CN102212622B - Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3) - Google Patents
Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3) Download PDFInfo
- Publication number
- CN102212622B CN102212622B CN 201110122089 CN201110122089A CN102212622B CN 102212622 B CN102212622 B CN 102212622B CN 201110122089 CN201110122089 CN 201110122089 CN 201110122089 A CN201110122089 A CN 201110122089A CN 102212622 B CN102212622 B CN 102212622B
- Authority
- CN
- China
- Prior art keywords
- pcr
- parainfluenza virus
- bovine parainfluenza
- reverse transcription
- reagent
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a reverse transcription-polymerase chain reaction (RT-PCR) detection method and a kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3), and belongs to the field of biomedical detection. The invention adopts the technical scheme that the method comprises acquisition of a sample, extraction of virus RNA, design optimization of a specific primer, RT-PCR amplification, agarose gel electrophoresis and result judgment; the specific primer is designed for a specific fragment of BPIV-3 nucleoprotein (NP) genes, and the upstream and downstream sequences of the specific primer are respectively P1: 5'-GGATGTTTGGGAGTGATCTTGAGTA-3' and P2: 5'-TGTGTTGAAAAATGAAGCAAGACCT-3'; the quick RT-PCR diagnosing kit for detecting the BPIV-3 comprises a sample virus RNA extracting reagent, a reverse transcription reagent, a PCR reagent, a positive reference substance and a negative reference substance; and by verifying, the method is sensitive and specific and has application prospect.
Description
Technical field
The invention belongs to biomedical detection field, relate to the microorganism molecular biology for detection.Be specifically related to a kind of RT-PCR detection method for quick diagnosis bovine parainfluenza virus 3 types.More relate to the application of the RT-PCR test kit of quick diagnosis bovine parainfluenza virus 3 types.
Background technology
(Bovine parainfluenza virus 3, BPIV-3) infection causes bovine parainfluenza, can be divided into the calf type and become the ox type by bovine parainfluenza virus 3 types.The calf type claims again calf region pneumonia, is a kind of contagious disease of invasion and attack calf in 2 thoughtful age several months, take heating, expiratory dyspnea, stream slurries, mucus or purulent rhinorrhea and cough as feature.Be born in the ox after the transportation because this disease is multiple, this virus claims again transporting hot virus.Bovine parainfluenza virus 3 types belong to paramyxovirus section paramyxovirus and belong to the member, and the RNA viruses of cyst membrane is arranged for the negative thigh of strand.
Since nineteen fifty-nine Reisinger etc. since the Niu Tizhong of the U.S. is separated to BPIV-3, France, USSR (Union of Soviet Socialist Republics), Japan, Denmark, Canada, Australia, Panama and the state such as Italian also isolate this virus in succession.Because the respiratory symptom that multiple cause of disease causes is similar to this disease, this sick Accurate Diagnosis method seems extremely important.The diagnosis research of relevant BPIV-3 report is few in the world, and relating to main method has:
(1) serodiagnosis comprises complement fixation test, neutralization experiment, aggegation experiment, immunodiffusion(ID), precipitation experiment, immuno-electrophoresis, immunofluorescence dyeing, double-antibody sandwich elisa and immunoelectronmicroscopy etc.In these methods, generally admitted and the having of widespread use: complement fixation test, indirect hemagglutination test.Agar diffusion experiment, neutralization experiment.But because long reaction time, material is many, and the preparatory period is long, detects to have obvious hysteresis quality, can only be qualitative can not be quantitative, and repeated relatively poor.
(2) biological experiment comprises experimentation on animals, egg inoculation and cell cultures.There are the problems such as not high, the consuming time length of sensitivity in this detection method, and has anticomplementary activity in some sample, and impact detects effect.The experimentation on animals cost is high, and expends a large amount of manpower and materials, and economic benefit is lower.
By contrast, molecular biology method has clear superiority, and is good, highly sensitive such as RT-PCR method specificity, not only can detect the viral nucleic acid in the viable cell, nucleic acid fragment that also can the direct-detection deactivation; Sample can derive from nose swab, throat swab, respiratory secretions etc., and can distinguish from different influenza strains, and this is that immunological method is irreplaceable.Other molecular biology methods have the fluorescence quantitative RT-PCR kit patent No. (200910062399.5), for target gene HN gene, M gene or F gene etc. are arranged.
Summary of the invention
The industry demand that at first develops in a healthy way for present cattle-raising, first purpose of the present invention is to utilize the RT-PCR technology to set up a kind of quick nucleic acid detection method for bovine parainfluenza virus 3 types, the method is sensitiveer, is easier to operation.To providing technical support for the quick diagnosis of bovine parainfluenza and epidemiology survey monitoring and prevention and control; Another object of the present invention is by system optimization reaction conditions and technique, prepares that corresponding diagnostic kit detects for clinical cause of disease and the bovine parainfluenza prevention and control provide product.
Realize concrete technical scheme of the present invention, be the RT-PCR method of described detection bovine parainfluenza virus 3 types, step comprises the collection of sample, the extraction of viral RNA, and the design optimization of Auele Specific Primer, the RT-PCR amplification, agarose gel electrophoresis and result judge; It is characterized in that: described Auele Specific Primer is that its upstream and downstream sequence is respectively for the specificity section design of bovine parainfluenza virus 3 type nucleoprotein genes (NP):
P1：5’-GGATGTTTGGGAGTGATCTTGAGTA-3’；
P2：5’-TGTGTTGAAAAATGAAGCAAGACCT-3’；
Two-step approach is adopted in described RT-PCR amplification, add the random primer 5 ' that length is 9bp-NNNNNNNNN-3 ' during reverse transcription, the specificity upstream and downstream primer P1 and the P2 that in reaction tubes, add above-mentioned bovine parainfluenza virus 3 types during the PCR reaction, RT-PCR reverse transcription reaction system is 20 μ L, comprise: 8 μ L RNA, 3 μ L DEPC process deionized water, 1 μ L random primer, behind 65 ℃ of 5min, put on ice rapidly.Add subsequently 5 * RT, 4 μ L damping fluids, 10 μ M dNTPs, 2 μ L, 0.5 μ M RNA enzyme inhibitors, 1 μ L, 25U/ μ L M-MLV 1 μ L, the continuation reaction conditions is: 30 ℃ of 30s, and 99 ℃ of 5min, 42 ℃ of 20min, 4 ℃ of 5min obtain cDNA;
The PCR system is 20 μ L, comprising: sterilization deionized water 12.5 μ L, 10 * PCR buffer2 μ L, 5U/ μ L Taq archaeal dna polymerase 0.5 μ L, dNTPs 1 μ L, bovine parainfluenza virus 3 type upstream and downstream primer P1, each 1 μ L of P2, cDNA sample 2 μ L.The PCR response procedures is: 94 ℃ of denaturation 5min; 94 ℃ of 30s, 58 ℃～62 ℃ 30s, 72 ℃ of 1min circulate 30 times; 72 ℃ are extended 5min, and 16 ℃ of 10min get PCR product 6 μ L and carry out 1% agarose electrophoresis, and positive sample and positive reference substance size all occurs and be the PCR product band of 425bp, and negative sample and negative control product are without the amplified production band.
The RT-PCR quick diagnosis reagent kit of detection bovine parainfluenza virus 3 types of the present invention comprises: the sample viral RNA extracts reagent, reverse transcription reagent, PCR reagent, positive reference substance and negative control product; It is characterized in that: described reverse transcription reagent processes water by 5 times of RT damping fluids, dNTPs, RNA enzyme inhibitors, M-MLV, DEPC and random primer forms, storage temperature-20 ℃.The reverse transcription reaction system is: 8 μ L RNA, 4 μ L, 5 * RT damping fluid, 3 μ L DEPC process deionized water, 10 μ M dNTPs (Takara), 2 μ L, 0.5 μ MRNA enzyme inhibitors (Toyoba), 1 μ L, 25U/ μ LM-MLV 1 μ L (Toyoba), 1 μ L random primer; Described PCR reagent is that storage temperature is-20 ℃ by sterilization deionized water, 10 * PCR damping fluid, Taq archaeal dna polymerase, dNTPs and cDNA template.The PCR reaction system comprises: sterilization deionized water 12.5 μ l, 10 * PCR damping fluid (Fermantas), 2 μ L, 5U/ μ LTaq archaeal dna polymerase (Fermantas) 0.5 μ l, 2.5mmol/L dNTPs (Fermantas) 1 μ L, each 1 μ L of bovine parainfluenza virus 3 type specificity upstream and downstream primer P1 and P2.
The present invention has obvious superiority compared with prior art.At first, this research of the present invention is take the nucleoprotein gene (NP) of BPIV-3 as target gene, the critical function of NP is the genome that is wrapped in virus, be combined with the RNA of virus and polymerase and form complex body (ribonucleoprotein complexes, RNPs), the other NP of Transcription and replication that is beneficial to virus has the nuclear translocation function, effect is very important for the RNA of virus enters nucleus, can enter host cell by receptor-mediated endocytosis, fusion through coating and host cell membrane, RNPs is released into cytoplasm, is transported to nucleus again.Therefore, the present invention chooses the NP gene as the RT-PCR main candidate of BPIV-3, set up should virus the RT-PCR detection method, and confirm that the method is sensitive, special, has application prospect.
The present invention with the nucleoprotein gene NP of BPIV-3 set up should virus the RT-PCR quick diagnosis reagent kit, because this test kit is sensitive, special, quick, can save time for the detection of cause of disease, reduce testing cost, reduce unnecessary financial loss, be highly suitable for laboratory quick diagnosis and the epidemiological study of bovine parainfluenza virus 3 types.
Description of drawings
Fig. 1: specific detection result's 1% agarose gel electrophoresis of the present invention detects figure
RT-PCR has detected bovine parainfluenza virus 3 types, bovine viral diarrhea virus, infectious bovine rhinotrachetis, ox syncytial virus and Pestivirus suis, Mycoplasma bovis, colon bacillus, ox pasteurellosis bacillus capsular serotype A type, Salmonella typhimurium and MDBK cell RNA among Fig. 1.Each swimming lane is followed successively by M.DL2000 ladder from left to right; 1. bovine parainfluenza virus 3 types; 2. Bovine Viral Diarrhoea-Mucosal Disease; 3. infectious bovine rhinotrachetis virus; 4. ox syncytial virus; 5. Pestivirus suis; 6. Mycoplasma bovis; 7. ox pasteurella multocida; 8. Salmonella typhimurium; 9. intestinal bacteria; 10.MDBK cell negative control; 11. blank.
Only have bovine parainfluenza virus 3 types can amplify the purpose band of expection size (425bp), show that specificity of the present invention is good.
Fig. 2: sensitivity detected result of the present invention detects figure by 1% agarose gel electrophoresis
Among Fig. 2 with bovine parainfluenza virus 3 type Reference strains from 10
9TCID
50/ 0.1mL begins 10 times and increases progressively dilution, carries out the RT-PCR amplification.Swimming lane is followed successively by M.DL2000 Ladder from left to right; 1.10
9TCID
50/ 0.1mL; 2.10
8TCID
50/ 0.1mL; 3.10
7TCID
50/ 0.1mL; 4.10
6TCID
50/ 0.1mL; 5.10
5TCID
50/ 0.1mL; 6.10
4TCID
50/ 0.1mL; 7.10
3TCID
50/ 0.1mL; 8.10
2TCID
50/ 0.1mL; 9.10
1TCID
50/ 0.1mL; 10.10
0TCID
50/ 0.1mL; 11.10
-1TCID
50/ 0.1mL; 12.10
-2TCID
50/ 0.1mL; 13.10
-3TCID
50/ 0.1mL; 14. cell RNA negative control; 15. bovine parainfluenza virus 3 type positive controls; 16. blank.
Result's demonstration, the template minimum detectable concentration of the method is 10
-3TCID
50/ 0.1mL.
Fig. 3: the electrophoresis result figure of positive control pipe and negative control pipe PCR product
Among the figure from left to right swimming lane be followed successively by: M.DL2000 ladder; 2. positive control; 3. negative control; 4. blank.
Fig. 4: different annealing temperature expanding effect figure in the test kit PCR reaction process
Among the figure from left to right swimming lane be followed successively by: M.DL2000 ladder; 1～10 annealing temperature is respectively 56.5 ℃, and 57.3 ℃, 58.1 ℃, 58.9 ℃, 59.7 ℃, 60.5 ℃, 61.3 ℃, 62.1 ℃, 62.9 ℃, 63.7 ℃.
Fig. 5: Auele Specific Primer optimal concentration figure in the test kit PCR reaction process
Among the figure from left to right swimming lane be followed successively by: M.DL2000 ladder; 1～5 primer concentration is 1 μ mol/L respectively, 0.8 μ mol/L, 0.6 μ mol/L, 0.5 μ mol/L, 0.4 μ mol/L.
Embodiment
Case study on implementation one:
The acquisition and processing of 1 sample
Gather nose swab or the throat swab sample of disease ox, above sample is added sterile saline prepare 1: 5 suspension, 4000r/min, centrifugal 10min gets supernatant for subsequent use.
The extraction of 2 sample RNA templates
(1) get step 1 supernatant 330 μ L and add the 1.5mL centrifuge tube, add 1mTRIZOL, abundant mixing, room temperature is placed 10min.
(2) add chloroform by 200 μ L chloroform/mL Trizol, room temperature is placed 15min (annotating: forbid the vortex oscillation device, in order to avoid the genomic dna fracture) behind the thermal agitation mixing; 4 ℃, the centrifugal 15min of 12,000g.Draw the upper strata water, to another centrifuge tube, (annotate: do not draw intermediate interface; If extract simultaneously DNA and protein, then keep lower floor's phenol and be stored in mutually 4 ℃ of refrigerators, if only carry RNA, then abandon lower floor's phenol phase).
(3) press 0.5mL Virahol/mLTrizol and add the Virahol mixing, room temperature is placed 5-10min.4 ℃, the centrifugal 10min of 12,000g abandons supernatant, and RNA is sunken to the pipe end.
(4) add 75% ethanol by 1mL 75% ethanol/mL Trizol, gentle vibration centrifuge tube, precipitation suspends.4 ℃, the centrifugal 5min of 8,000g abandons supernatant as far as possible.
(5) room temperature is dried or vacuum-drying 5-10min.Annotate: the RNA sample is too not dry, otherwise is difficult to dissolving.
(6) add 40 μ L DEPC and process water, then prepare reverse transcription.
3 use RT-PCR dedicated kit of the present invention to carry out following test
(1) reverse transcription reaction
Reaction system is 20 μ L, adds successively in the PCR reaction tubes, adds 3 μ L DEPC and processes water, 1 μ L reverse transcription random primer and 8 μ L RNA templates.Behind 65 ℃ of 5min, put on ice rapidly.Add successively again 4 μ L, 5 * RT damping fluid, 2 μ L dNTPs (10 μ M), 1 μ L RNA enzyme inhibitors (0.5 μ M), 1 μ L M-MLV (25U/ μ L), 30 ℃ of 30s, 99 ℃ of 5min, 42 ℃ of 20min, 4 ℃ of 5min, obtain cDNA ,-20 ℃ of preservations are with the template as further PCR.
Establish simultaneously a positive control and a negative control, reaction system and reaction conditions are the same, and different is to replace sample RNA with the positive reference substance in this test kit and negative control product respectively.
(2) PCR reaction
Reaction system is 20 μ L, in the reverse transcription PCR pipe, add successively sterilization deionized water 12.5 μ L behind the reverse transcription reaction,, 10 * PCR Dream buffer, 2 μ L, Dream Taq archaeal dna polymerase (5U/ μ L) 0.5 μ L, dNTPs (2.5mmol/L) 1 μ L, bovine parainfluenza virus 3 type specificity upstream and downstream primer P1 and each 1 μ L of P2.
The PCR response procedures is: 94 ℃ of denaturation 5min; 94 ℃ of 30s, 58 ℃～62 ℃ 30s, 72 ℃ of 1min circulate 30 times; 72 ℃ are extended 5min, 16 ℃ of 10min.
(3) agarose gel electrophoresis detects
Respectively get 6 μ LPCR products and DL2000 Marker, respectively with 2 μ L sample-loading buffer mixings after 1 application of sample in 1% sepharose, 100V electrophoresis 30～45min utilizes gel imaging system to take a picture, the analytical electrophoresis result.
(4) result judges
At first check the electrophoresis result of positive control pipe and negative control pipe PCR product, should produce a size behind the positive pipe PCR product electrophoresis is the band of 425bp, and does not have band in the negative tube.And then see the result of sample hose, if produce the band of 425bp, be judged to be bovine parainfluenza virus 3 types and infect; If without any band, then be judged to feminine gender, do not infect this virus.
Above result shows, No. 1 point sample hole contrast be positive reference substance, No. 2 corresponding negative control product in point sample hole, No. 3 be blank (deionized water).
Case study on implementation two: the reaction condition optimization of the RT-PCR test kit of bovine parainfluenza virus 3 types
The result:
Fig. 4: be followed successively by from left to right M.DL2000 ladder; 1～10 annealing temperature is respectively 56.5 ℃, and 57.3 ℃, 58.1 ℃, 58.9 ℃, 59.7 ℃, 60.5 ℃, 61.3 ℃, 62.1 ℃, 62.9 ℃, 63.7 ℃.The suitableeest annealing temperature of Image Display this test kit PCR is 58.1 ℃.
Fig. 5: be followed successively by from left to right M.DL2000 ladder; 1～5 primer concentration is 1 μ mol/L respectively, 0.8 μ mol/L, 0.6 μ mol/L, 0.5 μ mol/L, 0.4 μ mol/L.The Auele Specific Primer optimal concentration is 1 μ mol/L in this test kit of Image Display PCR reaction process.
Claims (1)
1. the RT-PCR quick detection kit of bovine parainfluenza virus 3 types comprises: sample viral RNA extraction reagent, reverse transcription reagent, PCR reagent, positive reference substance and negative control product; It is characterized in that: described reverse transcription reagent processes water by 5 times of RT damping fluids, dNTPs, RNA enzyme inhibitors, M-MLV, DEPC and random primer forms, storage temperature-20 ℃, the reverse transcription reaction system is: 8 μ L RNA, 4 μ L, 5 * RT damping fluid, 3 μ L DEPC process deionized water, 10 μ M dNTPs2 μ L, 1 μ L RNA enzyme inhibitors, 25U/ μ L M-MLV1 μ L, 0.5 μ M random primer, 1 μ L; Described PCR reagent is comprised of sterilization deionized water, 10 * PCR damping fluid, Taq archaeal dna polymerase, dNTPs, cDNA template, bovine parainfluenza virus 3 type specificity upstream and downstream primer P1 and P2, and storage temperature is-20 ℃; The PCR reaction system comprises: sterilization deionized water 12.5 μ l, 10 * PCR damping fluid, 2 μ L, 5U/ μ L Taq archaeal dna polymerase 0.5 μ l, 2.5mmol/L dNTPs 1 μ L, cDNA template 2 μ L, each 1 μ L of bovine parainfluenza virus 3 type specificity upstream and downstream primer P1 and P2, wherein bovine parainfluenza virus 3 type specificity upstream and downstream primer P1 and P2 are
P1:5’-GGATGTTTGGGAGTGATCTTGAGTA-3’
P2:5’-TGTGTTGAAAAATGAAGCAAGACCT-3’。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110122089 CN102212622B (en) | 2011-05-12 | 2011-05-12 | Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3) |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110122089 CN102212622B (en) | 2011-05-12 | 2011-05-12 | Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3) |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102212622A CN102212622A (en) | 2011-10-12 |
| CN102212622B true CN102212622B (en) | 2013-02-27 |
Family
ID=44744153
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110122089 Expired - Fee Related CN102212622B (en) | 2011-05-12 | 2011-05-12 | Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3) |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102212622B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468828A (en) * | 2013-09-16 | 2013-12-25 | 上海卓润生物科技有限公司 | PCR (Polymerase Chain Reaction) method for simultaneously detecting four bovine infectious disease RNA (Ribose Nucleic Acid) viruses by single tube |
| CN104391112A (en) * | 2014-05-27 | 2015-03-04 | 中国农业科学院特产研究所 | Expression protein detecting bovine parainfluenza virus 3 antibody and ELISA kit |
| CN106906307A (en) * | 2017-03-10 | 2017-06-30 | 中国农业科学院特产研究所 | The type of bovine parainfluenza virus 3 virus nano PCR detection kits and preparation method thereof |
| CN107988431A (en) * | 2017-12-13 | 2018-05-04 | 广西壮族自治区兽医研究所 | A kind of RT-PCR amplimers of quick detection 3 type of bovine parainfluenza virus and its application |
| CN110564858A (en) * | 2019-10-29 | 2019-12-13 | 成都益安博生物技术有限公司 | RT-PCR kit for predicting curative effect of immunodetection point regulation type medicines and prediction method thereof |
-
2011
- 2011-05-12 CN CN 201110122089 patent/CN102212622B/en not_active Expired - Fee Related
Non-Patent Citations (8)
| Title |
|---|
| BPIV-3和BVDV双重RT-PCR快速检测方法的建立;刘晓乐等;《动物医学进展》;20111130;第32卷(第11期);第1-5页 * |
| 刘晓乐等.BPIV-3和BVDV双重RT-PCR快速检测方法的建立.《动物医学进展》.2011,第32卷(第11期), |
| 刘晓乐等.牛副流感病毒3型RT-PCR检测方法的建立.《中国奶牛》.2011,(第22期), |
| 刘鹏等.牛副流感病毒3型的分离鉴定.《微生物学通报》.2009,第36卷(第9期), |
| 周玉龙等.牛副流感病毒3型的分离鉴定及感染牛抗体消长规律的研究.《中国***共患病学报》.2011,第27卷(第1期), |
| 牛副流感病毒3型RT-PCR检测方法的建立;刘晓乐等;《中国奶牛》;20111130(第22期);第1-4页 * |
| 牛副流感病毒3型的分离鉴定;刘鹏等;《微生物学通报》;20090920;第36卷(第9期);第1384-1389页 * |
| 牛副流感病毒3型的分离鉴定及感染牛抗体消长规律的研究;周玉龙等;《中国***共患病学报》;20110131;第27卷(第1期);第23-28页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102212622A (en) | 2011-10-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102212622B (en) | Reverse transcription-polymerase chain reaction (RT-PCR) detection method and kit for quickly diagnosing bovine parainfluenza virus 3 (BPIV-3) | |
| CN103275862B (en) | Fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) kit for detecting influenza A virus subtype H7N9 | |
| CN103509882B (en) | The fluorescence quantification PCR primer of Porcine epidemic diarrhea virus and probe | |
| CN103981286B (en) | Differentiate GeXP rapid detection kit and the primer sets thereof of 8 kinds of virus diseases of pigs | |
| CN102732638A (en) | Method for single-tube multiplex fluorescent polymerase chain reaction (PCR) detection of human coronavirus OC43, 229E, NL63, HKU1 and SARS, and primers, probes and kit adopted by the method | |
| CN101942525B (en) | One-tube method with multiplex detection for human Influenza A and B and new Influenza A H1N1 virus and kit | |
| CN107385111A (en) | The real-time fluorescence quantitative PCR detection primer and its kit of a kind of goose astrovirus | |
| CN103243179B (en) | Shell-type PCR (polymerase chain reaction) amplification primer of porcine epidemic diarrhea virus and application thereof | |
| CN104846125A (en) | Fluorescent RT-PCR (reverse transcription-polymerase chain reaction) primers, probe and kit for detecting MERS (Middle East Respiratory Syndrome), and detection method thereof | |
| CN103757139A (en) | Canine distemper virus and canine parvovirus duplex TaqMan-MGB fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method thereof | |
| CN106636472B (en) | Complete set of reagent and method for detecting avian influenza virus and chicken parvovirus | |
| CN107475456A (en) | PEDV quick determination methods and its kit based on isothermal reverse transcription recombinase polymeric enzymatic amplification method | |
| CN105543414A (en) | Respiratory syncytial virus A/B subtype multiplex fluorescence quantitative PCR detection primer set and probe set and reagent kit and preparation method thereof | |
| CN105755124A (en) | Method for detecting salmonella with fluorescence method on basis of enzymatic remediation isothermal cycle amplification | |
| CN106011315A (en) | RT-PCR discriminating diagnosis primers for type 1 and new serotype duck hepatitis virus | |
| CN104651534A (en) | Porcine circovivus loop-mediated isothermal amplification kit and application thereof | |
| CN104498629A (en) | Duplex real-time fluorescence quantitative PCR (polymerase chain reaction) detection kit for H3N2 subtype avian influenza virus (AIV) | |
| CN105154588A (en) | Primer pair for detecting caprine parainfluenza virus type 3 (CPIV3) and peste des petits ruminants virus (PPRV) | |
| CN104630387B (en) | The LAMP detection kit of Porcine epidemic diarrhea virus | |
| CN104263857B (en) | A kind of nano PCR kit of quick detection mink enteritis virus and its application | |
| CN110643741A (en) | Palimam serogroup virus group specificity and serotype specificity RT-PCR detection primer and kit | |
| CN110438260A (en) | A kind of African swine fever virus nucleic acid test strips detection kit | |
| CN105603120A (en) | GeXP multiple rapid detection primers for detection of bluetongue virus, bovine viral diarrhea virus and foot and mouth disease virus and detection method | |
| CN102534052B (en) | Nucleic-acid sequence-based amplification (NASBA) method for detecting swine influenza virus (SIV) | |
| CN101724712B (en) | Animal insect-borne disease multi-RT-PCR distinguishing and detecting reagent as well as preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130227 Termination date: 20130512 |