CN104830853A - Detection kit for identifying porcine reproductive and respiratory syndrome virus and application thereof - Google Patents

Abstract

The invention relates to a detection kit for identifying a porcine reproductive and respiratory syndrome virus and an application thereof. The kit comprises a pair of PRRSV specific detection primers and a detection system. The detection verifies that the kit is capable of rapidly, conveniently, effectively, high-specifically and high-sensitively detecting the PRRSV; the PRRSV TJM-F92 strain, the American classic type strain and the variant strain (highly pathogenic porcine reproductive and respiratory syndrome virus) can be distinguished through one pair of primers; and the reagent is low in cost, convenient to operate and is suitable for identifying the PRRSV by the import and export quarantine and professional laboratory.

Description

A kind of porcine reproductive and respiratory syndrome virus differentiates detection kit and application

Technical field

The invention belongs to animal virus technical field of molecular biological detection, relate to a kind of differential diagnosis PRRSV TJM-F92 strain, the primer of the classical strain of american type and variant and test kit and application thereof.

Background technology

Pig breeding and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) be commonly called as " pig blue-ear disease ", by PRRSV(Porcine reproductive and respiratory syndrome virus, PRRSV) a kind of transmissible disease that is feature with farrowing sow breeding difficulty and piglet respiratory symptom caused is one of main epidemic disease causing pig farm breeding difficulty at present.First this disease is broken out in the U.S., propagates into all over the world subsequently, causes huge financial loss to world's pig industry.In China, Guo Baoqing equals in the aborted fetus body of Beijing area outbreak of epidemic, to be separated to PRRSV first in 1996, thus confirms the existence of this disease in China.

In recent years, restructuring of constantly developing in PRRSV epidemiological process at home, new epidemic isolates gets more and more, and what these changes brought is that the difficulty of viral prevention and control continues to increase the strengthen continuously with virulence.Through the evolution of nearly 20 years, current domestic epidemic isolates is made to be not merely the classical strain of american type PRRSV.Within 2006, betide the highly pathogenic PRRS of China, the epidemic isolates (variant) of domestic a large amount of highly pathogenic PRRSV is caused to occur, the type strain major part NSP2 genetically deficient 90 bases, occupied more than 70% of domestic isolated strain at present, and himself is also in continuation variation.Simultaneously, the restructuring of the PRRSV gene of different sources causes a large amount of new strains and middle strain to produce, comprise the strain that the recombinant strain between wild poison, the recombinant strain of vaccine strain and street strain and vaccine strain develop and come, finally cause the new strain of PRRSV to emerge in an endless stream.

Because PRRSV Viral isolation method wastes time and energy, the PCR method reported at present can only distinguish the PRRSV of 1-2 type.For carrying out Etiological and research more accurately, obtain more cause of disease information, research and development fast, accurately can differentiate that the RT-PCR detection method of PRRSV classical strains and dissimilar variation strain is imperative.

So far, about the detection of PRRSV, existing patented technology publishes, but the content of these technology and present patent application exists larger difference.Briefing is as follows:

(1) Chinese Patent Application No. is 200710076592.5, and disclose a kind of primer for detecting highly pathogenic PRRSV nucleotide fragments and probe sequence, sequence is:

Upstream primer PVpf:5 '-CCCAAGCTGATGACACCTTT-3 '

Downstream primer PVpr:5 '-AATCCAGAGGCTCATCCTGGT-3 '

Probe PVpb:5 '-CGCGTAGAACTGTGACAACAACGCTGA-3 '

Whether the method uses fluorescence quantifying PCR method can detect containing highly pathogenic PRRSV in sample, but cannot detect whether contain classical strain.

(2) Chinese Patent Application No. is 200810032141.6, and disclose a kind of highly pathogenic PRRSV RT-PCR quick detection kit, primer sequence is:

PrimerP1：5’-CGTAGAACTGTGACAACAAC-3’

PrimerP2：5’-TGAGTATTTTGGGCGTGTGAT-3’

The method uses single stage method PCR method can detect in sample whether contain highly pathogenic PRRSV, cannot judge whether containing the classical strain of PRRSV.

(3) Chinese Patent Application No. is 200910077704.8 disclose the classical strain of PRRSV and variant dual real-time fluorescent RT differentiates detection method.

The primer and the probe that detect the classical strain of PRRS are:

Upstream primer PRRSV-F2:5 '-CGCACCAGATGGRACCTACTT-3 '

Downstream primer PRRSV-R3:5 '-ACGGTGTTCAGTGAGGGCTTT-3 '

Probe PRRSV-P:5 '-CCAGTCAACGCAGCG-3 '

The primer and the probe that detect the classical strain of PRRS are:

Upstream primer H-PRRSV-F2:5 '-AGTGGGTCGGCACCAGTT-3 '

Downstream primer H-PRRSV-R12:5 '-GCAGACAAATCCAGAGGCTCAT-3 '

Probe H-PRRSV-P:5 '-CGTAGAACTGTGACAAC-3 '

Whether the method primary first-order equation can differentiate the classical strain of PRRSV and variant, but cannot distinguish in sample containing PRRSVTJM-F92 strain.

(4) Chinese Patent Application No. 201310189880.7 discloses the RT-PCR kit detecting the popular virus of pig blue-ear disease and the Tianjin strain of HP-PRRS virus.Primer sequence is:

Upstream primer Pa:5 '-CGTCCGTGTCATCAAGCAGCTC-3 '

Downstream primer Pc:5 '-AAAAGAGGCACAATAGAGTAAAAGG-3 '

Whether the method primary first-order equation just can detect containing this pathogenic PRRSV Tianjin strain (TJ strain) and the popular virus of PRRSV (comprising the classical strain of highly pathogenic PRRSV Strain in Jiangxi, Strain Hunan and PRRSV) in sample, but PRRSV variant (comprising highly pathogenic PRRSV Strain in Jiangxi, Strain Hunan and similar strain thereof) and the classical strain of PRRSV can not be differentiated by the method further.

In sum, whether above-mentioned four kinds of patented methods all can not detect containing PRRSV classical strains, TJM-F92 strain and other high-pathogenicity porcine reproductive and variant in sample through primary first-order equation simultaneously, and three are differentiated.

Summary of the invention

The invention discloses a kind of PRRSV and differentiate detection kit, the primer of this test kit is different from and four of above-mentioned announcement patents, only need an amplified reaction whether just can detect in sample containing PRRSV TJM-F92, classical strain and variant (comprising high-pathogenicity blue ear disease virus) simultaneously, its reagent cost is low, simple operation, practical, be suitable for basic unit and specialized laboratory to the Rapid identification of virus strain.

Therefore, the invention provides a kind of discriminating Auele Specific Primer group for american type PRRSV TJM-F92 strain, classical strain and variant, it comprises:

PRRS-F 5’-TTCCTGCACCGCGTAGAACTGTG-3’ SEQ ID NO：1

PRRS-R 5’-CTATTTTCCCGAGGATGCGTGGA-3’ SEQ ID NO：2

The test kit that the Auele Specific Primer group that the present invention further discloses and adopts american type PRRSV TJM-F92 strain, the discriminating of classical strain and variant (comprising high-pathogenicity blue ear disease virus) detects is made, is characterized in that the composed as follows of it:

(1) PCR reaction solution (PB buffer) 700 μ L

Contain in PCR reaction solution: 10 × PCR(Mg ²⁺plus) buffer 2.5 μ L, dNTPs (2.5mM) 2.0 μ L, PRRS-F(100 pmol/ μ L) 1.0 μ L, PRRS-R(100 pmol/ μ L) 1.0 μ L

(2) ultrapure water 1.5mL

(3) taq enzyme (5U/ μ L) 60 μ L

(4) primer wherein refers to:

PRRS-F 5’-TTCCTGCACCGCGTAGAACTGTG-3’ SEQ ID NO：1

PRRS-R 5’-CTATTTTCCCGAGGATGCGTGGA-3’ SEQ ID NO：2

The present invention further discloses the using method of the discriminating detection kit for detecting PRRSV, comprises the steps:

(1) amplification reaction system: the cumulative volume of amplified reaction is 25 μ L, its various composition and final concentration are respectively: PCR reaction solution (PB buffer) 6.5 μ L, taq enzyme (5U/ μ L) 0.5 μ L, measuring samples cDNA 3 ~ 7 μ L, ultrapure water 11 ~ 15 μ L.

(2) amplified reaction process: 94 DEG C of denaturation 5 min; 94 DEG C of 30 s, 59 DEG C of 30 s, 72 DEG C of 30 min, carry out 30 circulations; 72 DEG C extend 10 min, and 4 DEG C are terminated reaction.

(3) amplified production detects: amplified reaction terminates, and gets amplified production 5 ~ 7 μ L and carries out gel electrophoresis in 1% agarose, voltage 100V, time 20 ~ 30min, electrophoretic image judged result.

(4) result judges:

1) when the clip size of amplified production is 717bp, containing the classical strain of PRRSV in sample;

2) when the clip size of amplified production is 628bp, containing PRRSV variant in sample;

3) when the clip size of amplified production is 267bp, containing PRRSV TJM-F92 strain in sample.

4) when without amplified production or when there is the amplified band of other clip size, sample detection result is negative.

5) as containing above 2 ~ 3 kinds of amplified fragments, then 2 ~ 3 kinds of virus strain may be contained in sample.

The positively effect had for PRRSV differential diagnosis kit disclosed by the invention is:

(1) the present invention has sensitivity, special, the many advantages such as favorable repeatability, only has amplified reaction to the nucleic acid of PRRSV, and to Pestivirus suis, PRV (Pseudorabies virus), pig parvoviral etc. without amplification, the susceptibility of test kit can reach 10 ²tCID ₅₀.Test kit is with low cost, simple operating steps, is applicable to apply.

(2) conventional PCR method generally can only differentiate a kind of PRRSV, and the present invention uses one group of primer can differentiate the virus of american type PRRSV TJM-F92 strain, classical strain and variant 3 type, testing cost and the detection time of virus strain qualification therefore can be reduced.Only need a reaction can identify three kinds of PRRSV simultaneously, highly sensitive, high specificity, with low cost, there is applications well prospect.

(3) the present invention provides new technique means for China PRRSV detects to a certain extent, can be used for the inspection and quarantine of China live pig intermediate links PRRSV, the epidemic situation monitoring of cultivating link and rapid differential diagnosis and pig farm to the purification of this epidemic disease, significant to the prevention and control level of the PRRSV improving China pig farm.

Accompanying drawing explanation

Fig. 1 is test kit sensitivity test result figure; From left to right, M:100bp Ladder is followed successively by; 1: blank; 2:10 ⁵tCID ₅₀pRRSV; 3:10 ⁴tCID ₅pRRSV ₀; 4:10 ³tCID ₅₀pRRSV; 5:10 ²tCID ₅₀pRRSV; 6:10 ¹tCID ₅₀pRRSV; 7:10 ⁰tCID ₅₀pRRSV;

Fig. 2 is test kit specific outcome figure; From left to right, M:100bp Ladder is followed successively by; 1: porcine circovirus 2 type (PCV2); 2: swine influenza virus (SIV); 3: pig parvoviral (PPV); 4: PRV (Pseudorabies virus) (PRV); 5: Pestivirus suis (CSFV); 6: streptococcus suis 2-type (SS2); 7: intestinal bacteria (E.coli); 8: actinobacillus pleuropneumoniae (App); 9: haemophilus parasuis (HPS); 10: eggs crack detection (P. Multocida); 11: streptococcus aureus (S. aureus); 12:PRRSV CH1R strain; 13: blank;

Fig. 3 is for differentiating the classical strain of american type PRRSV, variant and TJM-F92 strain RT-pcr amplification test-results figure; From left to right, M:100bp Ladder is followed successively by; 1:PRRSV VR2332 strain; 2:PRRS vaccine R98 strain (Rui Pu Biotechnology Ltd.); 3:PRRS vaccine CH1R strain (Pulaike Biological Engineering Co., Ltd.); 4:PRRS vaccine CH1R strain (Shanghai Hai Li Biotechnology Ltd.); 5:PRRS vaccine CH1R strain (Harbin Wei Ke biotechnology development company); 6:PRRS vaccine HUN4-F112 strain (Jilin decent job biological products limited-liability company); 7:PRRS vaccine JXA1R strain (Jilin Jing Xiang animal pharmaceutical estate company limited); 8:PRRS vaccine JXA1R strain (Wuhan Chopper Biology Co., Ltd.); 9:PRRS vaccine TJM-F92 strain (Xinjiang Tiankang Raise Livestock Biotechnology Co., Ltd, externally on sale); 10: blank.

Embodiment

In order to explain implementation method of the present invention more fully, provide the embodiment differentiating detection kit for pig PRRSV.These embodiments are only explain instead of limit the scope of the invention.Reagent raw material wherein used all has commercially available.PRRSV of the present invention differentiates that detection kit primer sets sequence is in table 1.

Table 1

Embodiment 1

Test kit sensitivity test

The RNA of 1, PRRSV TJM-F92 strain (originating commercially available) extracts:

Be 10 by the PRRSV TJM-F92 strain gradient dilution of cultivation ⁵tCID ₅₀, 10 ⁴tCID ₅, 10 ³tCID ₅₀, 10 ²tCID ₅₀, 10 ¹tCID ₅₀, 10 ⁰tCID ₅₀.Application Trizol method extracts viral RNA,

Method is as follows.

(1) every 200 μ L sample solutions add 600 μ L Trizol Reagent, and vortex oscillation 15s, room temperature leaves standstill 5min.

(2) add the chloroform of 200 μ L in centrifuge tube, turn upside down after mixing, room temperature leaves standstill 5min.

(3) 12000rpm 4 DEG C of centrifugal 10min, get supernatant liquid and are placed in new centrifuge tube (during absorption, the careful not wrong albumen inhaled in point aspect, againsts an ancient piece of jade, round, flat and with a hole in its centre as far as possible and inhale, change a rifle head each time).Add 800 μ L Virahols (containing 0.01%DEPC), turn upside down 1 ~ 2 time after lid lid, again mark to new centrifuge tube, room temperature places 15min.

(4) 12000rpm 4 DEG C of centrifugal 10min, supernatant discarded, tips upside down on thieving paper and sucks remaining liq, and precipitation is the RNA of extraction.

(5) washing of RNA: add ethanol (containing 0.01%DEPC) the 500 μ L of 75%, mixing of turning upside down after airtight, 12000rpm 4 DEG C of centrifugal 10min.Supernatant discarded, back-off sucks remaining liq on thieving paper.

(6) drying of RNA: brief centrifugation with centrifugal go out unnecessary 75% ethanol, inclination centrifuge tube, carefully adherently sucks remaining ethanol, and uncap the 3min that dries in the air.

(7) dissolving of RNA: add 20 μ LDEPC water dissolution RNA, directly do after mark next step test or-80 DEG C save backup.

2, cDNA synthesis

Adopt the cDNA synthetic system of 20.0 μ L, i.e. PRRSV RNA template 11 μ L, 5 × RT Buffer 5.0 μ L, Random Primers(50pmol/ μ L) 1.0 μ L, dNTP Mixture 2.0 μ L, Rnase Inhibitor(40 U/ μ L) 0.5 μ L, MLV Reverse Transcriptase XL(5U/ μ L) 0.5 μ L.

Response procedures: 25 DEG C of 10 min, 42 DEG C of 60 min, 99 DEG C of 5 min, 4 DEG C of 5 min, after cDNA end of synthesis, carry out pcr amplification or-20 DEG C save backup.

3, PCR reaction amplification: detection kit carries out pcr amplification to use PRRSV to differentiate.

The cumulative volume of amplified reaction is 25 μ L, and its various composition and final concentration are respectively: PCR reaction solution (PB buffer) 6.5 μ L, taq enzyme (5U/ μ L) 0.5 μ L, measuring samples cDNA 5 μ L, ultrapure water 13 μ L.

Response procedures: 94 DEG C of denaturation 5 min; 94 DEG C of 30 s, 59 DEG C of 30 s, 72 DEG C of 30 min, carry out 30 circulations; 72 DEG C extend 10 min, and 16 DEG C are terminated reaction.

4, electrophoresis: amplified reaction terminates, gets amplified production 5 μ L and carries out gel electrophoresis in 1% agarose, voltage 100V, time 20 ~ 30min, electrophoretic image judged result.

5, result judges: carry out RT-pcr amplification to PRRSV TJM-F92 strain, and result display TJM-F92 strain can increase 10 ²tCID ₅₀virus, is shown in Fig. 1.

Embodiment 2

Test kit specific test:

1, control strain table 2, comprising:

2, virus and DNA of bacteria are extracted: the cell/tissue DNA extraction kit adopting Tian Gen biotech firm, and method is with reference to specification sheets.

(1) get bacterium liquid 1ml 12,000rpm centrifugal 5 minutes, remove supernatant, add 200 μ L GA solution in precipitation, resuspended mixing.

(2) 20 μ L Proteinase K Solution are added, mixing.65 DEG C of water-baths 1 hour, period put upside down biased sample 2-3 time.Take out from water-bath, brief centrifugation is to remove the globule of cap wall.

(3) add 200 μ L damping fluid GB, fully put upside down mixing, place 10 minutes for 70 DEG C, solution strain is limpid, and brief centrifugation is to remove the globule of cap wall.

(4) add people 200 μ L dehydrated alcohol, fully vibration mixing 15 seconds, now may occur flocks, brief centrifugation is to remove the globule of cap wall.

(5) all add in an adsorption column CB3 (adsorption column puts into collection tube) by previous step gained solution and flocks, centrifugal 30 seconds of 12,000rpm, outwells waste liquid, is put back in collection tube by adsorption column CB3.

(6) in adsorption column CB3, add 500 μ L damping fluid GD (please first check whether before use and added dehydrated alcohol), centrifugal 30 seconds of 12,000rpm, outwells waste liquid, adsorption column CB3 is put into collection tube.

(7) in adsorption column CB3, add 700 μ L rinsing liquid PW (please first check whether before use and added dehydrated alcohol), centrifugal 30 seconds of 12,000rpm, outwells waste liquid, adsorption column CB3 is put into collection tube.

(8) in adsorption column CB3, add 500 μ L rinsing liquid PW, centrifugal 30 seconds of 12,000rpm, outwells waste liquid.

(9) put back in collection tube by adsorption column CB3, centrifugal 2 minutes of 12,000rpm, outwells waste liquid.Adsorption column CB3 is placed in room temperature and places 3 minutes, thoroughly to dry rinsing liquid remaining in sorbing material.

(10) adsorption column CB3 is proceeded in a clean centrifuge tube, the TE(80 DEG C of water-bath preheating of the unsettled dropping in middle part to adsorption film 50-200 μ L elution buffer), room temperature places 3 minutes, and 12, centrifugal 2 minutes of 000rpm, by solution collection in centrifuge tube.

(11) add in adsorption column CB3 again by the centrifugal solution obtained, room temperature places 2 minutes, and centrifugal 2 minutes of 12,000rpm, by solution collection in centrifuge tube.

3, the extraction of viral RNA: carry out RNA extraction according to Trizol method, concrete with reference to example 1.

4, cDNA synthesis

Adopt the cDNA synthetic system of 20.0 μ L, i.e. PRRSV RNA template 11 μ L, 5 × RT Buffer 5.0 μ L, Random Primers(50pmol/ μ L) 1.0 μ L, dNTP Mixture 2.0 μ L, Rnase Inhibitor(40 U/ μ L) 0.5 μ L, MLV Reverse Transcriptase XL(5U/ μ L) 0.5 μ L.

Response procedures: 25 DEG C of 10 min, 42 DEG C of 60 min, 99 DEG C of 5 min, 4 DEG C of 5 min, after cDNA end of synthesis, carry out pcr amplification or-20 DEG C save backup.

5, PCR reaction amplification

The cumulative volume of amplified reaction is 25 μ L, and its various composition and final concentration are respectively: PCR reaction solution (PB buffer) 6.5 μ L, taq enzyme (5U/ μ L) 0.5 μ L, measuring samples cDNA 3 μ L, ultrapure water 15 μ L.

Response procedures: 94 DEG C of denaturation 5 min; 94 DEG C of 30 s, 59 DEG C of 30 s, 72 DEG C of 30 min, carry out 30 circulations; 72 DEG C extend 10 min, and 4 DEG C are terminated reaction.

6, electrophoresis:

Amplified reaction terminates, and gets amplified production 5 μ L and carries out gel electrophoresis in 1% agarose, voltage 100V, time 20 ~ 30min, electrophoretic image judged result.

7, result judges:

(1) when the clip size of amplified production is 717bp, containing the classical strain of PRRSV in sample;

(2) when the clip size of amplified production is 628bp, containing PRRSV variant in sample;

(3) when the clip size of amplified production is 267bp, containing PRRSV TJM-F92 strain in sample.

(4) when without amplified production or when there is the amplified band of other clip size, sample detection result is negative.

(5) as containing above 2 ~ 3 kinds of amplified fragments, then 2 ~ 3 kinds of virus strain may be contained in sample.

Result illustrates: as can be seen from Figure 2, only have PRRSVCH1R strain to amplify object fragment (717bp), and blank and other contrast strains all occur without band.Therefore, the test kit of this patent has good specificity, contrast bacterium (poison) strain can not increased except PRRSV.

Embodiment 3

Differentiate the classical strain of PRRSV american type, variant and the test of TJM-F92 strain RT-pcr amplification

1, control strain table 3, comprising:

2, the extraction of porcine reproductive and respiratory syndrome virus RNA: carry out according to Trizol method, reference example 1.

3, cDNA synthesis

Adopt the cDNA synthetic system of 20.0 μ L, i.e. PRRSV RNA template 11 μ L, 5 × RT Buffer 5.0 μ L, Random Primers(50pmol/ μ L) 1.0 μ L, dNTP Mixture 2.0 μ L, Rnase Inhibitor(40 U/ μ L) 0.5 μ L, MLV Reverse Transcriptase XL(5U/ μ L) 0.5 μ L.

Response procedures: 25 DEG C of 10 min, 42 DEG C of 60 min, 99 DEG C of 5 min, 4 DEG C of 5 min, after cDNA end of synthesis, carry out pcr amplification or-20 DEG C save backup.

4, PCR reaction amplification: detection kit carries out pcr amplification to use porcine reproductive and respiratory syndrome virus to differentiate.

The cumulative volume of amplified reaction is 25 μ L, and its various composition and final concentration are respectively: PCR reaction solution (PB buffer) 6.5 μ L, taq enzyme (5U/ μ L) 0.5 μ L, measuring samples cDNA 5 μ L, ultrapure water 11 ~ 13 μ L.

Response procedures: 94 DEG C of denaturation 5 min; 94 DEG C of 30 s, 59 DEG C of 30 s, 72 DEG C of 30 min, carry out 30 circulations; 72 DEG C extend 10 min, and 16 DEG C are terminated reaction.

5, electrophoresis: amplified reaction terminates, gets amplified production 5 μ L and carries out gel electrophoresis in 1% agarose, voltage 100V, time 20 ~ 30min, electrophoretic image judged result.

6, result judges:

(1) when the clip size of amplified production is 717bp, containing the classical strain of PRRSV in sample;

(2) when the clip size of amplified production is 628bp, containing PRRSV variant in sample;

(3) when the clip size of amplified production is 267bp, containing PRRSVTJM-F92 strain in sample.

(4) when without amplified production or when there is the amplified band of other clip size, sample detection result is negative.

(5) as containing above 2 ~ 3 kinds of amplified fragments, then 2 ~ 3 kinds of virus strain may be contained in sample.

3, result illustrates:

As can be seen from Figure 3, all there is 717bp amplified band in PRRSV CH-1R strain, R98 strain, VR2332 strain, is the classical strain of PRRSV; All there are 628 bp amplified bands in PRRSV JXA1R strain, HUN4-F112 strain, is PRRSV variant; There is 267bp amplified band in PRRSV TJM-F92.

Therefore, the primer of this patent and test kit is used can to identify the classical strain of PRRSV, variant and TJM-F92 strain.

After the preferred embodiment described in detail, be familiar with this technology personage can be well understood to, do not departing under above-mentioned claim and spirit and can carry out various change and amendment, all above embodiment is done according to technical spirit of the present invention any simple modification, equivalent variations and modification, all belong to the scope of technical solution of the present invention.And the present invention is not also by the restriction of example embodiment in specification sheets.

SEQUENCE LISTING

<110> Xinjiang Tiankang Raise Livestock Biotechnology Co., Ltd, Tianjin City Livestock Raising and Veterinary Inst.

<120> porcine reproductive and respiratory syndrome virus differentiates detection kit and application

 

<160> 2

 

<170> PatentIn version 3.5

 

<210> 1

<211> 23

<212> DNA

<213> artificial sequence

 

<400> 1

ttcctgcacc gcgtagaact gtg 23

 

<210> 2

<211> 23

<212> DNA

<213> artificial sequence

 

<400> 2

ctattttccc gaggatgcgt gga 23

 

Claims (5)

1., for the Auele Specific Primer that porcine reproductive and respiratory syndrome virus is differentiated, it is characterized in that it is made up of following primer:

PRRS-F TTCCTGCACCGCGTAGAACTGTG SEQ ID NO：1

PRRS-R CTATTTTCCCGAGGATGCGTGGA SEQ ID NO：2 。

2. contain the detection kit for the Auele Specific Primer group of porcine reproductive and respiratory syndrome virus discriminating described in claim 1, it is characterized in that the composed as follows of it:

(1) PCR reaction solution (PB buffer) 700 μ L;

Contain in PCR reaction solution: 10 × PCR(Mg ²⁺plus) buffer 2.5 μ L, dNTPs (2.5mM) 2.0 μ L, PRRS-F(100 pmol/ μ L) 1.0 μ L, PRRS-R(100 pmol/ μ L) 1.0 μ L

(2) ultrapure water 1.5mL;

(3) taq enzyme (5U/ μ L) 60 μ L;

Primer wherein refers to:

PRRS-F TTCCTGCACCGCGTAGAACTGTG SEQ ID NO：1

PRRS-R CTATTTTCCCGAGGATGCGTGGA SEQ ID NO：2 。

3. adopt test kit described in claim 2 for a discriminating detection method for the classical strain of porcine reproductive and respiratory syndrome virus, variant, TJM-F92 strain, it is characterized in that being undertaken by following step:

(1) amplification reaction system: the cumulative volume of amplified reaction is 25 μ L, its various composition and final concentration are respectively: PCR reaction solution (PB buffer) 6.5 μ L, taq enzyme (5U/ μ L) 0.5 μ L, measuring samples cDNA 3 ~ 7 μ L, ultrapure water 11 ~ 15 μ L;

(2) amplified reaction process: 94 DEG C of denaturation 5 min; 94 DEG C of 30 s, 59 DEG C of 30 s, 72 DEG C of 30 min, carry out 30 circulations; 72 DEG C extend 10 min, and 4 DEG C are terminated reaction;

(3) amplified production detects: amplified reaction terminates, and gets amplified production 5 ~ 7 μ L and carries out gel electrophoresis in 1% agarose, voltage 100V, time 20 ~ 30min, according to electrophoretic image judged result.

4. described in claim 1 for the application of Auele Specific Primer group in differentiating for the preparation of the classical strain of PRRSV, variant, TJM-F92 strain that porcine reproductive and respiratory syndrome virus is differentiated.

5., according to claim 3 for the discriminating detection kit of the classical strain of porcine reproductive and respiratory syndrome virus, variant, TJM-F92 strain, it is characterized in that described variant is the highly pathogenic PRRSV strain that is representative with Strain in Jiangxi (JXA1R strain) and Strain Hunan (HUN4-F112 strain).