CN113025747A - Porcine pseudorabies virus nucleic acid extraction-free fluorescent isothermal amplification detection kit - Google Patents
Porcine pseudorabies virus nucleic acid extraction-free fluorescent isothermal amplification detection kit Download PDFInfo
- Publication number
- CN113025747A CN113025747A CN201911358886.6A CN201911358886A CN113025747A CN 113025747 A CN113025747 A CN 113025747A CN 201911358886 A CN201911358886 A CN 201911358886A CN 113025747 A CN113025747 A CN 113025747A
- Authority
- CN
- China
- Prior art keywords
- isothermal amplification
- pseudorabies virus
- detection
- porcine pseudorabies
- nucleic acid
- 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.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 53
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 title claims abstract description 47
- 238000011901 isothermal amplification Methods 0.000 title claims abstract description 38
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 27
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 27
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 27
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 claims abstract description 20
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 claims abstract description 20
- 101150072564 gE gene Proteins 0.000 claims abstract description 11
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 32
- 239000013612 plasmid Substances 0.000 claims description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000013641 positive control Substances 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 13
- 238000000605 extraction Methods 0.000 claims description 9
- 239000013642 negative control Substances 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L magnesium sulphate Substances [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 7
- 239000002480 mineral oil Substances 0.000 claims description 7
- 235000010446 mineral oil Nutrition 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 7
- 238000012163 sequencing technique Methods 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 5
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 5
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 claims description 4
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 claims description 4
- 229960003237 betaine Drugs 0.000 claims description 4
- 210000004369 blood Anatomy 0.000 claims description 4
- 239000008280 blood Substances 0.000 claims description 4
- 238000007796 conventional method Methods 0.000 claims description 4
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 239000003795 chemical substances by application Substances 0.000 claims description 3
- 238000010367 cloning Methods 0.000 claims description 2
- 238000003018 immunoassay Methods 0.000 claims description 2
- 239000012528 membrane Substances 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- 239000011535 reaction buffer Substances 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 20
- 230000003321 amplification Effects 0.000 abstract description 18
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 18
- 108090000623 proteins and genes Proteins 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 7
- 238000013461 design Methods 0.000 abstract description 4
- 238000009395 breeding Methods 0.000 abstract description 2
- 230000001488 breeding effect Effects 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 238000003307 slaughter Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 13
- 239000000523 sample Substances 0.000 description 13
- 238000005516 engineering process Methods 0.000 description 7
- 238000004806 packaging method and process Methods 0.000 description 7
- 238000002372 labelling Methods 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 241000282898 Sus scrofa Species 0.000 description 4
- 229940031567 attenuated vaccine Drugs 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 239000005662 Paraffin oil Substances 0.000 description 3
- 108010052762 Suid herpesvirus 1 glycoprotein E Proteins 0.000 description 3
- 241000282887 Suidae Species 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 229940057995 liquid paraffin Drugs 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 208000009305 pseudorabies Diseases 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- NLMKTBGFQGKQEV-UHFFFAOYSA-N 2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-hexadecoxyethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethanol Chemical compound CCCCCCCCCCCCCCCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO NLMKTBGFQGKQEV-UHFFFAOYSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 239000013504 Triton X-100 Substances 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 241000701386 African swine fever virus Species 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000710777 Classical swine fever virus Species 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000710198 Foot-and-mouth disease virus Species 0.000 description 1
- 208000005342 Porcine Reproductive and Respiratory Syndrome Diseases 0.000 description 1
- 241001673669 Porcine circovirus 2 Species 0.000 description 1
- 241000702619 Porcine parvovirus Species 0.000 description 1
- 241001135989 Porcine reproductive and respiratory syndrome virus Species 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000013066 combination product Substances 0.000 description 1
- 229940127555 combination product Drugs 0.000 description 1
- 238000012864 cross contamination Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 239000003269 fluorescent indicator Substances 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 239000013062 quality control Sample Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—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
Abstract
The invention selects the gE gene highly conserved in porcine pseudorabies virus as a detection target gene, designs a group of specific primers, establishes a fluorescence isothermal amplification detection method for specificity of the porcine pseudorabies virus of nucleic acid in a hand-free sample, and assembles the detection method into a kit. The Bst DNA polymerase is adopted, so that the method has the advantages of high amplification efficiency and good specificity; the reaction result is indicated by adopting fluorescent dye, so that accurate judgment is facilitated; the DNA of a sample does not need to be extracted, so that the detection operation is simplified; the whole detection process is fast and only needs 50 min. And the invention can detect a plurality of samples at one time, and is suitable for the detection of a large number of samples. The used fluorescent constant-temperature amplification instrument has low cost; has the advantages of high amplification efficiency and good specificity; is suitable for being used in the field of pig breeding, quarantine, monitoring and slaughtering and basic level detection units.
Description
Technical Field
The invention relates to a porcine pseudorabies virus nucleic acid fluorescence isothermal amplification detection technology without sample nucleic acid extraction operation and a kit, belonging to the technical field of microbial detection.
Background
Porcine Pseudorabies (PR) is an acute infectious disease in pigs caused by porcine pseudorabies virus (PRV). The disease can present as a fulminant epidemic in the swinery. The disease is mainly manifested by the symptoms of nerve symptoms, vomiting, diarrhea and the like of pregnant sows, mummy fetuses and newborn piglets, and is one of important infectious diseases which endanger the global pig industry. At present, PRs are widely applied to various countries in the world to prevent and control PRs, and PRs are purified by applying porcine pseudorabies virus gE gene-deleted attenuated vaccines and a differential diagnosis method matched with the porcine pseudorabies virus gE gene-deleted attenuated vaccines in developed countries such as Europe and America. At present, the disease is widely prevalent in China, China adopts measures similar to those in China on the control of the disease, adopts gE gene deletion attenuated vaccines for immune control, and then adopts a detection method aiming at PRV gE genes to eliminate the interference of vaccine strains, thereby diagnosing PRV wild virus infection.
The PRV detection method commonly used at present is a common PCR or fluorescence PCR method. The common PCR detection operation is easy to cause the pollution of virus nucleic acid amplification products, thereby causing false positive results; the fluorescence PCR technique requires an expensive fluorescence PCR instrument, and usually requires more than 2 hours to complete the whole detection process from nucleic acid extraction. In recent years, the quarantine of the live pigs and meat products in China requires the development of pathogenic detection, and quarantine proofs are provided according to detection results, so that a porcine pseudorabies virus detection technology which is quicker, simpler, more convenient, more accurate and lower in cost and is suitable for being applied to quarantine fields is required.
The nucleic acid isothermal amplification technology is an emerging gene detection technology, and has many advantages compared with the widely applied common PCR and fluorescence PCR. The method does not need expensive instruments (constant temperature), is simple to operate, has high speed and high sensitivity, and is widely applied. However, the existing nucleic acid isothermal amplification method mainly depends on the visual observation of the color change of the reaction solution to judge the result, and has strong subjectivity and poor accuracy.
The method adopts the fluorescent agent to dye the double-stranded DNA amplification product, and uses a simple reader to detect the fluorescent signal to judge the result, so that the accuracy is improved compared with an eye observation method; the invention adopts the extraction-free reaction solution to process and release the virus DNA in the sample, directly carries out amplification reaction without extracting the DNA in the sample, reduces the operation steps and the cost, shortens the detection time, and eliminates the cross contamination in the nucleic acid extraction process; the invention designs a set of 6 specific primers aiming at the porcine pseudorabies virus gene, thereby ensuring the specificity of amplification.
The invention comprehensively applies the technologies and finally establishes a novel nucleic acid rapid detection method and a kit for the porcine pseudorabies virus. Compared with the PCR and fluorescence PCR detection method for porcine pseudorabies virus which is widely used at present, the PCR and fluorescence PCR detection method for porcine pseudorabies virus has the advantages of high detection speed, high sensitivity, simple and convenient operation, low reagent and equipment cost and the like, is suitable for being used in the field and basic units of live pig breeding, slaughtering, quarantine and monitoring, and has good application prospect.
Disclosure of Invention
The invention aims to establish a fluorescence isothermal amplification detection method and a kit for porcine pseudorabies virus without taking nucleic acid, so that the detection process is simple and easy to implement, the viral nucleic acid in a sample does not need to be extracted, the result is accurately interpreted, an expensive instrument is not used, the detection cost is reduced, the total detection time is shortened to 50 minutes, and a rapid, sensitive, specific and low-cost technical means is provided for PRV detection.
In order to realize the purpose, the invention comprehensively uses Bst DNA polymerase isothermal amplification technology, tissue sample nucleic acid hands-free technology and fluorescent indicator to establish a PRV fluorescent isothermal amplification detection method and develop a kit, and the specific contents are as follows:
1. designing isothermal amplification primers: referring to the nucleotide sequence of the porcine pseudorabies virus gene, conserved regions are screened, 6 oligonucleotides are designed and combined into a set of primers (each oligonucleotide is not used independently).
PRVGEF3 ACGAGCCCCGCTTCCACG
PRVGEB3 GTAGATGCAGGGCTCGTA
PRVGELF TCAGGTCGAACGTGTCCCCG
PRVGELB GCCACGCTGGACTGGTACT
PRVGEFIP TGTCCGAGACCACGCGCGTTTTGCGCTCGGCTTCCACT
PRVGEBIP GCGACTCGCGCGAGAACTTTTTCGTAGTACAGCAGGCACCG
2. Preparing a primer solution: diluting artificially synthesized primers PRVGEF3, PRVGEB3, RVGEFIP and PRVGEBIP to 20 mu mol/L by DEPC treated water, and diluting artificially synthesized primers PRVGELF and PRVGELB to 40 mu mol/L by DEPC treated water; the diluted primers are mixed uniformly according to the proportion of PRVGEF3: PRVGEB3: RVGEFIP: PRVGEBIP: PRVGELF: PRVGELB =1:1:8:8:2: 2.
3. Preparation of positive control: the PRV gE genome is cloned by a conventional method, a pMD20-T-PRGE vector recombinant plasmid is constructed, and the sequence of a target gene in the plasmid is determined. After the sequencing result is correct, the competent cells of E.coli Top10 were transformed, and the cells were cultured in an expanded manner to extract plasmids as positive controls.
4.10 × preparation of isothermal amplification extraction-free buffer: weighing Trisbase: 24.23g, KCl: 3.73g (NH4)2 SO4：7g，MgSO47H2O: 2.465g, Triton X-100: 5mL, Brij-58: 5g, DEPC treated water is added to 400mL, the pH value is adjusted to 8.8 (25 ℃) by hydrochloric acid, and the volume is adjusted to 500 mL. And (4) after autoclaving and cooling, storing at 2-8 ℃ for later use.
5. Preparing a fluorescence isothermal amplification extraction-free reaction solution: taking 2.5 mL of 10 times isothermal amplification extraction-free buffer solution, 5.5 mL of primer solution, 4 mL of 5 mol/L betaine solution, 3.5 mL of 10 mmol/L dNTP mix and 100 mmol/L MgSO41.5 mL of the solution, 0.5 mL of 0.1% SYBR GreenI solution and 4.5 mL of 0.1% DEPC treated water, mixing, filtering with a 0.45 μm filter membrane for sterilization (sterile low temperature in the whole process), packaging into small tubes, and labeling.
6. Preparation of Bst DNA polymerase: bst DNA polymerase was purchased from outsourced (NEB) at a concentration of 8U/. mu.L, 1000. mu.L/tube. Bst DNA polymerase was quantitatively dispensed aseptically and labeled.
7. Preparation of mineral oil: quantitatively packaging the liquid paraffin oil into small tubes, and labeling.
8. Preparation of negative control: 0.1% DEPC aqueous solution, aseptically packaging, and labeling.
9. The detection method comprises the following steps:
(1) in a 0.2 mL reaction tube, 22. mu.L of the fluorescence isothermal amplification immunoassay extraction reaction solution and 1.0. mu.L of Bst DNA polymerase were added to each tube, and 20. mu.L of mineral oil was added to each tube.
(2) Sample adding: adding 1: 5 diluting 2.0 μ L of the blood or tissue grinding fluid of the detected pig, and setting a positive control tube and a negative control tube. Covering the pipe cap tightly, performing instantaneous centrifugation, and putting into a special fluorescent constant-temperature amplification instrument or a common fluorescent PCR instrument.
(3) Optimized reaction parameters: FAM channel fluorescence signals are collected every 1min at 64 ℃ for 50min for 50 times. When the Tt (time threshold) value is absent and a characteristic amplification curve is absent, determining that the sample is negative; when the Tt value is less than or equal to 45.0 and a typical amplification curve appears, the sample is judged to be positive; when the Tt value is more than 45.0 and a typical amplification curve appears, the amplification curve should be redone, the result still appears, and the result is judged to be positive, otherwise, the result is judged to be negative.
10. The invention also relates to a kit which comprises the primer pair combination product, and preferably further comprises one or more of a fluorescence isothermal amplification non-extraction reaction solution (containing SYBR GreenI fluorescent agent), Bst DNA polymerase, a negative control and a positive control. Preferably, the Bst DNA polymerase can be added into the fluorescence isothermal amplification non-extraction reaction solution for integrated packaging. The kit of the invention also comprises a positive control and a negative control; the positive control is pMD20-T-PRGE recombinant plasmid; the negative control was no nucleic acid water. The kit provided by the invention can be used for detecting the pseudorabies virus and can be used for detecting the pseudorabies virus in blood and tissues of pigs.
11. The kit verification method comprises the following steps: (1) the positive control plasmid was diluted 10 fold-1～10-6The detection is carried out according to the method in 9, and the sensitivity of the kit is verified to reach 10-3. (2) The kit is used for detecting nucleic acid of classical swine fever virus, nucleic acid of foot-and-mouth disease virus, nucleic acid of porcine reproductive and respiratory syndrome virus, and verifying the specificity of the kit, and has no positive cross reaction.
The invention selects the gE gene with highly conserved porcine pseudorabies virus as a detection target gene, designs a specific primer and establishes a PRV specific fluorescence isothermal amplification detection method. The Bst DNA polymerase is adopted, so that the method has the advantages of high amplification efficiency and good specificity; the reaction result is indicated by adopting fluorescent dye, so that accurate judgment is facilitated; the DNA of a sample does not need to be extracted, so that the detection operation is simplified; the whole detection process is fast and only needs 50 min. And the invention can detect a plurality of samples at one time, and is suitable for the detection of a large number of samples.
Detailed Description
The present invention is described in further detail below, and the examples are only for explaining the present invention and are not intended to limit the scope of the present invention.
Example 1 was carried out: primer design and construction of porcine pseudorabies virus gE gene recombinant plasmid
1. Designing a primer: according to the gE gene sequence of 26 representative strains of the porcine pseudorabies virus recorded in GenBank (see Table 1), a conserved region is screened, 16 primers in total are designed, and comparison and test screening are carried out.
TABLE 1 reference strains of porcine pseudorabies virus
The nucleotide sequence of the finally determined isothermal amplification primers is as follows:
PRVGEF3 ACGAGCCCCGCTTCCACG
PRVGEB3 GTAGATGCAGGGCTCGTA
PRVGELF TCAGGTCGAACGTGTCCCCG
PRVGELB GCCACGCTGGACTGGTACT
PRVGEFIP TGTCCGAGACCACGCGCGTTTTGCGCTCGGCTTCCACT
PRVGEBIP GCGACTCGCGCGAGAACTTTTTCGTAGTACAGCAGGCACCG
construction of pMD20-T-PRGE recombinant plasmid: according to the published gE gene sequence of the porcine pseudorabies virus, Shanghai Yuanjun Biometrics GmbH is entrusted to synthesize the GE gene, and the GE gene is cloned into a pMD20-T vector to form a pMD20-T-PRGE plasmid. The Top10 bacterial competent cells (purchased product) were transformed by the conventional method, and the cloned bacteria were selected on an ampicillin-resistant agar plate and identified by PCR. And (3) PCR reaction system: a25. mu.L reaction system contained 2.5. mu.L of 10 XPCR buffer, 2. mu.L of 2.5mmol/L dNTPs, 0.5. mu.L each of 10. mu. mol/L upstream and downstream primers PRGEF (5'-TTTGGATCCTCGCACACACCGGGGTTGAGA-3') and PRGER (5'-TTTAAGCTTGGCGGTGGGCATGTCGGAATG-3'), 0.5. mu.L of 5U/. mu.L Taq enzyme, and 1. mu.L bacterial suspension. PCR reaction procedure: 94 ℃/5 min; 94 ℃/30sec, 54 ℃/30sec, 72 ℃/2 min, 30 cycles; finally, extension is carried out for 10 min at 72 ℃. And (4) extracting plasmids from the bacteria liquid which is identified as positive by PCR, sending the plasmids to a sequencing company for sequencing, and comparing the sequencing result with the sequence of the reference template to be consistent.
Example 2 establishment of Fluorogenic isothermal amplification reaction System for porcine pseudorabies Virus
By sequencing the gE gene of the porcine pseudorabies virusCarrying out comparison analysis, designing and screening primers, primarily establishing a fluorescence isothermal amplification detection method, and detecting the concentration and Mg of the primers of the detection method2+The optimal conditions of the detection method are determined by optimally selecting reaction conditions such as concentration, SYBR GreenI fluorescent dye dosage, Bst DNA polymerase dosage, reaction temperature and the like:
the reaction system is 25 mu L in total and comprises three parts of fluorescent isothermal amplification reaction liquid, Bst DNA polymerase and a template. Wherein one reaction solution for detection comprises 2.5. mu.L of 10 × Thermopolbuffer, 5.5. mu.L of primer solution, 4. mu.L of 5 mol/L betaine solution, 3.5. mu.L of dNTP Mix (10 mmol/L), and 1.5. mu.L of MgSO4(100 mmol/L), 0.5 mu L SYBR GreenI solution and 4.5 mu L DEPC treated water; one Bst DNA polymerase tested consisted of 1. mu.L of Bst DNA polymerase (8U/. mu.L); one template tested consisted of 2.0 μ L of sample; finally, 20. mu.L of mineral oil (liquid paraffin oil) was added to 25. mu.L of the reaction system. A fluorescence isothermal amplification apparatus or a commonly used fluorescence PCR apparatus may be used.
Finally, the amplification conditions of the porcine pseudorabies virus fluorescence isothermal amplification detection method are determined to be 64 ℃, 50min, and fluorescence is collected once every minute. When the Tt (time threshold) value is absent and a characteristic amplification curve is absent, determining that the sample is negative; when the Tt value is less than or equal to 45.0 and a typical amplification curve appears, the sample is judged to be positive; when the Tt value is more than 45.0 and a typical amplification curve appears, the amplification curve should be redone, the result still appears, and the result is judged to be positive, otherwise, the result is judged to be negative.
Example 3 Assembly of the nucleic acid extraction-free fluorescent isothermal amplification detection kit for porcine pseudorabies virus
1. The components of the kit are prepared according to the following method:
(1) preparing a primer solution: artificially synthesized primers PRVGEF3, PRVGEB3, PRVGEFIP and PRVGEBIP were diluted to 20. mu. mol/L with DEPC treated water, and artificially synthesized primers PRVGELF and PRVGELB were diluted to 40. mu. mol/L with DEPC treated water. The diluted primers are mixed evenly according to the proportion of PRVGEF3, PRVGEB3, PRVGEFIP, PRVGEBIP, PRVGELF and PRVGELB =1:1:8:8:2: 2.
(2) Preparation of positive control: cloning gE gene of porcine pseudorabies virus by a conventional method, constructing pMD20-T-PRGE recombinant plasmid, and carrying out sequence determination on target gene in the plasmid. And (3) after the sequencing result is correct, transforming the recombinant plasmid bacteria obtained by the escherichia coli, carrying out amplification culture on the recombinant plasmid bacteria, and extracting the recombinant plasmid. The content of the recombinant plasmid is measured by a Nanodrop2000 instrument, the copy number of gE gene DNA in each microliter volume is calculated, the gE gene DNA is diluted to 104 copy number/microliter by TE buffer solution, and sterile quantitative subpackaging is carried out according to the specification of 200 microliter/tube to be used as a positive control of the kit.
(3) Preparation of 10 × isothermal amplification extraction-free buffer: weighing Trisbase: 24.23g, KCl: 3.73g, (NH4)2SO 4: 7g, MgSO47H 2O: 2.465g, Triton X-100: 5mL, Brij-58: 5g, DEPC treated water is added to 400mL, the pH value is adjusted to 8.8 (25 ℃) by hydrochloric acid, and the volume is adjusted to 500 mL. And (5) sterilizing at high pressure, cooling, and storing at 2-8 ℃ for later use.
(4) Preparing a fluorescence isothermal amplification extraction-free reaction solution: 2.5 mL of 10 × isothermal amplification extraction-free buffer solution, 5.5 mL of primer solution, 4 mL of 5 mol/L betaine solution, 3.5 mL of 10 mmol/L dNTP mix, 1.5 mL of 100 mmol/L MgSO4 solution, 0.5 mL of 0.1% SYBR GreenI solution and 4.5 mL of 0.1% DEPC treated water are mixed uniformly (sterile at low temperature throughout the process) and filtered through a 0.45 μm filter for sterilization, and the Mixture is packaged into small tubes and labeled.
(5) Preparation of Bst DNA polymerase: bst DNA polymerase was purchased from outsourced (NEB Co.), 8U/. mu.L, 1000. mu.L/tube. Bst DNA polymerase was quantitatively dispensed aseptically and labeled.
(6) Preparation of mineral oil: quantitatively packaging the liquid paraffin oil into small tubes, and labeling.
(7) Preparation of negative control: 0.1% DEPC aqueous solution, aseptically packaging, and labeling.
The kit is assembled according to the following requirements:
the fluorescence isothermal amplification non-extraction reaction solution, Bst DNA polymerase, mineral oil, negative control, positive control and the like are packaged by an external packaging box according to the quantity required by the table 2, and labels (including identification names, batch numbers, production dates, storage periods, production unit information and the like) are added.
TABLE 2 List of fluorescent isothermal amplification detection kit (extraction-free) for porcine pseudorabies virus
Example 4: sensitivity verification of porcine pseudorabies virus nucleic acid extraction-free fluorescent isothermal amplification detection kit
pMD20-T-PRGE recombinant plasmid (DNA content 10) was treated with DEPC to treat water4Copy/. mu.L) as a sensitive quality control sample at 1:10, 1:100, 1:1000, 1:10000, 1:100000 dilutions. Detection and determination were carried out according to the "usage and determination" of the kit. The detection results of the quality control samples at 1:10, 1:100 and 1:1000 are positive, positive or negative at 1:10000 and negative at 1: 100000. 40 samples which are clinically confirmed to be positive to the porcine pseudorabies virus are detected, including porcine blood, lymph node and spleen tissues, and the detection results are positive.
Example 5: specificity test of porcine pseudorabies virus nucleic acid extraction-free fluorescent isothermal amplification detection kit
The kit is used for detecting the nucleic acid of highly pathogenic porcine reproductive and respiratory syndrome vaccine, swine fever freeze-dried attenuated vaccine, porcine parvovirus, porcine circovirus type 2, African swine fever virus nucleic acid sample, normal porcine lymph node and 10% suspension of spleen by adopting 3 batches of porcine pseudorabies virus fluorescent isothermal amplification detection kit, and has no positive cross reaction. 60 samples which are clinically diagnosed and identified as negative are detected, the results are all negative, and the specificity is 100%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Beijing Senkang Biotechnology development Co., Ltd
<120> fluorescent isothermal amplification detection kit for nucleic acid extraction-free of porcine pseudorabies virus
<130> 2019
<141> 2019-12-23
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> PRV sequence
<400> 1
acgagccccg cttccacg 18
<210> 2
<211> 18
<212> DNA
<213> PRV sequence
<400> 2
gtagatgcag ggctcgta 18
<210> 3
<211> 20
<212> DNA
<213> PRV sequence
<400> 3
tcaggtcgaa cgtgtccccg 20
<210> 4
<211> 19
<212> DNA
<213> PRV sequence
<400> 4
gccacgctgg actggtact 19
<210> 5
<211> 38
<212> DNA
<213> PRV sequence
<400> 5
tgtccgagac cacgcgcgtt ttgcgctcgg cttccact 38
<210> 6
<211> 41
<212> DNA
<213> PRV sequence
<400> 6
gcgactcgcg cgagaacttt ttcgtagtac agcaggcacc g 41
Claims (4)
1. A fluorescence isothermal amplification kit for rapidly detecting nucleic acid extraction of porcine pseudorabies virus comprises a composition of 6 specific primers, and the nucleotide sequence of the kit is as follows:
PRVGEF3 ACGAGCCCCGCTTCCACG; SEQ ID NO.1;
PRVGEB3 GTAGATGCAGGGCTCGTA; SEQ ID NO.2;
PRVGELF TCAGGTCGAACGTGTCCCCG; SEQ ID NO.3;
PRVGELB GCCACGCTGGACTGGTACT; SEQ ID NO.4;
PRVGEFIP TGTCCGAGACCACGCGCGTTTTGCGCTCGGCTTCCACT; SEQ ID NO.5;
PRVGEBIP GCGACTCGCGCGAGAACTTTTTCGTAGTACAGCAGGCACCG; SEQ ID NO.6。
2. the kit according to claim 1, further comprising one or more of a fluorescence isothermal amplification immunoassay extraction reaction solution (containing SYBR GreenI fluorescent staining agent), Bst DNA polymerase, a negative control, a positive control and mineral oil.
3. A preparation method of a porcine pseudorabies virus nucleic acid extraction-free fluorescent isothermal amplification detection kit specifically comprises the following steps:
preparing a primer solution: synthesizing primers according to the nucleotide sequence of claim 1, wherein the artificially synthesized primers PRVGEF3, PRVGEB3, PRVGEFIP and PRVGEBIP are diluted to 20 mu mol/L by DEPC treated water, and the artificially synthesized primers PRVGELF and PRVGELB are diluted to 40 mu mol/L by DEPC treated water; uniformly mixing the diluted primers according to the proportion of PRVGEF3: PRVGEB3: PRVGEFIP: PRVGEBIP: PRVGELF: PRVGELB =1:1:8:8:2: 2;
preparing a fluorescence isothermal amplification extraction-free reaction solution: 0.5 mL of 0.1% SYBR GreenI solution, 2.5 mL of 10-fold concentration reaction buffer solution, 5.5 mL of primer solution, 4 mL of 5 mol/L betaine solution, 3.5 mL of 10 mmol/L dNTP mix, and 100 mmol/L MgSO41.5 mL of the solution and 4.5 mL of 0.1 percent DEPC treated water are mixed uniformly, sterilized by a filter membrane and subpackaged into small tubes;
preparation of Bst DNA polymerase: the concentration is 8U/muL, and the mixture is subpackaged according to 1000 muL/tube aseptically;
preparation of positive control: cloning gE gene of porcine pseudorabies virus by a conventional method, constructing pMD20-T-PRGE vector plasmid, and carrying out sequence determination on the gE gene in the plasmid; after the sequencing result is correct, Top10 bacteria competent cells are transformed, expanded culture is carried out, and plasmids are extracted as positive control;
the optimized reaction conditions are as follows: adding 22. mu.L of fluorescence isothermal amplification extraction-free reaction solution and 1.0. mu.L of Bst DNA polymerase into a reaction tube, adding 20. mu.L of mineral oil, and adding 2.0. mu.L of sample (without pre-extracting nucleic acid); the reaction condition parameters are 64 ℃ and 50min, and the fluorescence signal is collected once per minute.
4. Use of the kit according to claim 1 and claim 2 for the detection of porcine pseudorabies virus in porcine blood and porcine tissue.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911358886.6A CN113025747A (en) | 2019-12-25 | 2019-12-25 | Porcine pseudorabies virus nucleic acid extraction-free fluorescent isothermal amplification detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911358886.6A CN113025747A (en) | 2019-12-25 | 2019-12-25 | Porcine pseudorabies virus nucleic acid extraction-free fluorescent isothermal amplification detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113025747A true CN113025747A (en) | 2021-06-25 |
Family
ID=76458561
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911358886.6A Pending CN113025747A (en) | 2019-12-25 | 2019-12-25 | Porcine pseudorabies virus nucleic acid extraction-free fluorescent isothermal amplification detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113025747A (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1710101A (en) * | 2005-06-23 | 2005-12-21 | 武汉大学 | Fluorescent quantitative DCR kit for detecting swine pseudo rabies virus and its use |
| CN101775443A (en) * | 2010-01-06 | 2010-07-14 | 天津市畜牧兽医研究所 | LAMP kit for detecting PRV and preparation method thereof |
| CN105112565A (en) * | 2015-09-07 | 2015-12-02 | 山东省农业科学院畜牧兽医研究所 | LAMP detection method for detecting pseudorabies virus GE gene |
| CN106755591A (en) * | 2017-02-06 | 2017-05-31 | 广东省农业科学院动物卫生研究所 | Detect intersection isothermal duplication primer sets, kit and the application of Pseudorabies virus street strain |
| CN107034316A (en) * | 2017-06-19 | 2017-08-11 | 北京博奥晶典生物技术有限公司 | The system and its special LAMP primer of 6 boars virus are detected simultaneously |
| CN107502682A (en) * | 2017-10-17 | 2017-12-22 | 河南牧业经济学院 | A kind of preparation and its application process for being used to detect the genetic chip of the pseudo- mad sick dog of pig |
| CN109207438A (en) * | 2018-08-20 | 2019-01-15 | 北京康谷生物科技有限公司 | Porcine pseudorabies virus velogen strain and its preparing the application in inactivated vaccine |
| CN109457054A (en) * | 2018-12-29 | 2019-03-12 | 博奥生物集团有限公司 | LAMP detection primer and its application, the detection reagent and method of specific detection porcine pseudorabies virus |
| CN110229932A (en) * | 2019-06-10 | 2019-09-13 | 北京森康生物技术开发有限公司 | African swine fever virus nucleic acid is hands-free to take fluorescence isothermal amplification detection kit |
-
2019
- 2019-12-25 CN CN201911358886.6A patent/CN113025747A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1710101A (en) * | 2005-06-23 | 2005-12-21 | 武汉大学 | Fluorescent quantitative DCR kit for detecting swine pseudo rabies virus and its use |
| CN101775443A (en) * | 2010-01-06 | 2010-07-14 | 天津市畜牧兽医研究所 | LAMP kit for detecting PRV and preparation method thereof |
| CN105112565A (en) * | 2015-09-07 | 2015-12-02 | 山东省农业科学院畜牧兽医研究所 | LAMP detection method for detecting pseudorabies virus GE gene |
| CN106755591A (en) * | 2017-02-06 | 2017-05-31 | 广东省农业科学院动物卫生研究所 | Detect intersection isothermal duplication primer sets, kit and the application of Pseudorabies virus street strain |
| CN107034316A (en) * | 2017-06-19 | 2017-08-11 | 北京博奥晶典生物技术有限公司 | The system and its special LAMP primer of 6 boars virus are detected simultaneously |
| CN107502682A (en) * | 2017-10-17 | 2017-12-22 | 河南牧业经济学院 | A kind of preparation and its application process for being used to detect the genetic chip of the pseudo- mad sick dog of pig |
| CN109207438A (en) * | 2018-08-20 | 2019-01-15 | 北京康谷生物科技有限公司 | Porcine pseudorabies virus velogen strain and its preparing the application in inactivated vaccine |
| CN109457054A (en) * | 2018-12-29 | 2019-03-12 | 博奥生物集团有限公司 | LAMP detection primer and its application, the detection reagent and method of specific detection porcine pseudorabies virus |
| CN110229932A (en) * | 2019-06-10 | 2019-09-13 | 北京森康生物技术开发有限公司 | African swine fever virus nucleic acid is hands-free to take fluorescence isothermal amplification detection kit |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110229932B (en) | African swine fever virus nucleic acid extraction-free fluorescent isothermal amplification detection kit | |
| CN110760620A (en) | Classical swine fever virus and African classical swine fever virus dual-fluorescence PCR detection reagent, kit and detection method | |
| CN103305638B (en) | Dual real-time fluorescence PCR (Polymerase Chain Reaction) detection primer pair, probes, kit and detection method for type 1 and type 2 porcine circovirus | |
| CN111172321B (en) | Fluorescent PCR detection kit for identifying African swine fever infection and immunity | |
| CN108504778B (en) | Kit that is a kind of while detecting porcine circovirus 2 type and porcine pseudorabies virus and application | |
| CN111286559B (en) | Primer, probe and kit for detecting African swine fever virus | |
| CN105400906A (en) | Triple PCR primer set capable of simultaneously detecting porcine circovirus II, porcine pseudorabies virus and porcine parvovirus and application thereof | |
| CN112739833A (en) | Primer pair, probe and kit for detecting SARS-CoV-2 by utilizing nested RPA technology and application thereof | |
| CN109593883B (en) | Porcine circovirus multiplex real-time fluorescent PCR detection primer pair, probe and prepared kit | |
| CN113186312B (en) | Molecular marker for distinguishing Brucella A19 vaccine strain and wild strain | |
| CN110699485A (en) | RPA primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus | |
| CN110607398B (en) | RT-LAMP kit for fluorescent visual rapid detection of porcine epidemic diarrhea virus | |
| CN116814857A (en) | Cat parvovirus and kit thereof and fluorescent recombinase polymerase amplification method | |
| CN112063763A (en) | Loop-mediated isothermal amplification primer group for detecting porcine circovirus type 4, kit and application | |
| CN113025747A (en) | Porcine pseudorabies virus nucleic acid extraction-free fluorescent isothermal amplification detection kit | |
| WO2001072964A2 (en) | Detection of viable agents | |
| CN112522446A (en) | Detection primer pair and kit for wild strain of porcine pseudorabies virus | |
| CN113122656A (en) | Foot-and-mouth disease virus nucleic acid extraction-free fluorescent isothermal amplification detection kit | |
| CN112725525A (en) | Swine fever virus nucleic acid extraction-free fluorescent isothermal amplification detection kit | |
| CN111500774A (en) | Epidemic hemorrhagic disease virus and serotype identification RT-PCR kit | |
| CN112063757A (en) | Primer and kit for detecting African swine fever virus and application of primer and kit | |
| CN113549709A (en) | Primer pair, probe and kit for detecting SARS-CoV-2 by utilizing nested RPA technology and application thereof | |
| CN113846182B (en) | Kit for rapidly and visually detecting PCV3 and detection method thereof | |
| CN110423846A (en) | A kind of primer combination, detection method and the kit of the LAMP for the parting detection that can distinguish porcine circovirus 2 type and 3 types | |
| US20080176245A1 (en) | Detection of viable agents |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210625 |
|
| WD01 | Invention patent application deemed withdrawn after publication |