CN104195265A - PCR-HRM primer and method for rapidly distinguishing wild strain and vaccine strain of canine parvovirus - Google Patents
PCR-HRM primer and method for rapidly distinguishing wild strain and vaccine strain of canine parvovirus Download PDFInfo
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- CN104195265A CN104195265A CN201410419410.XA CN201410419410A CN104195265A CN 104195265 A CN104195265 A CN 104195265A CN 201410419410 A CN201410419410 A CN 201410419410A CN 104195265 A CN104195265 A CN 104195265A
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- 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
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Abstract
The invention discloses a PCR-HRM primer and a method for rapidly distinguishing a wild strain and a vaccine strain of canine parvovirus. The method comprises the following steps of: extracting a virus DNA from a sample as a template, carrying out PCR amplification by utilizing designed two pairs of specific primers and fluorescent saturated dye, carrying out HRM analysis on the detected sample respectively with a wild strain standard sample and a vaccine strain standard sample as contrast, and determining the type of the canine parvovirus. With the adoption of the method, the operation is simple, and only is the fluorescent saturated dye added before the PCR; the detection speed is high, the flux is high, the whole operation process only needs 3 hours, and cell culture of viruses is not needed, and thus the time needed by distinguishing detection is greatly shortened; the cost is low, a specific probe is not needed, and the fluorescent saturated dye is cheap and easily obtained; as the accuracy is high, the specificity and the repeatability are good, and analysis can be accurately and rapidly carried out with high flux, the method is beneficial to popularizing and applying in clinical practices.
Description
Technical field
The present invention relates to the discrimination method of virus vaccines Du Yu street strain, be specifically related to a kind of PCR-HRM primer and the method for quick differentiation canine parvovirus street strain and vaccine strain.
Background technology
Canine parvovirus (Canine parvovirus, CPV) is the little DNA virus of strand, is the main pathogen that causes dog acute hemorrhagic gastro-enteritis and pup acute myocarditis.1977, Eugster and Nairn were separated to canine parvovirus first from the sick dog ight soil of trouble hemorrhagic enteritis, and called after CPV-2.From the people such as Eugster first separated obtain CPV-2 since, passing in time of CPV antigenicity and constantly changing, constantly have new CPV genotype and antigenic type to occur, and worldwide wide-scale distribution is popular.At present known CPV genotype comprises CPV-2a, CPV-2b and CPV-2c, and archetype CPV-2 genotype is replaced by emerging antigenic variants, and archetype CPV-2 strain only with the form of attenuated live vaccines in the middle of the prevention of canine parvovirus.At present, domestic for preventing the vaccine strain of canine parvovirus to mainly contain: the CPVint attenuated live vaccines strain of the CPVpf attenuated live vaccines strain of Pfizer Dong Bao company and Dutch INTERVU CO, above two low virulent strains that vaccine strain is all original CPV-2 strains.
Development along with domestic pet industry, in at present clinical, with attenuated live vaccines, prevent canine parvovirus in a large number, although vaccination can prevent the popular of canine parvovirus, there is the phenomenon that reports that the dog sickness rate of inoculation canine parvovirus attenuated live vaccines strain increases.In addition, vaccination has also brought certain difficulty to the promptly and accurately diagnosis of disease.For example, after the dog that suffers from canine distemper only inoculates attenuated live vaccines, owing to canine parvovirus vaccine strain being detected in the middle of ight soil, mistaken diagnosis is the intestinal tract disease that canine parvovirus causes, thereby affects its disease therapeuticing effect.Therefore, distinguish fast vaccine strain and street strain quick and precisely the diagnosing and thering is certain clinical meaning the safety evaluation of vaccine strain and disease of canine parvovirus.And distinguish at present canine parvovirus vaccine strain, be fluorescence quantitative PCR method (MGB probe) with the main method of wild poison, although this method can accurately be distinguished canine parvovirus vaccine Zhu Yu street strain, but need simultaneously three pairs or more probe expensive, limit its practical application aborning.Therefore, be badly in need of at present the method for the differentiation canine parvovirus vaccine Zhu Yu street strain that a kind of operation is relatively simple and easy, detected result is reliable and testing cost is cheap.
Summary of the invention
In order to solve the problem of above-mentioned existence, the present invention has set up PCR-HRM primer and the method for a kind of quick differentiation canine parvovirus vaccine Zhu Yu street strain, the method operation is simple and easy, quick, detected result is reliable and testing cost is cheap, is conducive to apply in clinical practice.
The object of the present invention is to provide the PCR-HRM primer of a kind of quick differentiation canine parvovirus vaccine Zhu Yu street strain.
Another object of the present invention is to provide the PCR-HRM method of a kind of quick differentiation canine parvovirus vaccine Zhu Yu street strain.
The technical solution used in the present invention is:
A PCR-HRM primer for quick differentiation canine parvovirus street strain and vaccine strain, its nucleotide sequence is as follows:
Primer P1:TGGAAATCACAGCAAACTC(SEQ ID NO:1) or its Nucleotide complementary sequence,
Primer P2:AGTCTTGGTTTTAAGTCAGTATC(SEQ ID NO:2) or its Nucleotide complementary,
Primer P3:TGAAAATTATAGAAGAGTGGT(SEQ ID NO:3) or its Nucleotide complementary sequence,
Primer P4:CGTTAACTGCAGTTTTATCCA(SEQ ID NO:4) or its Nucleotide complementary sequence.
A PCR-HRM method for quick differentiation canine parvovirus street strain and vaccine strain, comprises the following steps:
1) from sample, extract viral DNA;
2) take DNA as template, with primer pair P1 claimed in claim 1 and P2, carry out pre-amplified reaction and obtain pre-amplified production;
3) using pre-amplified production as DNA profiling, with primer pair P3 claimed in claim 1, P4 and fluorescence saturable dye, carry out PCR-HRM amplified reaction and obtain amplified production;
4) amplified production is carried out to HRM analysis, determine Virus Type.
Further, above-mentioned steps 2) in the pre-amplification reaction system of PCR be:
Premix Ex-Taq 5.0μl
Primer P1 0.5 μ l
Primer P2 0.5 μ l
Template 1.0 μ l
ddH
2O 3.0μl。
Further, above-mentioned steps 2) in pre-amplification response procedures be: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s; Circulate 35 times; 72 ℃ are extended 8min eventually.
Further, above-mentioned steps 3) in PCR-HRM amplification reaction system be:
Premix Ex-Taq 5.0μl
Primer P3 0.5 μ l
Primer P4 0.5 μ l
Template 1.0 μ l
Fluorescence saturable dye 1 μ l
ddH
2O 2.0μl。
Further, fluorescence saturable dye described above is Eva green dyestuff.
Further, above-mentioned steps 3) in PCR-HRM amplified reaction program be: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s; Circulate 35 times; 68 ℃ to the 80 ℃ speed with 0.3 ℃/step are carried out melting curve analysis.
Further, above-mentioned steps 4) the concrete analysis process that described in, HRM analyzes is: 1) take vaccine strain CPVpf standard model during as contrast, if its gene type the value of the confidence (GCP) is greater than 95%, be judged to be vaccine strain CPVpf;
2) take vaccine strain CPVint standard model as when contrast, if its gene type the value of the confidence (GCP) is greater than 95%, be judged to be vaccine strain CPVint;
3), when YiCPV street strain standard model is for contrast, if being greater than 95%, its gene type the value of the confidence (GCP) is judged to be CPV street strain.
The invention has the beneficial effects as follows:
1) the present invention has set up PCR-HRM primer and the method for a kind of quick differentiation canine parvovirus vaccine Zhu Yu street strain first, simple to operate: only to need to add fluorescence saturable dye before PCR reaction; Fast and the high-throughput of detection speed: all operations were process only needs 3 hours, does not need viral cell cultures, has greatly shortened somatotype required time; Expense is low, does not need specific probe, and fluorescence saturable dye is cheap and easy to get; Accuracy is high, specificity good, reproducible, can be accurately, fast, analyze high-throughput, be conducive to apply in clinical practice.
2) PCR-HRM primer of the present invention, all has amplification well to canine parvovirus vaccine strain (comprising CPVpf and CPVint vaccine strain) with street strain, contributes to improve the efficiency of PCR, reduces the time that virus is differentiated somatotype.
3) PCR-HRM primer specificity of the present invention is good, except can be with canine parvovirus vaccine Zhu Yu street strain be combined, be not combined with other common dog viroid DNA, specific amplification canine parvovirus DNA, is conducive to improve the exactness of the present invention to gene type assay.
Accompanying drawing explanation
Fig. 1 is the standard model HRM of canine parvovirus vaccine Zhu Yu street strain stdn melting curve;
Fig. 2 is the standard model HRM of canine parvovirus vaccine Zhu Yu street strain peak type melting curve;
Fig. 3 is the clinical sample HRM of canine parvovirus vaccine Zhu Yu street strain stdn melting curve;
Fig. 4 is the clinical sample HRM of canine parvovirus vaccine Zhu Yu street strain peak type melting curve;
Fig. 5 is the canine parvovirus vaccine Zhu Yu PCR-HRM of street strain primer specificity gel electrophoresis figure.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited to this.
embodiment 1
(1) primer
1) the pre-amplimer of PCR
The primer pair P1 and the P2 that according to canine parvovirus VP2 protein gene order, design amplification canine parvovirus VP2 protein portion gene, its base sequence is as follows.
P1 :5'-TGGAAATCACAGCAAACTC -3' （SEQ ID NO：1），
P2:5'-AGTCTTGGTTTTAAGTCAGTATC-3'（SEQ ID NO：2）。
Wherein, primer P1 is on canine parvovirus VP2 protein gene 2962nd～2980 sites, and primer P2 is (accession number of this gene in Genbank is M38245) on canine parvovirus VP2 protein gene 4209th～4231 sites.
2) PCR-HRM primer:
The present invention is after a large amount of primers to designed screen, find to use combining of primer pair P3, P4 and primer pair P1, P2 the effect to PCR-HRM method differentiation canine parvovirus vaccine Zhu Yu street strain best, the base sequence of primer pair P3, P4 is as follows.
P3：5'-TGAAAATTATAGAAGAGTGGT-3'（SEQ ID NO：3），
P4：5'-CGTTAACTGCAGTTTTATCCA-3'（SEQ ID NO：4）。
Wherein, primer P3 is on canine parvovirus VP2 protein gene 3014th～3034 sites, and primer P4 is (accession number of this gene in Genbank is M38245) on canine parvovirus VP2 protein gene 3045th～3066 sites.
(2) preparation of standard model and PCR-HRM thereof analyze
1) extraction of canine parvovirus DNA:
With test kit MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0, extract the canine parvovirus DNA in samples.Samples can be that whole blood, ight soil, facies rectalis swab etc. are easy to obtain and the sample without grievous injury to animal body.
2) preparation of standard model:
In order to verify the inventive method feasibility and reliability, build standard positive sample simultaneously, for detecting, clinical sample afterwards provide HRM positive control, the present invention need preferentially prepare canine parvovirus vaccine Zhu He street strain positive criteria sample.The preparation process of standard model is as follows:
The DNA that learning from else's experience respectively order-checking is defined as CPVpf vaccine strain, CPVint vaccine strain and canine parvovirus street strain, as template, take respectively P1 and P2 to increase in advance as primer carries out PCR, and its pre-amplification reaction system is:
Premix Ex-Taq 5.0μl
Primer P1 0.5 μ l
Primer P2 0.5 μ l
Template 1.0 μ l
ddH
2O 3.0μl。
Pre-amplification response procedures is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s; Circulate 35 times; 72 ℃ are extended 8min eventually.
Pass through aforesaid method, obtain respectively the pre-amplified production of PCR of canine parvovirus vaccine strain CPVpf, vaccine strain CPVint and street strain, respectively by 100 times of the pre-amplified production dilutions of PCR, can obtain respectively the standard model of vaccine strain CPVpf, vaccine strain CPVint and street strain, as the positive control sample of follow-up study.
3) the PCR-HRM operation steps of positive criteria sample
The three kinds of positive criteria samples of above-mentioned acquisition of take are respectively DNA profiling, carry out respectively PCR-HRM amplified reaction and analysis;
PCR-HRM reaction system: 10 μ l
| ddH 2O | 2.0μl |
| Premix Ex-Taq | 5.0μl |
| Primer P3 | 0.5μl |
| Primer P4 | 0.5μl |
| DNA profiling | 1.0μl |
| Eva green dyestuff | 1.0μl |
| Total | 10μl |
PCR-HRM amplified reaction program:
95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s; Circulate 35 times; 68 ℃ to the 80 ℃ melting speed with 0.3 ℃/s are carried out melting curve analysis.
4) positive criteria sample P CR-HRM interpretation of result
Pcr amplification product is analyzed with Rotor-Gene Q analyser.The standard model HRM(of canine parvovirus vaccine Zhu Yu street strain high resolving power melting curve, High Resolution Melting curve) result is as shown in Figure 1 and Figure 2.
Fig. 1 is stdn melting curve figure, stdn melting curve figure demonstration, and CPVpf, 3 kinds of standard model melting curves of CPVint vaccine strain and street strain are separated from each other, and show that designed primer is suitable for HRM and analyzes.In order to eliminate the impact of artificial subjective factor on result of determination, utilize Rotor-Gene
tMq software version 2.0.2. software gene type is put letter parameter (GCP) its result is analyzed to (GCP value is more than or equal to 95% and is judged to be homologous genes type).Gene type result shows, CPVpf, CPVint vaccine strain and street strain's standard model (each standard model is 3 repetitions) gene type the value of the confidence (GCP) is respectively 99.95 ± 0.04%, 99.81 ± 0.15% and 99.10 ± 0.78%.
Fig. 2 is peak type melting curve figure, peak type melting curve figure demonstration, and three kinds of standard model melting curve shapes are similar, all have 1 to melt peak, show to cause that the major cause of GCP difference is the difference of three kinds of standard model melting temperature (Tm)s (Tm).Wherein, vaccine strain CPVpf melting temperature (Tm) is lower is 67.65 ± 0 ℃, and vaccine strain CPVint melting temperature (Tm) is higher is 68.59 ± 0.03 ℃, and it is 68.18 ± 0.02 ℃ between the two that street strain's melting temperature (Tm) is positioned at.
(3) clinical sample PCR-HRM analyzes
1) from sample, extract viral DNA: method is with DNA extraction method in above-mentioned (2);
2) take the viral DNA extracting is template, carries out PCR and increases in advance, and pre-amplification reaction system is:
Premix Ex-Taq 5.0μl
Primer P1 0.5 μ l
Primer P2 0.5 μ l
Template 1.0 μ l
ddH
2O 3.0μl。
Pre-amplification response procedures is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s; Circulate 35 times; 72 ℃ are extended 8min eventually.
3) the PCR product that increases in advance dilutes 100 afterwards as DNA profiling, carries out PCR-HRM amplified reaction:
Amplification reaction system is:
Premix Ex-Taq 5.0μl
Primer P3 0.5 μ l
Primer P4 0.5 μ l
Template 1.0 μ l
Eva green dyestuff 1.0 μ l
ddH
2O 2.0μl。
PCR-HRM amplified reaction program is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s; Circulate 35 times; 72 ℃ are extended 8min eventually; 68 ℃ to the 80 ℃ speed with 0.3 ℃/step are carried out melting curve analysis.
4) amplified production is carried out to HRM analysis, determine viral genotype.
The present invention has detected 40 parts of clinical samples, and the result of PCR-HRM as shown in Figure 3, Figure 4.
From the stdn melting curve figure shown in Fig. 3, can find out, when difference YiCPV street strain, CPVpf vaccine strain and CPVint vaccine strain standard model are in contrast time (the GCP parameter value of establishing is judged to be homologous genes type for its value is more than or equal to 90%), in 40 parts of clinical samples, 38 duplicate samples somatotypes are street strain, and its GCP value is 98.11 ± 1.06%; 2 parts of clinical sample somatotypes are CPVint vaccine strain, and its GCP value is 99.61 ± 0.22%; In 40 parts of clinical samples without CPVpf vaccine strain.
Fig. 4 is 40 parts of clinical sample peak type melting curve figure, CPV street strain, and CPVint vaccine strain and CPVpf vaccine strain Tm value are respectively 68.16 ± 0.04 ℃, 68.60 ± 0.03 ℃ and 67.65 ± 0 ℃.
(4) specificity experiment
The PCR-HRM method of below the present invention being set up is made specific detection.
Extract respectively other common dog viral DNAs, as extract canine distemper (Canine distemper virus, CDV), hepatitis infectiosa canis virus-2(Canine adenovirus type 2, CAV-2) and canine parainfluenza virus (Canine parainfluenza virus, CPIV) DNA is respectively as pcr template, with the PCR method in above-mentioned (3), carry out respectively PCR reaction, PCR product is carried out to gel electrophoresis analysis, and be analyzed with CPVpf, CPVint vaccine strain and street strain's positive PCR product, electrophoresis result is as shown in Figure 5.
M in Fig. 5 is Marker(DL1000 DNA marker), swimming lane 1-7 is respectively 1:CPVpf vaccine strain, 2:CPVint vaccine strain, 3:CPV street strain, 4:CDV, 5:CAV-2,6:CPIV, 7: negative control, gel electrophoresis result shows, canine parvovirus vaccine Zhu Yu street strain positive has object band in 50bp left and right, and electrophoretic band does not all appear in other sample, shows that the high HRM of being suitable for of designed primer specificity analyzes.
Animal health institute of <110> Guangdong Academy of Agricultural Sciences
<120> PCR-HRM primer and method of distinguishing fast canine parvovirus street strain and vaccine strain
<130>
<160> 4
<170> PatentIn version 3.5
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<211> 19
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The artificial primer of <213>
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The artificial primer of <213>
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agtcttggtt ttaagtcagt atc 23
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The artificial primer of <213>
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tgaaaattat agaagagtgg t 21
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The artificial primer of <213>
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cgttaactgc agttttatcc a 21
Claims (8)
1. distinguish fast a PCR-HRM primer for canine parvovirus street strain and vaccine strain, its nucleotide sequence is as follows:
Primer P1:TGGAAATCACAGCAAACTC(SEQ ID NO:1) or its Nucleotide complementary sequence,
Primer P2:AGTCTTGGTTTTAAGTCAGTATC(SEQ ID NO:2) or its Nucleotide complementary,
Primer P3:TGAAAATTATAGAAGAGTGGT(SEQ ID NO:3) or its Nucleotide complementary sequence,
Primer P4:CGTTAACTGCAGTTTTATCCA(SEQ ID NO:4) or its Nucleotide complementary sequence.
2. distinguish fast a PCR-HRM method for canine parvovirus street strain and vaccine strain, it is characterized in that: comprise the following steps:
1) from sample, extract viral DNA;
2) take DNA as template, with primer pair P1 claimed in claim 1 and P2, carry out pre-amplified reaction and obtain pre-amplified production;
3) using pre-amplified production as DNA profiling, with primer pair P3 claimed in claim 1, P4 and fluorescence saturable dye, carry out PCR-HRM amplified reaction and obtain amplified production;
4) amplified production is carried out to HRM analysis, determine Virus Type.
3. method according to claim 2, is characterized in that: step 2) in the pre-amplification reaction system of PCR be:
Premix Ex-Taq 5.0μl
Primer P1 0.5 μ l
Primer P2 0.5 μ l
Template 1.0 μ l
ddH
2O 3.0μl。
4. method according to claim 2, is characterized in that: step 2) in pre-amplification response procedures be: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 55 ℃ of annealing 30s, 72 ℃ are extended 30s; Circulate 35 times; 72 ℃ are extended 8min eventually.
5. method according to claim 2, is characterized in that: the PCR-HRM amplification reaction system in step 3) is:
Premix Ex-Taq 5.0μl
Primer P3 0.5 μ l
Primer P4 0.5 μ l
Template 1.0 μ l
Fluorescence saturable dye 1 μ l
ddH
2O 2.0μl。
6. according to the method described in claim 2 or 5, it is characterized in that: described fluorescence saturable dye is Eva green dyestuff.
7. method according to claim 2, is characterized in that: the PCR-HRM amplified reaction program in step 3) is: 95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 30s; Circulate 35 times; 68 ℃ to the 80 ℃ speed with 0.3 ℃/step are carried out melting curve analysis.
8. method according to claim 2, it is characterized in that: the concrete analysis process that HRM described in step 4) analyzes is: 1) take vaccine strain CPVpf standard model during as contrast, if its gene type the value of the confidence (GCP) is greater than 95%, be judged to be vaccine strain CPVpf;
2) take vaccine strain CPVint standard model as when contrast, if its gene type the value of the confidence (GCP) is greater than 95%, be judged to be vaccine strain CPVint;
3), when YiCPV street strain standard model is for contrast, if being greater than 95%, its gene type the value of the confidence (GCP) is judged to be CPV street strain.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410419410.XA CN104195265B (en) | 2014-08-22 | 2014-08-22 | The PCR-HRM primer of a kind of quick differentiation canine parvovirus street strain and vaccine strain and method |
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| CN201410419410.XA CN104195265B (en) | 2014-08-22 | 2014-08-22 | The PCR-HRM primer of a kind of quick differentiation canine parvovirus street strain and vaccine strain and method |
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Cited By (4)
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| CN105200162A (en) * | 2015-08-06 | 2015-12-30 | 广东省农业科学院动物卫生研究所 | HRM detection method for rapidly distinguishing HP-PRRS live vaccine JXA1-R strains and wild strains and primer of HRM detection method |
| CN105838826A (en) * | 2016-05-05 | 2016-08-10 | 广东省农业科学院动物卫生研究所 | Two-color fluorescence PCR primer, probe and method for quickly distinguishing canine parvovirus vaccine strain from wild strain |
| CN107937615A (en) * | 2017-12-20 | 2018-04-20 | 广东省农业科学院动物卫生研究所 | For distinguishing the primer and probe of Latex agglutination test street strain and vaccine strain |
| CN108165666A (en) * | 2016-12-07 | 2018-06-15 | 克雷多生物医学私人有限公司 | Detection CPV2a, CPV2b and CPV2c and the method for differentiating wild type and vaccine type |
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Cited By (8)
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| CN105200162A (en) * | 2015-08-06 | 2015-12-30 | 广东省农业科学院动物卫生研究所 | HRM detection method for rapidly distinguishing HP-PRRS live vaccine JXA1-R strains and wild strains and primer of HRM detection method |
| CN105200162B (en) * | 2015-08-06 | 2018-05-25 | 广东省农业科学院动物卫生研究所 | A kind of JXA1-R plants of quick differentiation HP-PRRS live vaccines and the HRM detection methods and its primer of street strain |
| CN105838826A (en) * | 2016-05-05 | 2016-08-10 | 广东省农业科学院动物卫生研究所 | Two-color fluorescence PCR primer, probe and method for quickly distinguishing canine parvovirus vaccine strain from wild strain |
| CN105838826B (en) * | 2016-05-05 | 2019-12-24 | 广东省农业科学院动物卫生研究所 | Double-color fluorescent PCR primer, probe and method for rapidly distinguishing canine parvovirus vaccine strain and wild strain |
| CN108165666A (en) * | 2016-12-07 | 2018-06-15 | 克雷多生物医学私人有限公司 | Detection CPV2a, CPV2b and CPV2c and the method for differentiating wild type and vaccine type |
| CN108165666B (en) * | 2016-12-07 | 2021-05-28 | 克雷多生物医学私人有限公司 | Method for detecting CPV2a, CPV2b and CPV2c and identifying wild type and vaccine type |
| CN107937615A (en) * | 2017-12-20 | 2018-04-20 | 广东省农业科学院动物卫生研究所 | For distinguishing the primer and probe of Latex agglutination test street strain and vaccine strain |
| CN107937615B (en) * | 2017-12-20 | 2021-06-25 | 广东省农业科学院动物卫生研究所 | Primers and probes for distinguishing wild strains and vaccine strains of swine Japanese encephalitis virus |
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