A kind of quick differentiation HP-PRRS vaccine GDr180 strain and the primer of street strain, probe and
Method
Technical field
The present invention relates to the discrimination method of viral lived vaccine strain and street strain, be specifically related to a kind of height of quickly distinguishing and cause a disease
Property the detection method of Porcine reproductive and respiratory syndrome live vaccine GDr180 strain and street strain and primer and probe.
Background technology
Summer in 2006, high-pathogenicity porcine reproductive and respiration syndrome (High pathogenic are broken out at southern china
Porcine reproductive and respiratory syndrome, HP-PRRS) epidemic situation, current epidemic situation rapid onset, disease
Dead rate is high, cure rate is low, and affected the whole country within half a year various places, causes serious loss to China's pig industry.HP-PRRS is by pig
Reproductive and respiratory syndrome virus (porcine reproductive and respiratory syndrome virus,
PRRSV) the acute high lethal epidemic disease of one that variant causes, this variant is referred to as high-pathogenicity porcine reproductive and respiration syndrome
Virus (HP-PRRSV).China mainly takes the comprehensive prevention and control measure based on vaccine immunity at present, pig is implemented pressure and exempts from
Epidemic disease, the high-pathogenicity porcine reproductive of use and respiration syndrome vaccine are live vaccine (Strain in Jiangxi JXAl-R, Strain Hunan Hun4-
F112, Tianjin strain TJM-F92, Guangdong Strain GDr180).
Owing to highly pathogenic PRRS Vaccines classes is more, Chinese scholars and enterprise of raising pigs are to HP-PRRS epidemic disease in addition
Seedling uses the difference of viewpoint, and the use causing the current pig blue-ear disease vaccine of China is the most chaotic.Multiple vaccine strain uses increasing simultaneously
Having added the risk of viral restructuring, part vaccine has virulence to return strong probability simultaneously.So, morbidity pig is carried out Pathogen test,
Diagnosing pathogenic infection effectively, as soon as possible is vaccine strain or street strain, and control animal epidemic effective for pig farm subtracts to greatest extent
Few economic loss is significant.HP-PRRS vaccine strain GDr180 obtains, in May, 2015, the three new beasts of class that the Ministry of Agriculture issues
Medicine certificate of registry also lists.Differential diagnostic method for HP-PRRS vaccine strain GDr180 Yu street strain only has an employing MGB
(Zhang Wenli differentiates etc. highly pathogenic PRRSV GD vaccine strain and street strain Multiplex real-time PCR for the report of sonde method
The foundation [J] of method, China's Preventive Veterinary Medicine report. 2012,11 (34): 898-902).Although the method has relatively Gao Ling
Sensitivity and specificity, but the method relates to two MGB probes, relatively costly, it is difficult to universal use.So setting up a kind of cost
Low, highly sensitive, high specificity, promote easily, can quickly distinguish the detection side of HP-PRRSV live vaccine GDr180 strain and street strain
Method has great importance.
Fluorescence melting curve analysis technology (fluorescence melting curve analysis, FMCA) is a kind of
Combine Real-Time Fluorescent Quantitative PCR Technique (RT-PCR) and melting curve analysis (Melting Curve Analysis, MCA) skill
New genotyping diagnosis detection technique (Ahn JJ, Kim Y, Lee SY, Hong JY, the Kim GW, Hwang of art
SY. Fluorescence melting curve analysis using self-quenching dual-labeled
peptide nucleic acid probes for simultaneously identifying multiple DNA
Sequences. Anal Biochem. 2015,484:143-7.).
Summary of the invention
It is an object of the invention to provide a kind of quickly differentiation high-pathogenicity porcine reproductive and respiration syndrome live vaccine
The primer of GDr180 strain and street strain and probe.
It is an object of the invention to provide a kind of quickly differentiation high-pathogenicity porcine reproductive and respiration syndrome live vaccine
GDr180 strain and the method for street strain.
It is still another object of the present invention to provide a kind of quickly differentiation high-pathogenicity porcine reproductive and respiration syndrome live vaccine
GDr180 strain and the test kit of street strain.
The technical solution used in the present invention is:
A kind of quick differentiation high-pathogenicity porcine reproductive and respiration syndrome live vaccine strain GDr180 strain and the primer of street strain, its core
Nucleotide sequence is as follows:
Primer P1:5 '-GGTTGACATGCTGACTTG-3 ' (SEQ ID NO:1),
Primer P2:5 '-CACTGAACCAATGGTGAG-3 ' (SEQ ID NO:2).
A kind of quick differentiation high-pathogenicity porcine reproductive and respiration syndrome live vaccine GDr180 strain and the probe of street strain, its
Nucleotide sequence is as follows:
Probe P:5 '-CCCAAGGATGATTCTCGAGACACCG-3 ' (SEQ ID NO:3).
A kind of quick differentiation high-pathogenicity porcine reproductive and respiration syndrome live vaccine GDr180 strain and the test kit of street strain,
This test kit contains primer described above.
Further, this test kit is possibly together with probe described above.
A kind of quick differentiation high-pathogenicity porcine reproductive and the method for respiration syndrome live vaccine GDr180 strain with street strain, wrap
Include following steps:
1) from sample, viral nucleic acid is extracted;
2) with nucleic acid as template, with primer described above, P1, P2, probe P are carried out FMCA amplified reaction and obtain amplified production;
3) amplified production is carried out FMCA analysis, determine Virus Type.
Further, step 2) in FMCA amplification reaction system be:
Nucleic acid-templated 1 L
PrimeScript 1 Step Enzyme Mix 0.5 µL
2×1 Step Buffer 6.25µL
2.0Mm primer P1 0.5 L
10Mm primer P2 0.5 L
10Mm probe P 0.25 L
RNase Free ddH2O 3.5µL
Cumulative volume 12.5 L.
Further, step 2) in FMCA amplified reaction program be: 50 DEG C of reverse transcription 30min;95 DEG C of denaturations 2min;
95 DEG C of degeneration 20s, 55 DEG C of annealing 20s, 72 DEG C extend 20s;Circulate 55 times;98 DEG C of degeneration 2min, 40 DEG C of heterozygosis 2min, 55 DEG C are arrived
75 DEG C carry out melting curve analysis with the melting speed of 1 DEG C/s.
Further, the concrete analysis process that FMCA described in step 3) analyzes is:
With standard positive sample high-pathogenicity porcine reproductive and respiration syndrome live vaccine GDr180 as positive control, if measuring samples
With the absolute value of melting peaks Tm value less than 1.0 DEG C, is then judged to HP-PRRSV vaccine strain GDr180 between positive control;
With standard positive sample high-pathogenicity porcine reproductive and the poison GD strain of respiration syndrome open country as positive control, if measuring samples and sun
Property comparison between melting peaks Tm value absolute value be less than 1.0 DEG C, then be judged to HP-PRRSV street strain.
The invention has the beneficial effects as follows:
1) present invention establish first a kind of quickly distinguish high-pathogenicity porcine reproductive and respiration syndrome live vaccine GDr180 strain with
Melting curve analysis method, primer and the probe of street strain, simple to operate: only need to add probe before PCR reacts;Detection
Speed is fast and high flux: all operations process only needs 3 hours, greatly shortens typing required time;Expense is relatively low: only need
An average probe is wanted i.e. to can reach the mesh ground of Differential Diagnosis;Accuracy is high, specificity is good, reproducible, can accurately, soon
Speed, be analyzed with high throughput, be conducive to popularization and application in clinical practice.
2) the FMCA primer of the present invention, to HP-PRRSV vaccine strain GDr180 and street strain, all has well amplification property, has
Help improve the efficiency of PCR, reduce virus and differentiate the time of typing.
3) the probe P of the present invention has matching completely to vaccine strain GDr180, and HP-PRRSV street strain is only had one
The difference of base, is favorably improved analysis efficiency, reduces the time of Viral typing.
4) FMCA primer and the probe specificity of the present invention are good, are only combined with vaccine strain GDr180 and street strain, not with it
His common pig source is viral, such as swine fever virus (Classical swine fever virus, CSFV), PRV (Pseudorabies virus)
(Pseudorabies Virus, PRV), pig annulus 2 type (Porcine circovirus type 2, PCV2), the tiny disease of pig
The nucleic acid such as poison (Porcine parvovirus, PPV) combine, and specific amplification HP-PRRSV nucleic acid is conducive to improving the present invention
The correctness that HP-PRRSV vaccine strain GDr180 and street strain are analyzed.
5) present invention utilizes one-step method FMCA method to improve difference HP-PRRSV vaccine strain GDr180 and the standard of street strain
Really property and repeatability.The inventive method low cost, highly sensitive, high specificity, there is higher clinical practice promotional value.
Accompanying drawing explanation
Fig. 1 is GDr180 and GD standard sample FMCA peak type melting curve figure;
Fig. 2 is clinical sample FMCA peak type melting curve figure;Using standard substance GDr180 and GD as positive control;Red melting
Curve is HP-PRRSV vaccine strain GDr180 positive reference substance, and blue melting curve is HP-PRRSV street strain GD positive control
Product, other for clinical wild poison sample and water control sample;
Fig. 3 is FMCA method specific test result;Red melting curve is HP-PRRSV vaccine strain GDr180 positive reference substance,
Blue melting curve is HP-PRRSV street strain GD positive reference substance, other for CSFV, PRV, PCV2 and PPV sample of nucleic acid and
Water control sample;
Fig. 4 is plasmid positive sample FMCA method sensitivity test result;Wherein A is plasmid p-GDr180 gradient dilution amplification song
Line, B is plasmid p-GDr180 gradient dilution melting curve;C is plasmid p-GD gradient dilution amplification curve, and D is plasmid p-GD ladder
Degree dilution melting curve.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is further illustrated, but is not limited thereto.
The quick differentiation high-pathogenicity porcine reproductive of embodiment 1 one kinds and respiration syndrome live vaccine GDr180 strain and street strain
Primer and probe
1) FMCA primer:
After designed a large amount of primers are screened, find primer base sequences SEQ ID NO:1 and SEQ ID NO:2
The effect that FMCA method is distinguished HP-PRRSV vaccine GDr180 strain and street strain is best, and its base sequence is as follows.
P1:5 '-GGTTGACATGCTGACTTG-3 ' (SEQ ID NO:1),
P2:5 '-CACTGAACCAATGGTGAG-3 ' (SEQ ID NO:2).
2) probe
After designed a large amount of probes are screened, find that probe base sequence SEQ ID NO:3 is to FMCA method district
The effect dividing HP-PRRSV vaccine GDr180 strain and street strain is best, and its base sequence is as follows.
Probe P:5 '-FAM-CCCAAGGATGATTCTCGAGACACCG-BHQ1-3 ' (SEQ ID NO:3), or its nucleoside
Acid reverse complementary sequence.
The quick differentiation high-pathogenicity porcine reproductive of embodiment 2 one kinds and respiration syndrome live vaccine GDr180 strain and street strain
Detection method
The FMCA of standard sample analyzes
1) extraction of PRRSV standard substance nucleic acid:
Take HP-PRRSV vaccine GDr180 strain respectively, add 3mL PBS hydrochloric acid buffer solution and dissolve, take HP-PRRSV open country poison
GD strain cell culture fluid, takes 200 μ L respectively by the MiniBEST Viral RNA/DNA Extraction Kit of TAKARA company
The description of Ver.4.0 carries out nucleic acid extraction.
2) the FMCA operating procedure of positive criteria sample
In order to verify designed primer and the probe distinguishing ability to actual sample, this research and utilization HP-PRRSV vaccine strain
The poison GD strain of GDr180 and HP-PRRSV open country carries out FMCA analysis as standard sample.Extract nucleic acid with above-mentioned standard sample respectively to make
For nucleic acid-templated, carry out FMCA amplified reaction and analysis respectively.
FMCA analyzes:
With reference to PrimeScript One Step RT-PCR Kit product description, prepare FMCA reactant liquor by consisting of.
RT-PCR amplified reaction program is 50 DEG C of reverse transcription 30min;95 DEG C of denaturations 2min;95 DEG C of degeneration 20s, 55 DEG C are moved back
Fire 20s, 72 DEG C extend 20s;Circulate 55 times.
FMCA runs program: 98 DEG C of degeneration 2min, 40 DEG C of heterozygosis 2min, 55 DEG C to 75 DEG C are entered with the melting speed of 1 DEG C/s
Row melting curve analysis.FMCA result of the test LightCycler 96 SW 1.1 software is analyzed.
3) positive criteria sample F MCA interpretation of result
RT-PCR amplified production ROCHE LightCycler 96 analyser is analyzed.
Fig. 1 is GDr180 and GD standard sample FMCA peak type melting curve figure.Analysis result shows, melting curve analysis
Method can distinguish HP-PRRSV vaccine strain GDr180 and HP-PRRSV street strain.Wherein, GDr180 melting peaks Tm value is more a height of
68.42 ± 0.2 DEG C, GD melting peaks Tm value is relatively low is 63.65 ± 0.1 DEG C, both melting peaks Tm obvious differences (about 4.7 DEG C).
The quick differentiation high-pathogenicity porcine reproductive of embodiment 3 one kinds and respiration syndrome live vaccine GDr180 strain and street strain
Method
Clinical sample FMCA analyzes
1) from sample, viral nucleic acid is extracted: taking suspected infection respectively has the Pulmonis Sus domestica dirty tissue sample 2g of HP-PRRSV, adds 3mL
PBS hydrochloric acid buffer solution is ground, and ground homogenate 4000 × g is centrifuged 8 min, and draws centrifuged supernatant
Save backup to-80 DEG C;Or serum sample 200 μ L;Tissue sample homogenate and blood serum sample press TAKARA company
The description of MiniBEST Viral RNA/DNA Extraction Kit Ver.4.0 carries out nucleic acid extraction.
2) with extract viral nucleic acid as template, carry out FMCA analysis with the method described in above-described embodiment 2, result is divided
Analysis method is as follows:
With standard substance GDr180 as positive control, between measuring samples and positive control, the absolute value of melting peaks Tm value is less than
HP-PRRSV vaccine strain GDr180 it is judged to when 1.0 DEG C;
With standard substance GD as positive control, between measuring samples and positive control, the absolute value of melting peaks Tm value is less than 1.0 DEG C
Time be judged to HP-PRRSV street strain.
In order to verify the set up method distinguishing ability to actual clinical sample.Plant of Guangdong Province is collected by the present invention
To 10 parts of HP-PRRSV clinical samples carry out FMCA analysis, analysis result such as Fig. 2, there it can be seen that 10 parts of clinical samples
In detect 1 part of HP-PRRSV vaccine strain GDr180(9 sample altogether) and 9 parts of HP-PRRSV street strains (No. 1-8, No. 10 samples
This).1 part of HP-PRRSV vaccine strain GDr180(9 sample) melting peaks Tm value is 68.38 DEG C, with positive control sample GDr180
Melting peaks Tm value absolute value is less than 1 DEG C (0.04 DEG C), 9 parts of HP-PRRSV street strain (No. 1-8, No. 10 samples) melting peaks Tm values
It it is 64.02 ± 0.36 DEG C, with positive control sample GD melting peaks Tm value absolute value less than 1 DEG C (0.56 ± 0.36 DEG C).
The quick differentiation high-pathogenicity porcine reproductive of embodiment 4 one kinds and respiration syndrome live vaccine GDr180 strain and street strain
Method
Specificity experiments
The FMCA detection method set up the present invention below carries out specific detection.
Extract other Prevention of Common Occurrence Porcine Disease poison nucleic acid respectively, as swine fever virus (classical swine fever virus,
CSFV), PRV (Pseudorabies virus) (Pseudorabies Virus, PRV), pig annulus 2 type (Porcine circovirus type
2, PCV2), the nucleic acid of pig parvoviral (Porcine parvovirus, PPV) be template, described in above-described embodiment 3
Method carry out FMCA analysis, separately verify primer P1/P2 and probe P specificity, simultaneously with HP-PRRSV vaccine strain GDr180
Being positive control with the poison GD strain of HP-PRRSV open country, water carries out FMCA analysis as negative control.
Analysis result is as it is shown on figure 3, can be seen that from figure, and FMCA detection method energy specific amplification of the present invention goes out HP-
PRRSV also forms special melting peaks type, and other common virus, as CSFV, PRV, PCV2 and PPV sample is not all formed special
Melting peaks type, show that probe P that the present invention uses, primer P1/P2 specificity are high, be suitable as FMCA probe and primer.
The quick differentiation high-pathogenicity porcine reproductive of embodiment 5 one kinds and respiration syndrome live vaccine GDr180 strain and street strain
Detection method
Sensitivity test
The detection method set up the present invention below carries out sensitivity technique.
Respectively with HP-PRRSV vaccine strain GDr180 and HP-PRRSV open country poison GD strain Nsp3 gene constructed positive plasmid p-
Genes of interest fragment containing present invention detection in GDr180 and p-GD(Nsp3 gene), positive plasmid is carried out respectively quantitatively and
Carry out FMCA analysis after gradient dilution, detect detection method lowest detectable limit.
Wherein FMCA reaction is with reference to the Ex Taq Version 2.0 description preparation amplified reaction body of TAKARA company
System, for:
Pcr amplification reaction program is 95 DEG C of denaturations 2min;95 DEG C of degeneration 20s, 55 DEG C of annealing 20s, 72 DEG C extend 20s;Circulation
55 times.
Remaining is analyzed according to the method described in above-described embodiment 3.
Fig. 4 is plasmid positive sample FMCA method sensitivity test result;A is plasmid p-GDr180 gradient dilution amplification song
Line, B is plasmid p-GDr180 gradient dilution melting curve;C is plasmid p-GD gradient dilution amplification curve, and D is plasmid p-GD ladder
Degree dilution melting curve, the plasmid concentration that data 1 ~ 10 represent is followed successively by 1.0x109、1.0x108、1.0x107、1.0x106、
1.0x105、1.0x104、1.0x103、1.0x102、1.0x101、1.0x100copies/µL;Figure 4, it can be seen that the present invention
FMCA method is respectively 1.0x10 to positive plasmid p-GDr180 and p-GD lowest detectable limit0Copy/ L and 1.0x100copy/µ
L。
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 HP-PRRS vaccine GDr180 strain and the primer of street strain, probe and method
<130>
<160> 3
<170> PatentIn version 3.5
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ggttgacatg ctgacttg 18
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cactgaacca atggtgag 18
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