2021-11-25 | 2022-01-11 | 江苏立华牧业股份有限公司 | Reagent for fluorescence quantitative detection of PRRSV and detection method for PRRSV typing |
| CN114410843A (en) * | 2022-01-29 | 2022-04-29 | 龙岩学院 | Fluorescent quantitative detection primer and probe set for simultaneously identifying four types of strains |
| CN114438260A (en) * | 2022-01-25 | 2022-05-06 | 中国农业科学院兰州兽医研究所 | One-step multiplex RT-PCR (reverse transcription-polymerase chain reaction) detection method and kit for porcine reproductive and respiratory syndrome virus |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050170335A1 (en) * | 2003-10-09 | 2005-08-04 | Tetracore, Inc. | Detection of PRRSV |
| EP2370817A4 (en) * | 2008-11-26 | 2012-05-23 | Univ South Dakota | Identification of porcine reproductive and respiratory syndrome virus |
-
2022
- 2022-09-20 CN CN202211143428.2A patent/CN116144836B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101736094A (en) * | 2010-03-02 | 2010-06-16 | 福建省农业科学院畜牧兽医研究所 | Variant porcine reproductive and respiratory syndrome virus (PRRSV) TaqMan fluorescence quantitative RT-PCR detecting kit and application thereof |
| CN107475459A (en) * | 2017-09-30 | 2017-12-15 | 山东省农业科学院畜牧兽医研究所 | Differentiate the detection method of american type PRRSV classical strainses, variation strain and new virus class NADC30 strains simultaneously |
| KR20200069603A (en) * | 2018-12-07 | 2020-06-17 | 대한민국(농림축산식품부 농림축산검역본부장) | PNA probe for detecting Porcine reproductive and respiratory syndrome virus genetic type and a method for detecting Porcine reproductive and respiratory syndrome virus genetic type using the same |
| CN113913559A (en) * | 2021-11-25 | 2022-01-11 | 江苏立华牧业股份有限公司 | Reagent for fluorescence quantitative detection of PRRSV and detection method for PRRSV typing |
| CN114438260A (en) * | 2022-01-25 | 2022-05-06 | 中国农业科学院兰州兽医研究所 | One-step multiplex RT-PCR (reverse transcription-polymerase chain reaction) detection method and kit for porcine reproductive and respiratory syndrome virus |
| CN114410843A (en) * | 2022-01-29 | 2022-04-29 | 龙岩学院 | Fluorescent quantitative detection primer and probe set for simultaneously identifying four types of strains |
Non-Patent Citations (1)
| Title |
|---|
| 猪繁殖与呼吸障碍综合征病毒(PRRSV)检测方法的建立;苏晓鸥;李玉荣;乔俊文;赵德明;;河北农业大学学报(第01期);82-85 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116144836A (en) | 2023-05-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| CN111020062A (en) | Triple real-time fluorescent quantitative PCR kit for detecting African swine fever wild strain and gene deletion strain | |
| CN113462820A (en) | Multiplex RT-PCR primer probe set for real-time fluorescent quantitative detection of four porcine diarrhea viruses, kit and detection method thereof | |
| CN105648114B (en) | Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primer, probe and kit for detecting new variant type highly pathogenic porcine reproductive and respiratory syndrome virus and detection method | |
| CN113403430A (en) | Triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus, kit and application | |
| CN111471797B (en) | RT-RPA primer pair, probe, kit and detection method for detecting cat coronavirus | |
| CN114634996B (en) | Primer probe combination and kit for detecting bovine respiratory disease and application of primer probe combination and kit | |
| CN113913559B (en) | Reagent for fluorescence quantitative detection of PRRSV and detection method for PRRSV typing | |
| CN115976285A (en) | Quadruple fluorescent quantitative PCR detection kit for detecting African swine fever | |
| CN114350848B (en) | Dual fluorescent probe primer combination for identifying African swine fever type I strain and type II strain, kit and application thereof | |
| AU2021104086A4 (en) | Detection reagent for fluorescent duplex pcr for classical swine fever virus and african swine fever virus, kit, and detection method | |
| CN116004920B (en) | Fluorescence PCR detection method and kit for four different lineages of strains of porcine reproductive and respiratory syndrome | |
| CN112280899A (en) | Porcine astrovirus type 2 TaqMan fluorescent quantitative PCR kit and application thereof | |
| CN116814859A (en) | Primer probe composition, kit and method for identifying African swine fever virus genes I and II | |
| CN114438265B (en) | Nucleic acid composition, kit and detection method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kokumi virus | |
| CN116144836B (en) | Quadruple fluorescent quantitative RT-PCR (reverse transcription-polymerase chain reaction) detection primer set for PRRSV (porcine reproductive and respiratory syndrome virus) and detection method | |
| CN111593137A (en) | Fluorescent quantitative PCR detection reagent and kit for African swine fever virus | |
| CN112626278B (en) | Primer and probe for identifying canine distemper virus wild strain and vaccine strain and application | |
| CN112410466A (en) | Primer, probe and detection method for porcine circovirus type 2 and porcine circovirus type 4 dual real-time fluorescent quantitative PCR detection | |
| CN111235317A (en) | Primer composition, kit and method for detecting PRRSV (porcine reproductive and respiratory syndrome Virus) and PCV (porcine circovirus Virus) | |
| CN114752707B (en) | Primer probe group and application thereof, and kit for detecting African swine fever virus, swine fever virus and erysipelas | |
| CN118308537B (en) | Molecular marker for genotyping and pedigree identification of porcine reproductive and respiratory syndrome virus | |
| CN116814862B (en) | Identification and detection of African swine fever gene type I virulent virus, type I attenuated virus and type II virus | |
| CN114672595B (en) | Porcine viral diarrhea detection primer combination, detection kit and application | |
| AU2021105359A4 (en) | Universal triplex RT-nested PCR detection primer and method for porcine reproductive and respiratory syndrome virus |
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 |