CN104195265B - The PCR-HRM primer of a kind of quick differentiation canine parvovirus street strain and vaccine strain and method - Google Patents
The PCR-HRM primer of a kind of quick differentiation canine parvovirus street strain and vaccine strain and method Download PDFInfo
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Abstract
The invention discloses PCR-HRM primer and the method for a kind of quick differentiation canine parvovirus street strain and vaccine strain.The method for first extracting viral DNA as template from sample, 2 pairs of Auele Specific Primers designed by utilization and fluorescence saturable dye carry out pcr amplification, and in contrast HRM analysis is carried out to detection sample with street strain and vaccine strain standard substance respectively, determine the type of canine parvovirus.The present invention is simple to operate, adds fluorescence saturable dye before only needing PCR to react; Fast and the high-throughput of detection speed, all operations process only needs 3 hours, does not need the cell cultures of virus, greatly shortens to distinguish to detect 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 accurately, fast, analyze, be conducive to applying in clinical practice high-throughput.
Description
Technical field
The present invention relates to virus vaccines poison and the discrimination method of street strain, be specifically related to PCR-HRM primer and the method for a kind of quick differentiation canine parvovirus street strain and vaccine strain.
Background technology
Canine parvovirus (Canineparvovirus, CPV) is strand small DNA virus, is the main pathogen causing dog acute hemorrhagic gastro-enteritis and pup acute myocarditis.1977, Eugster and Nairn was separated to canine parvovirus first from the sick dog ight soil of suffering from hemorrhagic enteritis, and called after CPV-2.Be separated first from people such as Eugster and obtained since CPV-2, CPV antigenicity passing in time and constantly changing, constantly have new CPV genotype and antigenic type to occur, and worldwide wide-scale distribution is popular.CPV genotype known at present comprises CPV-2a, CPV-2b and CPV-2c, and archetype CPV-2 genotype is replaced by emerging antigenic variants, and archetype CPV-2 strain is only used for 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 CPVpf attenuated live vaccines strain of Pfizer Dong Bao company and the CPVint attenuated live vaccines strain of Dutch INTERVU CO, above two vaccine strains are all low virulent strains of original CPV-2 strain.
Along with the development of domestic pet industry, in clinical at present, a large amount of attenuated live vaccines that uses prevents canine parvovirus, although vaccination can prevent the popular of canine parvovirus, there is the phenomenon reporting that a dog sickness rate of inoculation canine parvovirus attenuated live vaccines strain increases.In addition, vaccination also brings certain difficulty to the promptly and accurately diagnosis of disease.Such as, after the dog suffering 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, thus affects its disease therapeuticing effect.Therefore, vaccine strain and the street strain quick and precisely diagnosis to the safety evaluation of vaccine strain and disease distinguishing canine parvovirus fast has certain clinical meaning.And the main method distinguishing canine parvovirus vaccine strain and open country poison is at present fluorescence quantitative PCR method (MGB probe), although this method can accurately distinguish canine parvovirus vaccine strain and street strain, but need simultaneously three to or more probe expensive, limit its practical application aborning.Therefore, a kind of operation of urgent need is at present relatively simple and easy, detected result reliable and the method for the differentiation canine parvovirus vaccine strain that testing cost is cheap and street strain.
Summary of the invention
In order to solve above-mentioned Problems existing, the present invention establishes PCR-HRM primer and the method for the strain of a kind of quick differentiation canine parvovirus vaccine and street strain, the method operation is simple and easy, quick, detected result reliable and testing cost is cheap, is conducive to applying in clinical practice.
The object of the present invention is to provide the PCR-HRM primer of the strain of a kind of quick differentiation canine parvovirus vaccine and street strain.
Another object of the present invention is to the PCR-HRM method that the strain of a kind of quick differentiation canine parvovirus vaccine and street strain are provided.
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(SEQIDNO:1) or its nucleotide complementary sequence,
Primer P2:AGTCTTGGTTTTAAGTCAGTATC(SEQIDNO:2) or its nucleotide complementary,
Primer P3:TGAAAATTATAGAAGAGTGGT(SEQIDNO:3) or its nucleotide complementary sequence,
Primer P4:CGTTAACTGCAGTTTTATCCA(SEQIDNO: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, viral DNA is extracted;
2) take DNA as template, carry out pre-amplified reaction with primer pair P1 and P2 according to claim 1 and obtain pre-amplified production;
3) using pre-amplified production as DNA profiling, carry out PCR-HRM amplified reaction with primer pair P3, P4 according to claim 1 and fluorescence saturable dye and obtain amplified production;
4) HRM analysis is carried out to amplified production, determine Virus Type.
Further, above-mentioned steps 2) in the pre-amplification reaction system of PCR be:
PremixEx-Taq5.0μl
Primer P10.5 μ l
Primer P20.5 μ l
Template 1.0 μ l
ddH
2O3.0μl。
Further, above-mentioned steps 2) in pre-amplification response procedures be: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s; Circulate 35 times; 72 DEG C of ends extend 8min.
Further, above-mentioned steps 3) in PCR-HRM amplification reaction system be:
PremixEx-Taq5.0μl
Primer P30.5 μ l
Primer P40.5 μ l
Template 1.0 μ l
Fluorescence saturable dye 1 μ l
ddH
2O2.0μl。
Further, fluorescence saturable dye described above is Evagreen dyestuff.
Further, above-mentioned steps 3) in PCR-HRM amplified reaction program be: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s; Circulate 35 times; 68 DEG C to 80 DEG C are carried out melting curve analysis with the speed of 0.3 DEG C/step.
Further, above-mentioned steps 4) described in the concrete analysis process analyzed of HRM be: 1) with vaccine strain CPVpf standard model for contrast time, if its gene type the value of the confidence (GCP) is greater than 95%, be judged to be vaccine strain CPVpf;
2) with vaccine strain CPVint standard model for contrast time, if its gene type the value of the confidence (GCP) is greater than 95%, be judged to be vaccine strain CPVint;
3) with CPV street strain standard model for contrast time, if its gene type the value of the confidence (GCP) is greater than 95%, be judged to be CPV street strain.
The invention has the beneficial effects as follows:
1) the present invention establishes PCR-HRM primer and the method for the strain of a kind of quick differentiation canine parvovirus vaccine and street strain first, simple to operate: add fluorescence saturable dye before only needing PCR reaction; Fast and the high-throughput of detection speed: all operations process only needs 3 hours, does not need the cell cultures of virus, greatly shortens 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 accurately, fast, analyze, be conducive to applying in clinical practice high-throughput.
2) PCR-HRM primer of the present invention, all has amplification well to canine parvovirus vaccine strain (comprising CPVpf and CPVint vaccine strain) and street strain, contributes to the efficiency improving PCR, reduce the time that virus differentiates somatotype.
3) PCR-HRM primer specificity of the present invention is good, except can being combined with street strain with canine parvovirus vaccine strain, uncommon with other dog viroid DNA is combined, specific amplification canine parvovirus DNA, is conducive to improving the present invention to the exactness of gene type assay.
Accompanying drawing explanation
Fig. 1 is canine parvovirus vaccine strain and street strain standard model HRM stdn melting curve;
Fig. 2 is canine parvovirus vaccine strain and street strain standard model HRM peak type melting curve;
Fig. 3 is canine parvovirus vaccine strain and street strain clinical sample HRM stdn melting curve;
Fig. 4 is canine parvovirus vaccine strain and street strain clinical sample HRM peak type melting curve;
Fig. 5 is canine parvovirus vaccine strain and street strain PCR-HRM primer specificity gel electrophoresis figure.
Embodiment
Below in conjunction with specific embodiment, the present invention is further illustrated, but be not limited thereto.
Embodiment 1
(1) primer
1) the pre-amplimer of PCR
Design primer pair P1 and P2 of amplification canine parvovirus VP2 protein portion gene according to canine parvovirus VP2 protein gene order, its base sequence is as follows.
P1:5'-TGGAAATCACAGCAAACTC-3'（SEQIDNO：1），
P2:5'-AGTCTTGGTTTTAAGTCAGTATC-3'（SEQIDNO：2）。
Wherein, primer P1 is on canine parvovirus VP2 protein gene 2962nd ~ 2980 site, and primer P2 is (accession number of this gene in Genbank is M38245) on canine parvovirus VP2 protein gene 4209th ~ 4231 site.
2) PCR-HRM primer:
The present invention is after screening designed a large amount of primers, find that the effect that the conbined usage of primer pair P3, P4 and primer pair P1, P2 distinguishes canine parvovirus vaccine strain and street strain to PCR-HRM method is best, the base sequence of primer pair P3, P4 is as follows.
P3：5'-TGAAAATTATAGAAGAGTGGT-3'（SEQIDNO：3），
P4：5'-CGTTAACTGCAGTTTTATCCA-3'（SEQIDNO：4）。
Wherein, primer P3 is on canine parvovirus VP2 protein gene 3014th ~ 3034 site, and primer P4 is (accession number of this gene in Genbank is M38245) on canine parvovirus VP2 protein gene 3045th ~ 3066 site.
(2) preparation of standard model and PCR-HRM thereof analyze
1) extraction of canine parvovirus DNA:
The canine parvovirus DNA in samples is extracted with test kit MiniBESTViralRNA/DNAExtractionKitVer.4.0.Samples can be whole blood, ight soil, facies rectalis swab etc. be easy to obtain and to the sample of animal body without grievous injury.
2) preparation of standard model:
In order to verify the inventive method feasibility and reliability, build standard positive sample, provide HRM positive control for clinical sample afterwards detects, the present invention preferentially need prepare canine parvovirus vaccine strain and street strain's positive criteria sample simultaneously.The preparation process of standard model is as follows:
Learning from else's experience respectively order-checking be defined as CPVpf vaccine strain, CPVint vaccine strain and canine parvovirus street strain DNA as template, increase in advance for primer carries out PCR with P1 and P2 respectively, its pre-amplification reaction system is:
PremixEx-Taq5.0μl
Primer P10.5 μ l
Primer P20.5 μ l
Template 1.0 μ l
ddH
2O3.0μl。
Pre-amplification response procedures is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s; Circulate 35 times; 72 DEG C of ends extend 8min.
Pass through aforesaid method, obtain the pre-amplified production of PCR of canine parvovirus vaccine strain CPVpf, vaccine strain CPVint and street strain respectively, respectively pre-for PCR amplified production is diluted 100 times, the standard model of vaccine strain CPVpf, vaccine strain CPVint and street strain can be obtained respectively, as the positive control sample of follow-up study.
3) the PCR-HRM operation steps of positive criteria sample
Respectively with three of above-mentioned acquisition kinds of positive criteria samples for DNA profiling, carry out PCR-HRM amplified reaction and analysis respectively;
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 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s; Circulate 35 times; 68 DEG C to 80 DEG C are carried out melting curve analysis with the melting speed of 0.3 DEG C/s.
4) positive criteria sample P CR-HRM interpretation of result
Pcr amplification product Rotor-GeneQ analyser is analyzed.Canine parvovirus vaccine strain and street strain standard model HRM(high resolving power melting curve, HighResolutionMeltingcurve) result is as shown in Figure 1 and Figure 2.
Fig. 1 is stdn melting curve figure, and stdn melting curve figure shows, and CPVpf, CPVint vaccine strain and street strain 3 kinds of standard model melting curves are separated from each other, and shows 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
tMqsoftwareversion2.0.2. software gene type confidence parameter (GCP) is analyzed (GCP value is more than or equal to 95% and is judged to be homologous genes type) its result.Genotypic results 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) are respectively 99.95 ± 0.04%, 99.81 ± 0.15% and 99.10 ± 0.78%.
Fig. 2 is peak type melting curve figure, and peak type melting curve figure shows, and three kinds of standard model melting curve shapes are similar, all has 1 to melt peak, shows to cause the major cause of GCP difference to be 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 DEG C, and vaccine strain CPVint melting temperature (Tm) is higher is 68.59 ± 0.03 DEG C, and it is 68.18 ± 0.02 DEG C between the two that street strain's melting temperature (Tm) is positioned at.
(3) clinical sample PCR-HRM analyzes
1) from sample, viral DNA is extracted: method is with DNA extraction method in above-mentioned (2);
2) with the viral DNA extracted for template, carry out PCR and increase in advance, pre-amplification reaction system is:
PremixEx-Taq5.0μl
Primer P10.5 μ l
Primer P20.5 μ l
Template 1.0 μ l
ddH
2O3.0μl。
Pre-amplification response procedures is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s; Circulate 35 times; 72 DEG C of ends extend 8min.
3) as DNA profiling after pre-amplification PCR primer dilutes 100, PCR-HRM amplified reaction is carried out:
Amplification reaction system is:
PremixEx-Taq5.0μl
Primer P30.5 μ l
Primer P40.5 μ l
Template 1.0 μ l
Evagreen dyestuff 1.0 μ l
ddH
2O2.0μl。
PCR-HRM amplified reaction program is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s; Circulate 35 times; 72 DEG C of ends extend 8min; 68 DEG C to 80 DEG C are carried out melting curve analysis with the speed of 0.3 DEG C/step.
4) HRM analysis is carried out to amplified production, determine the genotype of virus.
The present invention have detected 40 parts of clinical samples, and the result of PCR-HRM as shown in Figure 3, Figure 4.
Can find out from the stdn melting curve figure shown in Fig. 3, when respectively with CPV street strain, CPVpf vaccine strain and CPVint vaccine strain standard model in contrast time (GCP parameter sets value to be judged to be homologous genes type as its value is more than or equal to 90%), in 40 parts of clinical samples, 38 increment product 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%; Without CPVpf vaccine strain in 40 parts of clinical samples.
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 DEG C, 68.60 ± 0.03 DEG C and 67.65 ± 0 DEG C.
(4) specificity experiments
Specific detection is done to the PCR-HRM method that the present invention sets up below.
Extract other common dog viral DNAs respectively, as extracted canine distemper (Caninedistempervirus, CDV), hepatitis infectiosa canis virus-2(Canineadenovirustype2, and canine parainfluenza virus (Canineparainfluenzavirus CAV-2), CPIV) DNA is respectively as pcr template, PCR reaction is carried out respectively with the PCR method in above-mentioned (3), PCR primer is carried out gel electrophoresis analysis, and be analyzed with CPVpf, CPVint vaccine strain and street strain's positive PCR primer, electrophoresis result is as shown in Figure 5.
M in Fig. 5 is Marker(DL1000DNAmarker), 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 results shows, canine parvovirus vaccine strain and street strain's positive have object band at about 50bp, and electrophoretic band does not all appear in other sample, show that the high HRM that is suitable for of designed primer specificity analyzes.
<110> Institute of Animal Health,Guangdong Academy Of Agricultural Sciences
<120> mono-kind distinguishes PCR-HRM primer and the method for canine parvovirus street strain and vaccine strain fast
<130>
<160>4
<170>PatentInversion3.5
<210>1
<211>19
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The artificial primer of <213>
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tggaaatcacagcaaactc19
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The artificial primer of <213>
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agtcttggttttaagtcagtatc23
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The artificial primer of <213>
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tgaaaattatagaagagtggt21
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<212>DNA
The artificial primer of <213>
<400>4
cgttaactgcagttttatcca21
Claims (8)
1. distinguish a PCR-HRM primer for canine parvovirus street strain and vaccine strain fast, its nucleotide sequence is as follows:
Primer P1:TGGAAATCACAGCAAACTC(SEQIDNO:1),
Primer P2:AGTCTTGGTTTTAAGTCAGTATC(SEQIDNO:2),
Primer P3:TGAAAATTATAGAAGAGTGGT(SEQIDNO:3),
Primer P4:CGTTAACTGCAGTTTTATCCA(SEQIDNO:4).
2. distinguish a PCR-HRM method for canine parvovirus street strain and vaccine strain fast, it is characterized in that: comprise the following steps:
1) from sample, viral DNA is extracted;
2) take DNA as template, carry out pre-amplified reaction with primer pair P1 and P2 according to claim 1 and obtain pre-amplified production;
3) using pre-amplified production as DNA profiling, carry out PCR-HRM amplified reaction with primer pair P3, P4 according to claim 1 and fluorescence saturable dye and obtain amplified production;
4) HRM analysis is carried out to amplified production, determine Virus Type;
Aforesaid method is used for the Diagnosis and Treat of non-diseases.
3. method according to claim 2, is characterized in that: step 2) in the pre-amplification reaction system of PCR be:
PremixEx-Taq5.0μl
Primer P10.5 μ l
Primer P20.5 μ l
Template 1.0 μ l
ddH
2O3.0μl。
4. method according to claim 2, is characterized in that: step 2) in pre-amplification response procedures be: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 30s; Circulate 35 times; 72 DEG C of ends extend 8min.
5. method according to claim 2, is characterized in that: the PCR-HRM amplification reaction system in step 3) is:
PremixEx-Taq5.0μl
Primer P30.5 μ l
Primer P40.5 μ l
Template 1.0 μ l
Fluorescence saturable dye 1 μ l
ddH
2O2.0μl。
6. the method according to claim 2 or 5, is characterized in that: described fluorescence saturable dye is Evagreen dyestuff.
7. method according to claim 2, is characterized in that: the PCR-HRM amplified reaction program in step 3) is: 95 DEG C of denaturation 5min; 95 DEG C of sex change 30s, 58 DEG C of annealing 30s, 72 DEG C extend 30s; Circulate 35 times; 68 DEG C to 80 DEG C are carried out melting curve analysis with the speed of 0.3 DEG C/step.
8. method according to claim 2, is characterized in that: HRM described in step 4) analyze concrete analysis process be: 1) with vaccine strain CPVpf standard model for contrast time, if its gene type the value of the confidence is greater than 95%, be judged to be vaccine strain CPVpf;
2) with vaccine strain CPVint standard model for contrast time, if its gene type the value of the confidence is greater than 95%, be judged to be vaccine strain CPVint;
3) with CPV street strain standard model for contrast time, if its gene type the value of the confidence is greater than 95%, be judged to be CPV street strain.
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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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| 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 |
| 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 |
| US10233509B2 (en) * | 2016-12-07 | 2019-03-19 | Credo Biomedical Pte Ltd. | Method for detection CPV 2a, 2b, and 2c and for discrimination wild type from vaccine type |
| 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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| CN103320535A (en) * | 2013-06-27 | 2013-09-25 | 广东省农业科学院动物卫生研究所 | Method for identifying wild strain and vaccine strain of hog cholera virus |
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| PCR法检测犬细小病毒的研究进展;严云等;《广东畜牧兽医科技》;20071231;第32卷(第1期);第13-15页 * |
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