CN117511936A - Kit and method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR - Google Patents
Kit and method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR Download PDFInfo
- Publication number
- CN117511936A CN117511936A CN202410014399.2A CN202410014399A CN117511936A CN 117511936 A CN117511936 A CN 117511936A CN 202410014399 A CN202410014399 A CN 202410014399A CN 117511936 A CN117511936 A CN 117511936A
- Authority
- CN
- China
- Prior art keywords
- pcr
- respiratory syndrome
- kit
- porcine reproductive
- syndrome virus
- 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.)
- Granted
Links
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 title claims abstract description 55
- 238000003757 reverse transcription PCR Methods 0.000 title claims abstract description 42
- 230000001717 pathogenic effect Effects 0.000 title claims abstract description 35
- 238000000034 method Methods 0.000 title claims abstract description 16
- 239000000523 sample Substances 0.000 claims abstract description 46
- 238000012408 PCR amplification Methods 0.000 claims abstract description 13
- 238000011144 upstream manufacturing Methods 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 11
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 239000000203 mixture Substances 0.000 claims description 9
- 238000003908 quality control method Methods 0.000 claims description 8
- 238000010791 quenching Methods 0.000 claims description 8
- 230000000171 quenching effect Effects 0.000 claims description 8
- 239000002299 complementary DNA Substances 0.000 claims description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- 239000012295 chemical reaction liquid Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000003623 enhancer Substances 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- -1 BHQ-3 Chemical compound 0.000 claims description 4
- 102100034343 Integrase Human genes 0.000 claims description 4
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 4
- 108010006785 Taq Polymerase Proteins 0.000 claims description 4
- 239000000872 buffer Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 238000010839 reverse transcription Methods 0.000 claims description 4
- 239000003161 ribonuclease inhibitor Substances 0.000 claims description 4
- 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 claims description 4
- 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 3
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 239000005547 deoxyribonucleotide Substances 0.000 claims description 3
- 125000002637 deoxyribonucleotide group Chemical group 0.000 claims description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 claims description 3
- 229910001425 magnesium ion Inorganic materials 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000001226 triphosphate Substances 0.000 claims description 3
- 235000011178 triphosphate Nutrition 0.000 claims description 3
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 claims description 3
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 2
- PHIQHXFUZVPYII-ZCFIWIBFSA-O (R)-carnitinium Chemical compound C[N+](C)(C)C[C@H](O)CC(O)=O PHIQHXFUZVPYII-ZCFIWIBFSA-O 0.000 claims description 2
- WCKQPPQRFNHPRJ-UHFFFAOYSA-N 4-[[4-(dimethylamino)phenyl]diazenyl]benzoic acid Chemical compound C1=CC(N(C)C)=CC=C1N=NC1=CC=C(C(O)=O)C=C1 WCKQPPQRFNHPRJ-UHFFFAOYSA-N 0.000 claims description 2
- OKIZCWYLBDKLSU-UHFFFAOYSA-M N,N,N-Trimethylmethanaminium chloride Chemical compound [Cl-].C[N+](C)(C)C OKIZCWYLBDKLSU-UHFFFAOYSA-M 0.000 claims description 2
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 2
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 2
- 229960004203 carnitine Drugs 0.000 claims description 2
- 239000003599 detergent Substances 0.000 claims description 2
- 125000006853 reporter group Chemical group 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- MPLHNVLQVRSVEE-UHFFFAOYSA-N texas red Chemical compound [O-]S(=O)(=O)C1=CC(S(Cl)(=O)=O)=CC=C1C(C1=CC=2CCCN3CCCC(C=23)=C1O1)=C2C1=C(CCC1)C3=[N+]1CCCC3=C2 MPLHNVLQVRSVEE-UHFFFAOYSA-N 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 49
- 102000039446 nucleic acids Human genes 0.000 abstract description 11
- 108020004707 nucleic acids Proteins 0.000 abstract description 11
- 150000007523 nucleic acids Chemical class 0.000 abstract description 11
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000002474 experimental method Methods 0.000 description 19
- 230000003321 amplification Effects 0.000 description 14
- 238000003199 nucleic acid amplification method Methods 0.000 description 14
- 241000700605 Viruses Species 0.000 description 7
- 230000007918 pathogenicity Effects 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 description 4
- 238000000137 annealing Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 108090001074 Nucleocapsid Proteins Proteins 0.000 description 3
- 241000282898 Sus scrofa Species 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241000282887 Suidae Species 0.000 description 2
- 101710110895 Uncharacterized 7.3 kDa protein in cox-rep intergenic region Proteins 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 241000701386 African swine fever virus Species 0.000 description 1
- 241001533384 Circovirus Species 0.000 description 1
- 241000710777 Classical swine fever virus Species 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 238000007397 LAMP assay Methods 0.000 description 1
- 241001135549 Porcine epidemic diarrhea virus Species 0.000 description 1
- 241000702665 Porcine rotavirus Species 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- 241000711484 Transmissible gastroenteritis virus Species 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011897 real-time detection Methods 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 208000023504 respiratory system disease Diseases 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000003612 virological effect Effects 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
- C12Q1/686—Polymerase chain reaction [PCR]
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)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Virology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kit and a detection method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR, and belongs to the technical field of in-vitro nucleic acid detection. The kit comprises an upstream primer with a sequence shown as SEQ ID NO.1, a downstream primer with a sequence shown as SEQ ID NO.2 and a molecular beacon probe with a sequence shown as SEQ ID NO.3, and the invention also provides an MB-RT-PCR method for detecting the high-pathogenicity porcine reproductive and respiratory syndrome virus by using the kit, optimizes PCR amplification conditions, not only can rapidly detect the porcine reproductive and respiratory syndrome virus, but also has the characteristics of high sensitivity, strong specificity and wide detection range of the kit.
Description
Technical Field
The invention relates to the technical field of in-vitro nucleic acid detection, in particular to a kit and a detection method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR.
Background
Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is an acute infectious disease caused by porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome viruse, PRRSV) and mainly characterized by respiratory diseases of sows, pigs of various ages and particularly piglets, and has the characteristics of high morbidity, high mortality and low cure rate, and is found in all countries of mainly raising pigs worldwide. Currently, the HP-PRRSV detection method mainly comprises virus separation, conventional PCR, reverse transcription-loop-mediated isothermal amplification (RT-LAMP), fluorescent quantitative PT-PCR method and the like, and for example, a conventional PCR detection method kit is disclosed in a patent document CN 101343670A; the triple real-time fluorescent quantitative PCR technology is adopted in the patent document CN104745726A, so that the porcine reproductive and respiratory syndrome virus, the highly pathogenic porcine reproductive and respiratory syndrome virus and the swine fever virus can be rapidly detected. The methods are complex to operate, long in required time, strict in detection environment, incapable of quantifying viruses and incapable of determining the occurrence degree of diseases, and the HP-PRRSV detection methods are all designed based on the high-pathogenicity porcine reproductive and respiratory syndrome virus strain discovered in 2006, and PRRSV is continuously mutated, some mutation influences the biological characteristics of viruses, the infectivity and toxicity of the novel mutated HP-PRRSV are enhanced, and the detection method and the kit in the prior art cannot meet the detection of the HP-PRRSV in recent years. Although a fluorescent RT-PCR primer, a fluorescent probe and a detection method of a kit for detecting the novel variant highly pathogenic porcine reproductive and respiratory syndrome virus after 2012 in China are disclosed in the patent document CN105648114B, the detection method is only aimed at detecting the novel variant highly pathogenic porcine reproductive and respiratory syndrome virus in China between 2012 and 2020, and the detection range is limited.
Common virus detection is RT-PCR technology, and the labeling method is generally a fluorescent dye embedding method (SYBR Green I), a fluorescent probe method (TaqMan probe) and a molecular beacon method. Wherein the Molecular Beacon (MB) is a fluorescent probe with a stem-loop structure, when no target sequence exists, the molecular beacon probe is closed at a low temperature, the fluorescent group and the quenching group are close to each other and do not fluoresce, and when the target sequence exists, the molecular beacon forms a stable double-chain hybrid with the complementary target sequence at a low temperature, so that the fluorescent group and the quenching group are separated and fluoresce, the quantity of the molecular beacon combined with the template increases along with the increase of the circulation times, and the final fluorescence intensity is proportional to the amplified template quantity. The technology has extremely high specificity, simple and convenient operation and high sensitivity, and can also carry out real-time detection. Therefore, if the characteristic that the molecular beacon probe emits fluorescence intensity difference at different temperatures can be utilized, a kit capable of detecting the high-pathogenicity porcine reproductive and respiratory syndrome virus and a detection method using the kit are designed, so that the defects of low sensitivity, limited detection range and the like in the prior art can be overcome, and the kit is also beneficial to auxiliary diagnosis and epidemiological research of the novel variant high-pathogenicity porcine reproductive and respiratory syndrome virus.
Disclosure of Invention
Aiming at the defects of the prior art, the invention designs a kit capable of rapidly detecting the porcine reproductive and respiratory syndrome virus with high pathogenicity based on an MB-RT-PCR method, and simultaneously provides a detection method using the kit. The kit has the characteristics of strong specificity, high sensitivity and short time consumption.
In order to achieve the above purpose, the technical solution of the present invention is as follows:
the invention designs a kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR, which comprises MB-RT-PCR reaction liquid, enzyme mixed liquid, negative quality control product and positive quality control product, wherein the MB-RT-PCR reaction liquid comprises an upstream primer shown as SEQ ID NO.1, a downstream primer shown as SEQ ID NO.2 and a molecular beacon probe shown as SEQ ID NO. 3.
Specifically, the sequence of the upstream primer is 5'-GGCAAGCAGCAGAAGAGAA-3'; the sequence of the downstream primer is 5'-TTCCCGGTCCCTTGCCTCTG-3'; the molecular beacon probe is an MB probe, and the sequence is as follows: 5'-CAGCAA-GCCAGTCAATCAGCTGTGCCA-TTGCTG-3'.
The derivative sequences of the above primers and probes are also within the scope of the present invention, and include the complementary strand sequences of the primer sequences, or sequences obtained by extending one to five bases or deleting one to five bases from the 5 '-end and the 3' -end of the primer sequences.
In one embodiment, the volume ratio of the upstream primer, the downstream primer and the probe is (0.05-0.1): (0.05-0.1): (0.01:0.02).
Specifically, the volume ratio of the upstream primer to the downstream primer to the probe is 0.35:0.35:0.01.
In one embodiment, the 3 'end of the molecular beacon probe is marked with a fluorescence quenching group, and the 5' end is respectively marked with a fluorescence reporting group.
Specifically, the fluorescent reporter group is selected from FAM, CY5, ROX, VIC, or Texas Red; the fluorescence quenching group is selected from BHQ-1, BHQ-2, BHQ-3, DABCYL, DABSYL, TAMRA or TAMRA.
In one embodiment, the MB-RT-PCR reaction solution further comprises RT-PCR buffer solution, PCR enhancer, magnesium chloride, deoxyribonucleotide triphosphate mixture and buffer solution containing magnesium ions.
In one embodiment, the RT-PCR buffer comprises Tris-HCl with pH of 7.5-8.9 and concentration of 20 mM-80M, ammonium sulfate with concentration of 10 mM-5M, potassium chloride with concentration of 100 mM-4M and Triton X-100 with volume ratio of 0.01-0.3%, and specifically, the pH of Tris-HCl is 8.0.
In one embodiment, the PCR enhancer is selected from at least one of tetramethyl ammonium chloride, carnitine, trehalose, and the non-ionic detergent NP-40.
In one embodiment, the enzyme mixture contains Taq DNA polymerase 2U/. Mu.L-6U/. Mu.L, reverse transcriptase 5U/. Mu.L-12U/. Mu.L with high thermal stability, and RNase inhibitor 1U/. Mu.L-5U/. Mu.L.
Specifically, the enzyme mixture contains Taq polymerase 5U/. Mu.L, reverse transcriptase 10U/. Mu.L with high thermostability, and RNase inhibitor 2U/. Mu.L.
The invention also provides an MB-RT-PCR method for detecting the high pathogenicity porcine reproductive and respiratory syndrome virus, which comprises the following specific steps:
s1, extracting total RNA of a sample, and synthesizing single-stranded cDNA by reverse transcription;
s2, carrying out PCR amplification reaction on the template by using the cDNA synthesized in the step S1 as a template and utilizing the primer of the kit for detecting the high-pathogenicity porcine reproductive and respiratory syndrome virus, and detecting and analyzing after the reaction is finished.
Specifically, the cycle threshold Ct of the cDNA is compared with a standard curve to obtain the copy concentration of the gene fragment of the highly pathogenic porcine reproductive and respiratory syndrome virus in the cDNA.
In one example, the step S2 uses 25. Mu.L PCR amplification system, including 14. Mu.L LMB-RT-PCR reaction solution, 1. Mu.L enzyme mixture, and 10. Mu.L template.
In one embodiment, the PCR amplification reaction conditions are: 20min at 50 ℃;95 ℃ for 5min; 15s at 95 ℃, 30s at 53-60 ℃ and 40 cycles.
Specifically, the PCR amplification reaction conditions were: 20min at 50 ℃;95 ℃ for 5min; 15s at 95℃and 30s at 57℃for 40 cycles.
Compared with the prior art, the invention has the following beneficial effects:
the invention designs a group of primer pairs and molecular beacon probes aiming at a novel variant type high pathogenicity porcine reproductive and respiratory syndrome virus ORF7 encoding nucleocapsid protein N gene, wherein the gene covers variant strains at home and abroad IN recent years, and foreign strains comprise 2022 Severn isolate 01-5573/D2022, 2021 US isolate USA/IN105404/2021, 2022 Korean isolate 18R10-24-1, 2022 Korean isolate 20D160-1, 2021 Korean isolate K07-2273 and the like. Meanwhile, the invention designs a kit comprising the primer pair and the probe and capable of being used for detecting the novel variant highly pathogenic porcine reproductive and respiratory syndrome virus and a detection method. In addition, the invention optimizes the concentration of the primer and the probe and the PCR reaction condition, overcomes the defects of low sensitivity and poor repeatability of the detection result of the traditional kit, and is applicable to new variant strains in recent years and wide in detection range.
Drawings
In order to more clearly illustrate the technical solutions of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a molecular beacon probe;
FIG. 2 is a graph showing the comparison of amplification results of a novel variant highly pathogenic porcine reproductive and respiratory syndrome virus detection kit of the present invention with products 1 and 2 of other companies, wherein: the abscissa represents the number of amplification cycles, and the ordinate Rn represents the fluorescence intensity.
FIG. 3 is a graph of CY5 channel linear experimental data for the novel mutant highly pathogenic porcine reproductive and respiratory syndrome virus detection kit.
FIG. 4 is a schematic diagram of a CY5 channel standard curve for the novel variant highly pathogenic porcine reproductive and respiratory syndrome virus nucleic acid detection kit.
FIG. 5 is a diagram of experimental data of the detection limit of the novel variant highly pathogenic porcine reproductive and respiratory syndrome virus detection kit.
FIG. 6 is a diagram of the data of the specificity experiment of the novel mutant highly pathogenic porcine reproductive and respiratory syndrome virus detection kit.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. 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.
The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1: kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR
1. Primers and probes were designed and synthesized
According to the relative conserved region (SEQ ID NO. 4) of a nucleocapsid protein N gene (GenBank accession number: ON 954167.1) encoded by a novel variant highly pathogenic porcine reproductive and respiratory syndrome virus ORF7 ON the NCBI platform as a target region, an upstream Primer and a downstream Primer and an MB probe shown in FIG. 1 are designed by using Primer 5.0 software, and the specific nucleotide sequence is as follows:
upstream primer F1: 5'-GGCAAGCAGCAGAAGAGAA-3' (SEQ ID NO. 1);
downstream primer R1: 5'-TTCCCGGTCCCTTGCCTCTG-3' (SEQ ID NO. 2);
MB probe P1: 5'-CAGCAA-GCCAGTCAATCAGCTGTGCCA-TTGCTG-3' (SEQ ID NO. 3).
The 3 'end of the MB probe is marked with a fluorescence quenching group, and the 5' end of the MB probe is respectively provided with a fluorescence reporting group. The fluorescence report group for detecting the new variant highly pathogenic porcine reproductive and respiratory syndrome virus is CY5, and the fluorescence quenching group is BHQ-2.
Relatively conserved regions of nucleocapsid protein N gene: 5'-ATGCCAAATAACAACGGCAAGCAGCAGAAGAGAAAGAAGGGGGATGGCCAGCCAGTCAATCAGCTGTGCCAGATGCTGGGTAAGATCATCGCTCAGCAAAACCAGTCCAGAGGCAAGGGACCGGGAAAGAAAAATAAGAAGAAAAACCCGGAGAAGCCCCATTTTCCTCTAGCGACTGAAGATGATGTCAGACATCACTTTACCCCCAGTGAGCGGCAATTGTGTCTGTCGTCAATCCAGACCGCCTTTAATCAAGGCGCTGGGACTTGCACCCTGTCAGATTCAGGGAGGATAAGTTACACTGTGGAGTTTAGTTTGCCTACGCATCATACTGTGCGCCTGATCCGCGTCACAGCATCACCCTCAGCATGA-3' (SEQ ID NO. 4).
2. Kit composition for detecting highly pathogenic porcine reproductive and respiratory syndrome virus
The kit comprises MB-RT-PCR reaction liquid, enzyme mixed liquid, negative quality control product and positive quality control product.
Wherein the MB-RT-PCR reaction solution (single dose of 14. Mu.L) comprises the upstream primer F1 (40. Mu.M, 0.35. Mu.L) shown in SEQ ID NO.1, the downstream primer R1 (40. Mu.M, 0.35. Mu.L) shown in SEQ ID NO.2, and the MB probe P1 (40. Mu.M, 0.01. Mu.L) and the distributed water (8.29. Mu.L) shown in SEQ ID NO.3, which are all derived from Shanghai JieRui bioengineering Co., ltd.
The MB-RT-PCR reaction solution also comprises an RT-PCR buffer solution, a PCR enhancer, magnesium chloride, a deoxyribonucleotide triphosphate mixture and a buffer solution containing magnesium ions. Wherein the RT-PCR buffer consisted of 45M Tris-HCl (pH=8.0), 1M ammonium sulfate, 2M potassium chloride and Triton X-100 (0.1% by volume).
The enzyme cocktail (single dose 1. Mu.L) contained Taq polymerase 5U/. Mu.L, highly thermostable reverse transcriptase 10U/. Mu.L, and RNase inhibitor 2U/. Mu.L.
3. The present example also provides a method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus using the kit of example 1, comprising:
1. extraction of viral nucleic acid: a YXN-NB-43 complex sample nucleic acid extraction kit (from Guangzhou Yi-Biotechnology Co., ltd.) was used to extract highly pathogenic porcine reproductive and respiratory syndrome virus nucleic acid, and cDNA was synthesized by reverse transcription according to the kit instructions as a template for subsequent experiments.
Establishing and optimizing MB-RT-PCR test amplification conditions:
25. Mu.L MB-RT-PCR reaction system was used: the PCR amplification reaction of the template is carried out by using 14 mu L of MB-RT-PCR reaction liquid, 1 mu L of enzyme mixed liquid and 10 mu L of template and the kit for detecting the high-pathogenicity porcine reproductive and respiratory syndrome virus.
The primer volume, MB probe volume, and annealing temperature were optimized separately. The basic principle of the optimization is as follows: the method can effectively detect the highly pathogenic porcine reproductive and respiratory syndrome virus, and has low cost, short time consumption and the like.
(1) Setting 11 different primer probe ratios shown in the following table 1, performing a screening experiment of primer and probe volumes, and determining that the optimal primer and probe volume ratio is 0.35 after optimization: 0.01, i.e. the upstream primer: a downstream primer: the volume ratio of the probe is 0.35:0.35:0.01, and used in subsequent experiments. Experimental data are shown in table 1, where the primers: probes represent the upstream primer: a downstream primer: and (3) a probe.
TABLE 1 Experimental data sheet for screening the volume ratio of the optimal primer probe for PCR amplification
(2) Optimal annealing temperature screening:
the PCR reaction conditions were maintained for reverse transcription at 50℃for 20min, pre-denaturation at 95℃for 5min, denaturation at 95℃for 15s, and 40 cycles were performed, and the following 8 different Tm values were set for the screening experiments at the optimal annealing temperatures.
TABLE 2 Experimental data table for screening optimum Tm value for PCR amplification
As can be seen from Table 2, the optimum annealing temperature Tm value after screening was 57 ℃, i.e., the optimum PCR reaction conditions are shown in Table 3 below:
TABLE 3 PCR amplification reaction conditions Table
3. Result determination and analysis
And after the reaction is finished, collecting fluorescent signals, recording an amplification curve and an amplification cycle number Ct of the detection channel, and judging a detection result according to the amplification curve and the amplification cycle number. Fluorescence channel detection selection CY5, quality control and HP-PRRSV positive assay values are shown in tables 4, 5 below:
TABLE 4 quality control Table
TABLE 5 HP PRRSV positive judging value table
Interpretation of detection results:
negative: no CT value or CT > 37, and no typical S-type amplification curve, indicating no new variant highly pathogenic porcine reproductive and respiratory syndrome virus in the sample.
Positive: the CT value is less than or equal to 37, and a typical S-shaped amplification curve appears, which indicates that the new variant type high pathogenicity porcine reproductive and respiratory syndrome virus exists in the sample.
Suspicious: if Ct is more than 37 and less than or equal to 40 and a sample with a typical S-shaped amplification curve is an uncertain sample, the nucleic acid needs to be extracted again for detection, if the amplification curve of the recheck CY5 channel is S-shaped and Ct is more than 37 and less than or equal to 40, the sample is positive, otherwise, the sample is negative.
Invalidation: the detection holes have no CT value, which indicates that the experimental result is invalid, and the sample needs to be detected after nucleic acid extraction is carried out again.
Example 2: performance of detection kit for analyzing high-pathogenicity porcine reproductive and respiratory syndrome virus
The high pathogenicity porcine reproductive and respiratory syndrome virus detection kit and the detection method designed in the embodiment 1 are respectively used for comparison experiments, linear experiments, minimum detection limit experiments, precision detection and specificity detection, and the specific operations are as follows:
1. comparative experiments with other company products
The experiment tests the new variant type high pathogenicity porcine reproductive and respiratory syndrome virus detection kit of the example 1, and the product amplification results of other companies are compared, and the PCR amplification experiment is carried out by adopting the product 1 of a certain company 1 and the product 2 of a certain company 2.
As can be seen from fig. 2, the detection result of the kit is improved by about 5 Ct compared with other products of other companies under the condition of using the same template concentration, the repeatability is better, the experimental data are shown in the following table 6, and the experimental result is shown in fig. 2.
Table 6 comparative experimental data sheet for the kit and other company products
2. Linear experiments
The target probe of target gene is marked as CY5 fluorescent group, the linear reference product of the kit consists of positive plasmid containing target fragment, and the positive plasmid is subjected to 10 times gradient dilution by adopting TE buffer solution to obtain the following gradient solution (E8: 1.0X10) 8 copies/μL、E7：1.0×10 7 copies/μL、E6：1.0×10 6 copies/μL、E5：1.0×10 5 copies/μL、E4：1 .0×10 4 copies/μL、E3：1 .0×10 3 copies/μL、E2：1 .0×10 2 copies/μL、E1：1.0×10 1 copies/μL、0.5E1：1.0×10 1/2 copies/μL、0.25E1：1.0×10 1/4 cobies/. Mu.L) was used as a linear reference, 3 replicates each.
As can be seen from fig. 3, the kit is shown at E8: 1.0X10 8 copies/. Mu.L to E1: 1.0X10 1 The copies/. Mu.L has a good linear relation, and meanwhile, the linear equation fitted by the kit is as follows: y= -4.0804x+42.838, correlation coefficient R 2 =0.9998, where y represents Ct value and x represents the logarithmic value of positive reference. Experimental results referring to fig. 3, the standard curve referring to fig. 4.
3. Minimum detection limit experiment
The experiment verifies the minimum detection limit of the kit, and E8 in a linear reference is adopted: 1.0X10 8 copies/μL、E7：1.0×10 7 copies/μL、E6：1.0×10 6 copies/μL、E5：1.0×10 5 copies/μL、E4：1.0×10 4 copies/μL、E3：1.0×10 3 copies/μL、E2：1.0×10 2 copies/μL、E1：1.0×10 1 copies/μL、0.5E1：1.0×10 1/2 copies/μL、0.25E1：1.0×10 1/4 The positive plasmid at equal concentrations of copies/. Mu.L was used as a detection limit reference, and 10 replicates were performed for each concentration.
As can be seen from fig. 5, all of the detection limit references E8-E2 show amplification curves, which are positive data, and only 6 samples of the E1 reference amplify S-type curves, which do not meet the requirements, so the experiment shows that the detection limit of the kit is E2: 1.0X10 2 The lowest detected nucleic acid concentration was 8.9 copies/. Mu.L.
4. Precision experiments
The experiment verifies the precision in the batch of the kit, and E7 in the linear reference is adopted: 1.0X10 7 copies/μL、E6：1.0×10 5 copies/μL、E5：1.0×10 5 Three high-low concentration positive plasmids such as copies/. Mu.L are used as precision reference substances, 10 repeated samples are respectively made, and as a result, the variation coefficient of two concentration data is less than 2%, and the experimental results are shown in the following table 7.
TABLE 7 precision test results table
5. Specificity experiments
The common pathogen of the new variant highly pathogenic porcine reproductive and respiratory syndrome virus with the same infection position is used as a specific reference, an inactivated positive sample is adopted, and then DNA/RNA in the inactivated positive sample is extracted as a template by using YXN-NB-43 (a complex sample nucleic acid extraction kit) developed by Guangzhou Yi biological technology Co., ltd, and a specific experiment is carried out by using the detection method of the example 1. Wherein the specific reference is classical swine blue-ear virus, type 06 swine blue-ear virus, african swine fever virus, type 2 swine circovirus, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine foot-and-mouth disease virus, and porcine rotavirus respectively.
As can be seen from FIG. 6, none of these 8 specific references has a typical "S" type amplification curve, indicating that the kit has good specificity.
In conclusion, the kit designed by the invention can rapidly detect the novel variant type high pathogenicity porcine reproductive and respiratory syndrome virus by using an MB-RT-PCR method, has the characteristics of high sensitivity, strong specificity and good repeatability of detection results, and has the minimum detection limit of 1.0x10 2 The lowest detected nucleic acid concentration was 8.9 copies/. Mu.L.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.
Claims (10)
1. A kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR is characterized by comprising MB-RT-PCR reaction liquid, enzyme mixed liquid, negative quality control product and positive quality control product, wherein the MB-RT-PCR reaction liquid comprises an upstream primer with a nucleotide sequence shown as SEQ ID NO.1, a downstream primer with a nucleotide sequence shown as SEQ ID NO.2 and a molecular beacon probe shown as SEQ ID NO. 3.
2. The kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR according to claim 1, wherein the volume ratio of the upstream primer to the downstream primer to the probe is (0.05-0.1): (0.05-0.1): (0.01:0.02).
3. The kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR according to claim 2, wherein the 3 'end of the molecular beacon probe is marked with a fluorescence quenching group, and the 5' end is respectively marked with a fluorescence reporting group; the fluorescence reporter group is selected from FAM, CY5, ROX, VIC, or Texas Red, and the fluorescence quenching group is selected from BHQ-1, BHQ-2, BHQ-3, DABCYL, DABSYL, TAMRA, or TAMRA.
4. The kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR according to claim 1, wherein the MB-RT-PCR reaction solution further comprises an RT-PCR buffer, a PCR enhancer, magnesium chloride, a deoxyribonucleotide triphosphate mixture, and a buffer containing magnesium ions.
5. The kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR according to claim 4, wherein the RT-PCR buffer solution comprises Tris-HCl with pH of 7.5-8.9 and concentration of 20 mM-80M, 10 mM-5M ammonium sulfate, 100 mM-4M potassium chloride and Triton X-100 with volume ratio of 0.01-0.3%.
6. The kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR according to claim 4, wherein the PCR enhancer is at least one selected from the group consisting of tetramethyl ammonium chloride, carnitine, trehalose and non-ionic detergent NP-40.
7. The kit for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR according to claim 1, wherein the enzyme mixture comprises 2U/μL-6U/μL of Taq DNA polymerase, 5U/μL-12U/μL of reverse transcriptase with high thermal stability and 1U/μL-5U/μL of RNase inhibitor.
8. An MB-RT-PCR method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus, which is characterized by comprising the following specific steps:
s1, extracting total RNA of a sample, and synthesizing single-stranded cDNA by reverse transcription;
s2, carrying out PCR amplification reaction on the template by using the cDNA synthesized in the step S1 as the template and using the kit for detecting the high-pathogenicity porcine reproductive and respiratory syndrome virus according to claim 1, and detecting and analyzing after the reaction is finished.
9. The method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus MB-RT-PCR according to claim 8, wherein the step S2 adopts a 25. Mu.L PCR amplification system comprising 14. Mu.L MB-RT-PCR reaction solution, 1. Mu.L enzyme mixture solution and 10. Mu.L template.
10. The MB-RT-PCR method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus according to claim 8, wherein the PCR amplification reaction conditions are: 20min at 50 ℃;95 ℃ for 5min; 15s at 95 ℃, 30s at 53-60 ℃ and 40 cycles.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410014399.2A CN117511936B (en) | 2024-01-05 | 2024-01-05 | Kit and method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410014399.2A CN117511936B (en) | 2024-01-05 | 2024-01-05 | Kit and method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN117511936A true CN117511936A (en) | 2024-02-06 |
| CN117511936B CN117511936B (en) | 2024-04-09 |
Family
ID=89749789
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202410014399.2A Active CN117511936B (en) | 2024-01-05 | 2024-01-05 | Kit and method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117511936B (en) |
Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343672A (en) * | 2008-07-09 | 2009-01-14 | 中国农业大学 | Detection reagent kit for porcine propagate and breath complex virus and uses thereof |
| KR20110027871A (en) * | 2009-09-11 | 2011-03-17 | 주식회사 제노바이오텍 | Dna probe, gene chip and gene diagnosis kit for prdc pathogens and method of diagnosis using the same |
| CN103740864A (en) * | 2014-01-21 | 2014-04-23 | 北京亿森宝生物科技有限公司 | Fluorescent quantitative qRT-PCR detection method and detection kit for high-pathogenicity porcine reproductive and respiratory syndrome virus (PRRSV) |
| CN105648114A (en) * | 2016-02-22 | 2016-06-08 | 深圳市易瑞生物技术有限公司 | Fluorescence RT-PCR (reverse transcription-polymerase chain reaction) primers, probe and kit for detecting new-variant HP-PRRSV (high pathogenic porcine reproductive and respiratory syndrome virus) of sample as well as detection method |
| CN110885902A (en) * | 2018-09-10 | 2020-03-17 | 北京亿森宝生物科技有限公司 | Freeze-drying microchip, kit and method for detecting porcine reproductive and respiratory syndrome virus and identifying highly pathogenic classical variant strain of porcine reproductive and respiratory syndrome virus |
| RU2019129560A (en) * | 2019-09-18 | 2021-03-18 | Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский ветеринарный институт патологии, фармокологии и терапии" (ФГБНУ "ВНИВИПФиТ") | Method for identification of porcine reproductive and respiratory syndrome virus based on real-time PCR |
| CN113046329A (en) * | 2020-11-10 | 2021-06-29 | 山东省滨州畜牧兽医研究院 | Porcine reproductive and respiratory syndrome virus chimeric recombinant PRRSV DIVA vaccine strain cDY56 |
| CN115852054A (en) * | 2022-11-30 | 2023-03-28 | 福建傲农生物科技集团股份有限公司 | Fluorescent probe primer and kit for porcine reproductive and respiratory syndrome European strain and application of fluorescent probe primer and kit |
| CN115976286A (en) * | 2023-02-15 | 2023-04-18 | 福建佰孟医学科技有限公司 | Universal porcine reproductive and respiratory syndrome virus detection primer composition, kit and detection method |
| CN116064954A (en) * | 2022-09-30 | 2023-05-05 | 广州奕昕生物科技有限公司 | Kit for detecting novel coronavirus and typing based on MB-RT-PCR and application thereof |
| US20230374614A1 (en) * | 2021-12-03 | 2023-11-23 | Army Medical University | Primers, kit and method for detecting of african swine fever virus |
-
2024
- 2024-01-05 CN CN202410014399.2A patent/CN117511936B/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101343672A (en) * | 2008-07-09 | 2009-01-14 | 中国农业大学 | Detection reagent kit for porcine propagate and breath complex virus and uses thereof |
| KR20110027871A (en) * | 2009-09-11 | 2011-03-17 | 주식회사 제노바이오텍 | Dna probe, gene chip and gene diagnosis kit for prdc pathogens and method of diagnosis using the same |
| CN103740864A (en) * | 2014-01-21 | 2014-04-23 | 北京亿森宝生物科技有限公司 | Fluorescent quantitative qRT-PCR detection method and detection kit for high-pathogenicity porcine reproductive and respiratory syndrome virus (PRRSV) |
| CN105648114A (en) * | 2016-02-22 | 2016-06-08 | 深圳市易瑞生物技术有限公司 | Fluorescence RT-PCR (reverse transcription-polymerase chain reaction) primers, probe and kit for detecting new-variant HP-PRRSV (high pathogenic porcine reproductive and respiratory syndrome virus) of sample as well as detection method |
| CN110885902A (en) * | 2018-09-10 | 2020-03-17 | 北京亿森宝生物科技有限公司 | Freeze-drying microchip, kit and method for detecting porcine reproductive and respiratory syndrome virus and identifying highly pathogenic classical variant strain of porcine reproductive and respiratory syndrome virus |
| RU2019129560A (en) * | 2019-09-18 | 2021-03-18 | Федеральное государственное бюджетное научное учреждение "Всероссийский научно-исследовательский ветеринарный институт патологии, фармокологии и терапии" (ФГБНУ "ВНИВИПФиТ") | Method for identification of porcine reproductive and respiratory syndrome virus based on real-time PCR |
| CN113046329A (en) * | 2020-11-10 | 2021-06-29 | 山东省滨州畜牧兽医研究院 | Porcine reproductive and respiratory syndrome virus chimeric recombinant PRRSV DIVA vaccine strain cDY56 |
| US20230374614A1 (en) * | 2021-12-03 | 2023-11-23 | Army Medical University | Primers, kit and method for detecting of african swine fever virus |
| CN116064954A (en) * | 2022-09-30 | 2023-05-05 | 广州奕昕生物科技有限公司 | Kit for detecting novel coronavirus and typing based on MB-RT-PCR and application thereof |
| CN115852054A (en) * | 2022-11-30 | 2023-03-28 | 福建傲农生物科技集团股份有限公司 | Fluorescent probe primer and kit for porcine reproductive and respiratory syndrome European strain and application of fluorescent probe primer and kit |
| CN115976286A (en) * | 2023-02-15 | 2023-04-18 | 福建佰孟医学科技有限公司 | Universal porcine reproductive and respiratory syndrome virus detection primer composition, kit and detection method |
Non-Patent Citations (4)
| Title |
|---|
| GO‑EUN SHIN等: "Genetic diversity of porcine reproductive and respiratory syndrome virus and evaluation of three one-step real-time RT-PCR assays in Korea", BMC VETERINARY RESEARCH, 30 August 2022 (2022-08-30), pages 1 - 2 * |
| JINGHUA PAN等: "Research Progress on the detection methods of porcine reproductive and respiratory syndrome virus", FRONTIERS IN MICROBIOLOGY, 9 March 2023 (2023-03-09), pages 1 - 12 * |
| WEI TIAN-CHAO等: "Evaluation of the Pathogenicity of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Variant in Piglets", AGRICULTURAL SCIENCES IN CHINA, vol. 10, no. 8, 31 August 2011 (2011-08-31), pages 1283 * |
| 周国华: "SNP检测技术与个体化药物治疗", 28 February 2015, 苏州大学出版社, pages: 24 - 37 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN117511936B (en) | 2024-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hodneland et al. | Sensitive and specific detection of Salmonid alphavirus using real-time PCR (TaqMan®) | |
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| CN111500791B (en) | Triple FQ-PCR detection method for porcine epidemic diarrhea virus, porcine delta coronavirus and porcine acute diarrhea syndrome coronavirus | |
| CN113403430A (en) | Triple fluorescent quantitative PCR primer group for detecting different types of porcine circovirus, kit and application | |
| CN107699635B (en) | Fluorescent RPA (reverse transcriptase amplification) detection method for porcine epidemic diarrhea virus | |
| 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 | |
| CN112029908A (en) | Detection kit for porcine coronavirus and acute diarrhea syndrome coronavirus | |
| CN117511936B (en) | Kit and method for detecting highly pathogenic porcine reproductive and respiratory syndrome virus based on MB-RT-PCR | |
| CN110438264B (en) | Method for detecting porcine epidemic diarrhea virus and B-type porcine enterovirus by using double real-time fluorescence quantitative RT-PCR | |
| CN116162734B (en) | Specific primer, probe and kit for rapidly detecting African swine fever wild strain and gene deletion strain | |
| CN114438265B (en) | Nucleic acid composition, kit and detection method for simultaneously detecting porcine delta coronavirus, reovirus and porcine kokumi virus | |
| AU2021104086A4 (en) | Detection reagent for fluorescent duplex pcr for classical swine fever virus and african swine fever virus, kit, and detection method | |
| CN110724763A (en) | Fluorescent quantitative PCR detection method for human adenovirus and bocavirus and application thereof | |
| CN111334613B (en) | RPA primer pair, probe, kit and detection method for detecting canine adenovirus | |
| CN114480726A (en) | Primer probe set, kit and detection method for African swine fever virus nucleic acid detection | |
| CN114807318A (en) | Composition, kit and method for detecting Vero cell genome DNA | |
| CN114540546A (en) | Primer probe set, kit and detection method for PRRSV and CSFV double fluorescence quantitative PCR detection | |
| CN113684318A (en) | Fluorescent PCR (polymerase chain reaction) detection kit for M protein gene of porcine reproductive and respiratory syndrome 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 | |
| US20240018610A1 (en) | Multiplex Assay for Simultaneous Detection of Equine Group A and B Rotaviruses and Genotyping of Equine Rotavirus A G3 And G14 | |
| CN113801966B (en) | Fluorescent quantitative PCR method and kit for detecting novel coronavirus subgenomic | |
| CN116144832A (en) | Rapid fluorescence PCR detection kit for enterovirus EV71 type and CoxA16 type | |
| CN118308537A (en) | Molecular marker for genotyping and pedigree identification of porcine reproductive and respiratory syndrome virus | |
| US20120122081A1 (en) | Differentiating picorna viruses, nucleic acids therefor, use thereof and bioassay methods employing them |
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 |