CN106048079A - A double-colour fluorescence detecting method, primers and probes for rapidly distinguishing Genotype II PRV, Genotype I PRV and a vaccine strain - Google Patents
A double-colour fluorescence detecting method, primers and probes for rapidly distinguishing Genotype II PRV, Genotype I PRV and a vaccine strain Download PDFInfo
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Abstract
A double-colour fluorescence detecting method, primers and probes for rapidly distinguishing a Genotype II porcine pseudorabies virus, a Genotype I porcine pseudorabies virus and a vaccine strain Bartha-K61 are disclosed. The method combines a real-time fluorescence PCR technique and a melting curve analysis technique. The Genotype II porcine pseudorabies virus, the Genotype I porcine pseudorabies virus and the vaccine strain Bartha-K61 are identified according to melting curve Tm value differences. Operation is simple, wherein identification detection of the two genotypes and the vaccine strain can be achieved by only one reaction. The detection speed is high in and the throughput is high. The total operation process can be finished in 3 hours. Virus cell culturing is not needed. Time for identification detection of the Genotype II porcine pseudorabies virus, the Genotype I porcine pseudorabies virus and the vaccine strain Bartha-K61 is greatly reduced. The method, the primers and the probes are high in accuracy, good in specificity, good in repeatability and capable of accurately and rapidly analyzing with a high throughput, and facilitate popularization and application in the clinical practice.
Description
Technical field
The present invention relates to virus different genotype, and the discrimination method of vaccine virus and street strain, be specifically related to a kind of quickly
Distinguish the double of PRV (Pseudorabies virus) (Pseudorabies virus, PRV) sinotype, American-European type and vaccine strain Bartha-K61
Color fluorescence detection method and test kit, the method is a kind of probe melting curve analysis based on two double labelling self-quenching probes
Technology, for PRV (Pseudorabies virus) sinotype, American-European type and the detection method of vaccine strain Bartha-K61.
Background technology
Since 2011, after the ground such as China North China, Central China, south China large-scale pig farm successively occurs that pseudorabies vaccine is crossed in immunity
Outburst pseudorabies, selected swine farms swinery open country poison antibody positive rate is up to 100%, and miscarriage occurs in in-pig 35%, and this is China
The extreme shock that live pig pig industry meets with.Vaccine virus immunization is the effective means currently controlling porcine pseudorabies, current China
Have 4 kinds of PRV commercialization attenuated vaccines (Bartha-K61 strain, Bucharest strain, HB-98 strain, and SA215 strain) Clinical practice,
Wherein Bartha-K61 strain market share is higher.Separately there is a kind of commercialization inactivated vaccine: Strain Ea also has sale.Inactivated vaccine is in peace
Full property aspect is higher than Attenuate vaccine, but can not breed in Pseudorabies virus latent tissue (such as nervi trigeminus etc.), to there being sense of hiding
The pig of dye can not play preventive effect；Pseudorabies gene-deleted vaccine virulence is more weak, immunogenicity preferable, but has virulence to return by force
Disease popularity, latent infection is caused to cause the danger dissipating poison, it is therefore necessary to set up supporting vaccine detection method.2015,
Tong Guangzhi etc. are by PRV full genome and the evolutionary analysis of 67 genes, it was demonstrated that PRV can be divided into two evolutionary branchings: American-European type
(Genotype II sees China, horse for (Genotype I sees the ground such as Europe, America, Australia, Asia) and sinotype
Come the ground such as West Asia), the representative strains of American-European type has Bartha, Becker, Kaplan, HS etc.；The representative strains of sinotype have Fa, Ea,
HeN1, JS, TJ etc. (Chao Ye,etal, &,Guang-Zhi Tong. Virology (2015) 483:32-43).Mesh
The epidemic isolates of front PRV (Pseudorabies virus) and sinotype strain Fa and Ea strain sibship relatively near (Xiuling Yu,etal, &, Kegong Tian. Emerging Infectious Diseases (2014) 20: 102-104；Yinbiao Wang,etal, &, Gaiping Zhang. Virus Genes (2015) 50:401 409), it is necessary to tracing detection clinic is
The no appearance having American-European type, so it is significant with the discriminating detection method of American-European type PRV (Pseudorabies virus) to set up sinotype.
The gpI(gE lacking all or part of gE gene according to marker vaccines and set up) antibody ELISA detection kit
And gB antibody ELISA detection kit, although with the use of reaching to identify the mesh ground that vaccine virus infects, but antibody test is deposited
In serious hysteresis quality, also cannot differentiate strain type simultaneously, be unfavorable for instructing further vaccine immunity, be the most currently also badly in need of one
Kind operation is relatively simple, testing result reliable and can high flux, quickly distinguish PRV (Pseudorabies virus) different genotype and vaccine
The method of strain.
Summary of the invention
In order to solve the problem of above-mentioned existence, the present invention establishes a kind of quickly differentiation PRV (Pseudorabies virus) sinotype, Europe
U.S. type and the two-color fluorescence PCR detection method of vaccine strain Bartha-K61, the method operation is simple, quick, high flux, detection knot
Fruit is reliable, is conducive to popularization and application in clinical practice.
It is an object of the invention to provide a kind of quickly differentiation PRV (Pseudorabies virus) sinotype, American-European type and vaccine strain
The two-color fluorescence PCR primer of Bartha-K61 and probe.
Another object of the present invention is to provide a kind of and quickly distinguish PRV (Pseudorabies virus) sinotype, American-European type and vaccine strain
The two-color fluorescence PCR method of Bartha-K61.
It is still another object of the present invention to provide a kind of quickly differentiation PRV (Pseudorabies virus) sinotype, American-European type and vaccine strain
The two-color fluorescence PCR test kit of Bartha-K61.
The technical solution used in the present invention is:
A kind of quick differentiation PRV (Pseudorabies virus) sinotype, American-European type and the two-color fluorescence PCR primer of vaccine strain Bartha-K61,
Its nucleotide sequence is as follows:
Primers F: 5'-GGGCGTCTACACGTGGCGC-3'(SEQ ID NO:1),
Primer R:5'-GTTGGTCACGAAGGCGGCGT-3'(SEQ ID NO:2).
A kind of quick differentiation PRV (Pseudorabies virus) sinotype, American-European type and the two-color fluorescence PCR of vaccine strain Bartha-K61
Probe, its nucleotide sequence is as follows:
Probe P1:5'-CGCAGCGCCAACGTCTCGCTCGTCCTGTAC-3'(SEQ ID NO:3),
Probe P2:5'-CCGAGTTCGGCCTGAGCGCGCCGCC-3'(SEQ ID NO:4).
Further, the fluorophor of described probe sequence 5 ' end labelling is a kind of in FAM, HEX, VIC, CY5, TET, visits
The quenching group of pin sequence 3 ' end labelling is a kind of in TAMRA, MGB, BHQ；And the fluorescent base of 5 ' the end labellings of probe P1 and P2
Group differs.
A kind of quick differentiation PRV (Pseudorabies virus) sinotype, American-European type and the two-color fluorescence PCR of vaccine strain Bartha-K61
Test kit, containing primer described above in this test kit.
Further, possibly together with probe described above in mentioned reagent box.
A kind of quick differentiation PRV (Pseudorabies virus) sinotype, American-European type and the two-color fluorescence PCR of vaccine strain Bartha-K61
Method, comprises the following steps:
1) from sample, viral DNA is extracted；
2) with extract DNA as template, with primer described above to F, R, and probe P1 described above and probe P2 carries out glimmering
Light pcr amplification reaction obtains amplified production；
3) amplified production is carried out melting curve analysis, determine the Virus Type in sample.
Further, step 2) in fluorescent PCR amplification reaction system be:
Premix Ex-Taq 5.0μl
Primer P1(1 μM) 0.4 μ l
Primer P2(10 μM) 0.4 μ l
Probe Probe1(10 μM) 0.2 μ l
Probe Probe2(10 μM) 0.2 μ l
Template 1.0 μ l
ddH2O 2.8μl。
Further, step 2) in fluorescent PCR amplified reaction program be: 95 DEG C of denaturations 5min；95 DEG C of degeneration 20s,
60 DEG C of annealing 20s, 72 DEG C extend 20s；Circulate 55 times.
Further, the melting curve analysis program in step 3) is: 95 DEG C of degeneration 10 sec；40 DEG C to 97 DEG C with 0.13
DEG C/speed of s, the fluorescence signal of 5 time/DEG C continuous acquisition probe P1 and P2, carry out melting curve analysis.
Further, the concrete analysis process of melting curve analysis described in step 3) is:
1) in the fluoroscopic examination result of probe P1, with Bartha-K61 standard sample for comparison time, if sample melting temperature and
When the absolute value of the Tm value of positive control Bartha-K61 melting temperature is less than 1.0 DEG C, then it is judged to American-European type pseudorabies
Virus；
2) in the fluoroscopic examination result of probe P1, during with HDDJ standard sample for comparison, if sample melting temperature and the positive are right
According to HDDJ melting temperature Tm value absolute value less than 1.0 DEG C time, then be judged to sinotype PRV (Pseudorabies virus)；
3) in the fluoroscopic examination result of probe P2, with Bartha-K61 standard sample for comparison time, if sample melting temperature and
When the absolute value of the Tm value of positive control Bartha-K61 melting temperature is less than 1.0 DEG C, then it is judged to Bartha-K61 vaccine
Strain.
The invention has the beneficial effects as follows:
1) present invention establishes a kind of quickly differentiation PRV (Pseudorabies virus) sinotype, American-European type and vaccine strain Bartha-K61 first
Two Colour Fluorescence detection method, primer and probe.Simple to operate: only to need a reaction can realize sinotype, American-European type and epidemic disease
The discriminating detection of Seedling strain Bartha-K61；Detection speed is fast and high flux: all operations process can complete in 3 hours, is not required to
The cell wanting virus is cultivated, and greatly shortens PRV (Pseudorabies virus) sinotype, American-European type strain and Bartha-K61 vaccine strain and reflects
Do not detect required time；Accuracy is high, specificity is good, reproducible, can accurately, quickly, be analyzed with high throughput, favorably
In popularization and application in clinical practice.
2) PCR primer of the present invention is to F/R, to PRV (Pseudorabies virus) open country poison sinotype, American-European type and vaccine strain
Bartha-K61 can specific amplification, be favorably improved the efficiency of PCR, reduce virus and differentiate the time of typing.Probe P1 can be special
The opposite sex and PRV (Pseudorabies virus) sinotype and American-European type nucleic acid sequences hybridization, specificity is preferable.Probe P2 can specificity and pig puppet
Rabies virus Bartha-K61 vaccine strain and other non-Bartha-K61 strain discrimination bit dot blot, specificity is preferable.Primers F/
R, probe P1 and P2, be not combined with other common pig breeding dysfunction correlated virus nucleic acid, is conducive to improving the present invention to result
The correctness analyzed.
4) a kind of quick differentiation PRV (Pseudorabies virus) sinotype of the present invention, American-European type and vaccine strain Bartha-K61's is double-colored
The lowest detectable limit of fluorescence detection method can reach single copy, and sensitivity is higher.
Accompanying drawing explanation
Fig. 1 is positive criteria sample gene evolution analysis chart, wherein ● for standardization sample sinotype HDDJ, ▲ for mark
Standardization sample sinotype Ea, ■ is standardization sample sinotype Fa, ◇ be standardization sample Bartha-K61 vaccine strain (simultaneously
Also it is American-European type), △ is standardization sample America and Europe's type HS；
Fig. 2 is normalized sample Two Colour Fluorescence detection method melting curve figure, and wherein A is that FAM and the HEX dual pathways detects simultaneously
Melting curve figure, B and C is respectively FAM and HEX single channel and detects melting curve figure respectively；Standard sample is Bartha-K61 epidemic disease
Seedling strain (being also simultaneously American-European type), non-Bartha-K61 strain HDDJ(is also sinotype simultaneously)；
Fig. 3 is Two Colour Fluorescence detection method specific test melting curve figure, and wherein A is that FAM and the HEX dual pathways detects simultaneously
Melting curve figure, B and C is respectively FAM and HEX single channel and detects melting curve figure respectively；
Fig. 4 is Two Colour Fluorescence detection method positive plasmid p-Bartha-K61 sensitivity test amplification curve diagram and melting curve
Figure, wherein A is that FAM and the HEX dual pathways detects amplification curve diagram simultaneously, and B is that FAM and the HEX dual pathways detects melting curve simultaneously
Figure, C and D is respectively FAM single channel and HEX single channel detects amplification curve diagram respectively；E and F is respectively FAM single channel and HEX
Single channel detects melting curve figure respectively；
Fig. 5 is Two Colour Fluorescence detection method positive plasmid p-HDDJ sensitivity test amplification curve diagram and melting curve figure, wherein
A is that FAM and the HEX dual pathways detects amplification curve diagram simultaneously, and B is that FAM and the HEX dual pathways detects melting curve figure, C and D simultaneously
It is respectively FAM single channel and HEX single channel detects amplification curve diagram respectively；E and F is respectively FAM single channel and HEX single channel divides
Jian Ce melting curve figure；
Fig. 6 is clinical sample Two Colour Fluorescence detection method melting curve figure, and wherein to be that FAM and the HEX dual pathways detects simultaneously molten for A
Solution curve figure, B and C is respectively FAM and HEX single channel and detects melting curve figure respectively；Standard sample Bartha-K61 vaccine strain
(being also American-European type) and non-Bartha-K61 strain HDDJ(are also sinotype simultaneously simultaneously) it is positive control, clinical sample 4,
Numbering is respectively INT, S, ZZ, LC；Result display sample INT and S is American-European type strain, ZZ and LC is sinotype strain, the most not
It it is Bartha-K61 vaccine strain.This result is through sequence verification.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated, but is not limited thereto.
Embodiment 1 primer and probe
After screening designed a large amount of primers and probe, discovery primer is to F, R, and probe P1 and P2 is to double-colored glimmering
The effect that light method distinguishes PRV (Pseudorabies virus) sinotype, American-European type and vaccine strain Bartha-K61 is best, and its base sequence is as follows
Shown in.
Primers F: 5'-GGGCGTCTACACGTGGCGC-3'(SEQ ID NO:1),
Primer R:5'-GTTGGTCACGAAGGCGGCGT-3'(SEQ ID NO:2),
Probe P1:5'-FAM-CGCAGCGCCAACGTCTCGCTCGTCCTGTAC-BHQ1-3'(SEQ ID NO:3),
Probe P2:5'-HEX-CCGAGTTCGGCCTGAGCGCGCCGCC-BHQ1-3'(SEQ ID NO:4).
The preparation of embodiment 2 standard sample, two-color fluorescence PCR amplification and melting curve analysis
1) extraction of PRV (Pseudorabies virus) DNA:
Take suspected infection respectively and have the samples of PRV, samples can be the lymph node of sick dead pig, brain, the heart, liver, spleen,
Lung, kidney, tonsil etc. are organized, or gather porcine blood serum or nose formula.It is standby that serum can directly take 200 μ l；Nose formula need to be dissolved in
In 1mL PBS hydrochloric acid buffer solution, take 200 μ l after standing 10-20min standby；The tissue samples of sick dead pig need to be centrifuged after grinding
Take supernatant 200 μ l standby；Bartha-K61 vaccine, adds 3mL PBS hydrochloric acid buffer solution and dissolves, take 200 μ L standby.Press
The description of the MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0 of TAKARA carries out nucleic acid extraction.
2) preparation of standard sample:
In order to verify the inventive method feasibility and reliability, build standard positive sample (correct through sequencing) simultaneously, for
Clinical sample detection afterwards provides positive control, and it is (same that the present invention need to first prepare PRV (Pseudorabies virus) Bartha-K61 vaccine strain
Time be also American-European type), Europe class strain HS, sinotype strain HDDJ, sinotype strain Fa, sinotype strain Ea positive criteria sample
Product.The preparation process of standard sample is as follows: take Bartha-K61 vaccine strain (be also American-European type) simultaneously, Europe class strain HS, in
State type strain HDDJ, sinotype strain Fa, sinotype strain Ea, respectively at ST cell (Pig testicular cell) Secondary Culture to state
Stable, take 200 μ l virus liquids and carry out extracting nucleic acid, as positive criteria product according to the extracting method of PRV (Pseudorabies virus) DNA.
By said method, obtain the PRV (Pseudorabies virus) Bartha-K61 vaccine strain containing genes of interest segment respectively
(being also American-European type), Europe class strain HS, sinotype strain HDDJ, sinotype strain Fa, sinotype strain Ea simultaneously.
For the genotype of the selected standard positive sample of checking, according to document report (Chao Ye,etal, &,Guang-
Zhi Tong. Virology (2015) 483:32-43), choose and there is the gene of typing feature accordingly do phylogenetic analysis,
As shown in Figure 1.
Fig. 1 is standardization sample gC gene evolution analysis chart, wherein ● for standardization sample sinotype HDDJ, ▲ for standard
Change sample sinotype Ea, ■ is standardization sample sinotype Fa, ◇ be standardization sample Bartha-K61 vaccine strain (while the most also
It is American-European type), △ is standardization sample America and Europe's type HS.Bartha-K61 vaccine strain, HS are American-European type as can be seen from Figure 1
(Genotype I), HDDJ, Fa, Ea are sinotype (Genotype II).
3) the fluorescent PCR operating procedure of positive criteria sample:
Respectively with three kinds of positive criteria samples of above-mentioned acquisition as DNA profiling, carry out fluorescent PCR amplified reaction respectively and melt song
Line analysis；
PCR reaction system:
ddH2O 2.8μl
Premix Ex-Taq 5.0μl
1 μM of primers F 0.4 μ l
10 μMs of primer R 0.4 μ l
10μM P1 0.2μl
10μM P2 0.2μl
Template 1.0 μ l
Cumulative volume 10 μ l.
Pcr amplification reaction program is as follows:
95 DEG C of denaturations 5min；95 DEG C of degeneration 20s, 60 DEG C of annealing 20s, 72 DEG C extend 20s；Circulate 55 times.
Melting curve analysis program is as follows:
95 DEG C of degeneration 10 sec；40 DEG C to 97 DEG C with the speed of 0.13 DEG C/s, 5 time/DEG C continuous acquisition FAM and HEX fluorescence signal,
Carry out melting curve analysis.
4) positive criteria sample melting curve analysis interpretation of result
Pcr amplification product LightCycler 96 analyser is analyzed.5 positive criteria samples of PRV (Pseudorabies virus) melt
Tracing analysis result is as shown in Figure 2.
Fig. 2 is positive criteria sample Two Colour Fluorescence detection method melting curve figure, and Fig. 2 A is Fig. 2 B and Fig. 2 C fluorescence curve
Stacking chart.
Fig. 2 B is FAM fluorescence channel analysis result (i.e. the fluoroscopic examination result of probe P1), there it can be seen that American-European type
The melting curve of strain Bartha-K61, HS and sinotype strain HDDJ, Fa, Ea standard sample is separated from each other, and shows designed
Primers F, R and probe P1 are suitable for PRV (Pseudorabies virus) sinotype and the melting curve analysis of American-European two kinds of genotype of type.Root
According to the difference of two kinds of positive criterias sample melting temperature (Tm), American-European type strain Bartha-K61, HS melting temperature is relatively low is
70.42 ± 0.15 DEG C, more a height of 76.8 ± 0.07 DEG C of sinotype strain HDDJ melting temperature (Fig. 2 A and Fig. 2 B).
Fig. 2 C is the analysis result (i.e. the fluoroscopic examination result of probe P2) of HEX fluorescence channel, there it can be seen that vaccine
Strain Bartha-K61 and non-Bartha-K61 strain HS, HDDJ, Fa, Ea standard sample melting curve are separated from each other, and show set
Meter primers F, R and probe P2 are suitable for porcine pseudorabies toxic vaccine strain Bartha-K61 and the melting of non-Bartha-K61 strain
Tracing analysis.According to the difference of two kinds of standard sample melting temperatures (Tm), vaccine strain Bartha-K61 melting temperature is relatively low is
71.66 DEG C, more a height of 76.08 ± 0.08 DEG C of non-Bartha-K61 strain melting temperature (Fig. 2 A and Fig. 2 C).
Embodiment 3 specificity experiments
The detection method set up the present invention below makees specific detection.
Extract other common pig breeding dysfunction disease correlated virus nucleic acid respectively, as extracted swine fever virus (classical
Swine fever virus, CSFV), Latex agglutination test (Japanese encephalitis virus, JEV), pig
Annulus 2 type (Porcine circovirus type 2, PCV 2), porcine reproductive and respiratory syndrome virus (Porcine
Reproductive and Respiratory Syndrome Virus, PRRSV), pig parvoviral (Porcine
Parvovirus, PPV), it is cDNA by the RNA reverse transcription of CSFV, JEV and PRRSV, with cDNA, PCV 2 He of virus above
The nucleic acid of PPV and water respectively as pcr template, are carried out point with the pcr amplification reaction in above-mentioned (2) and melting curve analysis method
Analysis, and same with PRV (Pseudorabies virus) Bartha-K61 vaccine strain (being also American-European type) and non-Bartha-K61 strain HDDJ(simultaneously
Time be also sinotype) positive criteria sample is analyzed, melting curve peak type figure is as shown in Figure 3.
It can be seen that detection method can only go out PRV (Pseudorabies virus) Bartha-by specific amplification from Fig. 3 A ~ C
K61 vaccine strain (be also American-European type) and non-Bartha-K61 strain HDDJ(are also sinotype simultaneously simultaneously) positive criteria sample is molten
Xie Feng, and other pig breeding dysfunction disease correlated virus, as CSFV, JEV, PRRSV, PCV 2 and PPV does not the most amplify spy
Different melting peaks.Show that primers F, R and probe P1 and P2 specificity are preferable, may be used for PRV (Pseudorabies virus) sinotype with American-European
Type, and the fluoroscopic examination of vaccine strain Bartha-K61.
Embodiment 4 sensitivity experiment
The detection method set up the present invention below makees sensitivity technique.
With embodiment 2 Plays product Bartha-K61 as template, primer P1 and P2 is amplimer, by extension amplification outcome
To PMD18T-Vector, building positive plasmid p-Bartha-K61, p-Bartha-K61 does detection of nucleic acids, convert plasmid
Number, does 10 times of gradient dilutions, forms 1.0x109、1.0x108、1.0x107、1.0x106、1.0x105、1.0x104、1.0x103、
1.0x102、1.0x101、1.0x100Copies/ μ l totally 10 gradients, by the fluorescent PCR amplified reaction in above-described embodiment 2 and
Melting curve analysis method is analyzed, and amplification curve diagram and melting curve peak type figure are as shown in Figure 4.
Fig. 4 is Two Colour Fluorescence detection method sensitivity test amplification curve diagram and the melting of positive plasmid p-Bartha-K61
Curve chart, wherein A is the amplification curve diagram that FAM and the HEX dual pathways detects simultaneously, i.e. the stacking chart of curve in C and D figure；C and D
It is respectively FAM single channel and the amplification curve diagram of HEX single channel detection；B is that the melting that FAM and the HEX dual pathways detects simultaneously is bent
Line chart, i.e. the stacking chart of curve in E and F figure；E and F is respectively FAM single channel and the melting curve figure of HEX single channel detection.From
In can be seen that this detection method presents the fluorescence signal of significantly decline along with the reduction of nucleic acid concentration, plasmid number is as little as
1.0 copy/ μ l, FAM passage and HEX passage can also detect corresponding fluorescence signal.
Positive plasmid p-HDDJ is built according to above-mentioned same method, 10 times of gradient dilutions, glimmering with in above-described embodiment 2
Light pcr amplification reaction and melting curve analysis method are analyzed, amplification curve diagram and melting curve peak type figure such as Fig. 5 institute
Show.
Fig. 5 is Two Colour Fluorescence detection method sensitivity test amplification curve diagram and the melting curve of positive plasmid p-HDDJ
Figure, wherein A is the amplification curve diagram that FAM and the HEX dual pathways detects simultaneously, i.e. the stacking chart of curve in C and D figure；C and D is respectively
The amplification curve diagram detected for FAM single channel and HEX single channel；B is the melting curve figure that FAM and the HEX dual pathways detects simultaneously,
The stacking chart of curve in i.e. E and F figure；E and F is respectively FAM single channel and the melting curve figure of HEX single channel detection.Therefrom may be used
To find out that this detection method presents the fluorescence signal of significantly decline, plasmid number as little as 1.0 along with the reduction of nucleic acid concentration
Copy/ μ l, FAM passage and HEX passage can also detect corresponding fluorescence signal.
Above-mentioned testing result illustrates that the inventive method sensitivity is higher.
The amplification of embodiment 5 clinical sample fluorescent PCR and melting curve analysis
1) from sample, viral nucleic acid is extracted: method, with above-described embodiment 2 amplifying nucleic acid extracting method, is extracted in 4 parts of clinical samples
Viral nucleic acid；
2) with extract viral nucleic acid as template, method is divided with fluorescent PCR amplified reaction and melting curve in above-described embodiment 2
Analysis；(also it is simultaneously American-European with the positive criteria product PRV (Pseudorabies virus) Bartha-K61 vaccine strain described in embodiment 2 simultaneously
Type) and non-Bartha-K61 strain HDDJ(be also simultaneously sinotype) as positive control.
3) clinical sample melting curve analysis interpretation of result
Fluorescent PCR amplified production LightCycler 96 analyser is analyzed.The present invention have detected 4 parts of clinical samples, molten
Solution curve analysis result is as shown in Figure 6.
Can be seen that from the clinical sample Two Colour Fluorescence detection method melting curve figure of Fig. 6, analyze FAM fluorescence channel (i.e.
The testing result of probe P1) time, using PRV America and Europe's type Bartha-K61 and sinotype HDDJ standard sample melting curve as comparison
Time, between measuring samples and positive control Bartha-K61, the absolute value of melting peaks Tm value is judged to America and Europe when being less than 1.0 DEG C
Type PRV；Between measuring samples and positive control HDDJ, the absolute value of melting peaks Tm value is judged to sinotype when being less than 1.0 DEG C
PRV；Result shows, in the 4 parts of clinical samples detected, numbering INT and S are American-European type PRV, and numbering ZZ and LC are sinotype PRV
(see Fig. 6 A and B).
When analyzing HEX fluorescence channel (i.e. the testing result of probe P2), with PRV vaccine strain Bartha-K61 and non-
When Bartha-K61 strain HDDJ standard sample melting curve is as comparison, between measuring samples and positive control Bartha-K61
It is judged to PRV vaccine strain Bartha-K61 when the absolute value of melting peaks Tm value is less than 1.0 DEG C；Measuring samples and positive control
Between HDDJ, the absolute value of melting peaks Tm value is judged to non-PRV vaccine Bartha-K61 when being less than 1.0 DEG C；Result shows, institute
In 4 parts of clinical samples of detection, INT, S, ZZ and LC are not the most PRV vaccine strain Bartha-K61.
Summary FAM passage and HEX passage result (i.e. the testing result of probe P1 and P2), it is determined that 4 parts of clinical samples
In numbered INT and S be American-European type PRV, ZZ and LC is sinotype PRV, is not the most PRV vaccine strain Bartha-K61.
This result is through sequence verification.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-described embodiment
Limit, the change made under other any spirit without departing from the present invention and principle, modify, substitute, combine, simplify,
All should be the substitute mode of equivalence, within being included in protection scope of the present invention.
<110>Institute of Animal Health,Guangdong Academy Of Agricultural Sciences
<120>a kind of quick differentiation PRV sinotype, American-European type and Two Colour Fluorescence detection method, primer and the spy of vaccine strain
Pin
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>artificial sequence
<400> 1
gggcgtctac acgtggcgc 19
<210> 2
<211> 20
<212> DNA
<213>artificial sequence
<400> 2
gttggtcacg aaggcggcgt 20
<210> 3
<211> 30
<212> DNA
<213>artificial sequence
<400> 3
cgcagcgcca acgtctcgct cgtcctgtac 30
<210> 4
<211> 25
<212> DNA
<213>artificial sequence
<400> 4
ccgagttcgg cctgagcgcg ccgcc 25
Claims (10)
1. the two-color fluorescence PCR of a quick differentiation PRV (Pseudorabies virus) sinotype, American-European type and vaccine strain Bartha-K61 draws
Thing, its nucleotide sequence is as follows:
Primers F: 5'-GGGCGTCTACACGTGGCGC-3'(SEQ ID NO:1),
Primer R:5'-GTTGGTCACGAAGGCGGCGT-3'(SEQ ID NO:2).
2. the two-color fluorescence PCR of a quick differentiation PRV (Pseudorabies virus) sinotype, American-European type and vaccine strain Bartha-K61 is visited
Pin, its nucleotide sequence is as follows:
Probe P1:5'-CGCAGCGCCAACGTCTCGCTCGTCCTGTAC-3'(SEQ ID NO:3),
Probe P2:5'-CCGAGTTCGGCCTGAGCGCGCCGCC-3'(SEQ ID NO:4).
Probe the most according to claim 2, it is characterised in that described probe sequence 5 ' end labelling fluorophor be FAM,
One in HEX, VIC, CY5, TET, the quenching group of probe sequence 3 ' end labelling is a kind of in TAMRA, MGB, BHQ；And probe
The fluorophor of 5 ' the end labellings of P1 and P2 differs.
4. the two-color fluorescence PCR examination of a quick differentiation PRV (Pseudorabies virus) sinotype, American-European type and vaccine strain Bartha-K61
Agent box, it is characterised in that containing the primer described in claim 1 in this test kit.
Test kit the most according to claim 4, it is characterised in that possibly together with described in Claims 2 or 3 in this test kit
Probe.
6. a quick differentiation PRV (Pseudorabies virus) sinotype, American-European type and the two-color fluorescence PCR side of vaccine strain Bartha-K61
Method, it is characterised in that comprise the following steps:
1) from sample, viral DNA is extracted；
2) with extract DNA as template, with the primer described in claim 1 to F, R, and probe P1 described in Claims 2 or 3 and
Probe P2 carries out fluorescent PCR amplified reaction and obtains amplified production；
3) amplified production is carried out melting curve analysis, determine the Virus Type in sample.
Method the most according to claim 6, it is characterised in that: step 2) in fluorescent PCR amplification reaction system be:
Premix Ex-Taq 5.0μl
1 μM of primer P1 0.4 μ l
10 μMs of primer P2 0.4 μ l
10 μMs of probe Probe1 0.2 μ l
10 μMs of probe Probe2 0.2 μ l
Template 1.0 μ l
ddH2O 2.8μl。
Method the most according to claim 6, it is characterised in that: step 2) in fluorescent PCR amplified reaction program be: 95 DEG C
Denaturation 5min；95 DEG C of degeneration 20s, 60 DEG C of annealing 20s, 72 DEG C extend 20s；Circulate 55 times.
Method the most according to claim 6, it is characterised in that: the melting curve analysis program in step 3) is: 95 DEG C of changes
Property 10 s；40 DEG C to 97 DEG C, with the speed of 0.13 DEG C/s, the fluorescence signal of 5 time/DEG C continuous acquisition probe P1 and P2, melt
Tracing analysis.
Method the most according to claim 6, it is characterised in that: the concrete analysis of melting curve analysis described in step 3)
Process is:
1) in the fluoroscopic examination result of probe P1, with Bartha-K61 standard sample for comparison time, if sample melting temperature and
When the absolute value of the Tm value of positive control Bartha-K61 melting temperature is less than 1.0 DEG C, then it is judged to American-European type pseudorabies
Virus；
2) in the fluoroscopic examination result of probe P1, during with HDDJ standard sample for comparison, if sample melting temperature and the positive are right
According to HDDJ melting temperature Tm value absolute value less than 1.0 DEG C time, then be judged to sinotype PRV (Pseudorabies virus)；
3) in the fluoroscopic examination result of probe P2, with Bartha-K61 standard sample for comparison time, if sample melting temperature and
When the absolute value of the Tm value of positive control Bartha-K61 melting temperature is less than 1.0 DEG C, then it is judged to Bartha-K61 vaccine
Strain.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561374A (en) * | 2014-12-18 | 2015-04-29 | 河南省动物疫病预防控制中心 | Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain |
| CN105483287A (en) * | 2015-12-22 | 2016-04-13 | 广东省实验动物监测所 | HRM detection method and primer for quickly differentiating porcine pseudorabies virus vaccine strain Bartha-K61 from other strains |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104561374A (en) * | 2014-12-18 | 2015-04-29 | 河南省动物疫病预防控制中心 | Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain |
| CN105483287A (en) * | 2015-12-22 | 2016-04-13 | 广东省实验动物监测所 | HRM detection method and primer for quickly differentiating porcine pseudorabies virus vaccine strain Bartha-K61 from other strains |
Non-Patent Citations (1)
| Title |
|---|
| 张志等: "伪狂犬病毒野毒株与疫苗株的实时荧光定量PCR鉴别方法的建立", 《中国兽医科学》 * |
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