CN103498008B - Method and kit for detecting porcine reproductive and respiratory syndrome virus live vaccine strain JXA1-R - Google Patents
Method and kit for detecting porcine reproductive and respiratory syndrome virus live vaccine strain JXA1-R Download PDFInfo
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- CN103498008B CN103498008B CN201310455113.6A CN201310455113A CN103498008B CN 103498008 B CN103498008 B CN 103498008B CN 201310455113 A CN201310455113 A CN 201310455113A CN 103498008 B CN103498008 B CN 103498008B
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Abstract
The invention discloses a method and a kit for detecting a porcine reproductive and respiratory syndrome virus live vaccine strain JXA1-R. The detection method comprises the following steps of designing a primer for synthesizing and amplifying a 3,195th to 3,682nd nucleotide fragment of an ORF1a of a strain JAX1-R by using an RFLP (restriction fragment length polymorphism) method, extracting RNA (ribonucleic acid) from a sample to be detected, amplifying the 3,195th to 3,682nd nucleotide fragment of the ORF1a of the strain JAX1-R by using an RT-PCR (reverse transcription-polymerase chain reaction) method, cutting an amplification product by using restriction endonuclease SacII, performing agarose gel electrophoresis on the product, and when a 487bp band exists, determining that the sample to be detected contains the porcine reproductive and respiratory syndrome virus live vaccine strain JXA1-R. The kit mainly comprises the primer and the restriction endonuclease SacII. According to the method and the kit, the method can be implemented through ordinary PCR and conventional apparatuses by simple steps, the detection time is shortened, and the detection cost is remarkably lowered.
Description
Technical field
The present invention relates to technical field of molecular biological detection, be specifically related to a kind of method and the test kit that detect pig blue-ear disease living vaccine JXA1-R strain.
Background technology
Highly pathogenic PRRS is (also known as porcine reproductive and respiratory syndrome virus by highly pathogenic pig blue-ear disease poison, porcine reproductive andrespiratory syndrome virus, PRRSV) the serious breeding difficulty of sow, wean pig growth retardation and the communicable disease of death that cause.This disease is since outburst in 06 year, huge financial loss is caused to domestic pig industry, current most pig farm uses PRRSV variant (JXA1-R) attenuated vaccine (i.e. pig blue-ear disease living vaccine JXA1-R strain, or abbreviation JXA1-R vaccine strain) to prevent this disease.Highly pathogenic PRRS and common pig blue-ear disease can use RT-PCR(reverse transcriptase polymerase chain reaction) carry out discriminating and detect, but JXA1-R vaccine strain and street strain cannot with existing PCR(polymerase chain reactions) method carries out discriminating and detects.JXA1-R vaccine strain can be bred and produce viremia in pig body, and this causes very large interference to the laboratory diagnosis of highly pathogenic PRRS, and the highly pathogenic PRRS poison being difficult to determine to detect is vaccine virus or wild poison.
Current existing JXA1-R vaccine strain differentiates that detection method has following two kinds:
Method one is the high-pathogenicity porcine reproductive and respiration syndrome living vaccine (JXA1-R strain) real-time fluorescence quantitative RT-PCR detection reagent kit that use Anheal Laboratories Co., Ltd to produce, based on fluorescent quantitative PCR technique, quantitative real time PCR Instrument reacts, when there being specific amplification curve, then be judged to be high-pathogenicity porcine reproductive and respiration syndrome vaccine poison (JXA1-R vaccine strain) positive, otherwise be judged to be feminine gender, general needs completes experiment in 3 hours.The core technology of the method comprises reaction system and response procedures two portions, and the key of reaction system is the nucleotide sequence of primer and probe and the configuration proportion with enzyme and salt thereof, and the ratio that all ingredients must be recommended in it be prepared; The key of response procedures controls best annealing and extension time well.Although the method is accurately quick, owing to testing required detection kit and quantitative real time PCR Instrument costly, cause testing cost higher.
Method two adopts sequencing, based on the clone of Nucleotide and sequential detection technology, is routine sequence analysis method.The reagent that the method needs comprises specificity amplification primer, nucleic acid extraction kit, general PCR kit, Nucleic acid purification kits, PMD-18T support agent box and DH5 α intestinal bacteria, the instrument needed comprises PCR instrument, whizzer, ultraviolet transilluminator, constant incubator, concrete operation step is the gene-specific fragments first using primer amplified measuring samples nucleic acid, goal gene fragment purification rear clone is entered PMD-18T carrier, again the carrier of having cloned is sent to order-checking company to check order, finally will record sequence and high-pathogenicity porcine reproductive and respiration syndrome vaccine poison (JXA1-R vaccine strain) to compare, higher with vaccine virus nucleic acid homology, and special site does not morph, then be judged to be JXA1-R strain, detect and once generally need 7 ~ 10 day time.There is the problem that cost is high equally, sense cycle is also longer in addition.
Summary of the invention
In order to solve the aforementioned problems in the prior, the invention provides a kind of method of simple and quick detection pig blue-ear disease living vaccine JXA1-R strain.
The full-length genome of PRRSV is about 15kb, and containing 9 open reading frame (ORFs), order is followed successively by 5 '-ORF1a-ORF1b-ORF2a-ORF2b-ORF-(3-7) 3 '.Each reading frame and adjacent reading frame all have a small amount of overlapping.Other strain nucleotide's sequences of the PRRSV that the NCBI that (brings into use JXA1-R strain attenuated vaccine for 2010) by JXA1-R vaccine strain and before 2010 by BLAST software delivers are compared, and find JXA1-R vaccine strain specific deletion restriction endonuclease in its ORF1a 3195 ~ 3682 gene orders
sacthe restriction enzyme site of II, the present invention utilizes restriction fragment length polymorphism (Restriction Fragment Length Polymorphism, RFLP) analytical technology principle, by the restriction enzyme site of this special disappearance, in conjunction with RT-PCR(Reverse Transcription-Polymerase Chain Reaction, reverse transcription PCR) method, carry out discriminating to highly pathogenic pig blue-ear disease JXA1-R vaccine strain to detect, accurately can differentiate JXA1-R vaccine strain and other PRRSV infection samples.
Concrete technical scheme of the present invention is as follows:
A kind of method detecting pig blue-ear disease living vaccine JXA1-R strain, adopt rflp analysis method, the primer of design and synthesis amplification pig blue-ear disease living vaccine JXA1-R strain ORF1a 3195 ~ 3682 nucleotide fragments, extract the RNA in measuring samples, pass through RT-PCR, by described primer amplification JXA1-R strain ORF1a 3195 ~ 3682 nucleotide fragments, amplified production and restriction endonuclease
saciI carries out endonuclease reaction, and digestion products occurs that after agarose gel electrophoresis the band of length 487bp is judged as in measuring samples containing pig blue-ear disease living vaccine JXA1-R strain.
As a kind of preferred version, in the method for above-mentioned detection pig blue-ear disease living vaccine JXA1-R strain, described RT-PCR is One step RT-PCR, and the nucleotide sequence of described primer is as shown in SEQ ID NO:1 ~ 2.
Present invention also offers a kind of test kit detecting pig blue-ear disease living vaccine JXA1-R strain, this test kit comprises the primer nucleotide sequences shown in SEQ ID NO:1 ~ 2 and restriction endonuclease
saciI.
As a kind of preferred version, the test kit of above-mentioned detection pig blue-ear disease living vaccine JXA1-R strain, is made up of One step RT-PCR reaction solution and endonuclease reaction liquid two group reagent, wherein:
One step RT-PCR reaction solution: the PCR damping fluid 5 μ L of 10 times of dilutions, the ThermoScript II 1 μ L of the archaeal dna polymerase 1 μ L of 5U/ μ L, 200U/ μ L, the RNA enzyme inhibitors 1 μ L of the dNTPs1 μ L of 10mM, 200U/ μ L, the MgCl of 25mM
28 μ L, the SEQ ID NO:1 of 20 μMs and each 1 μ L of the primer nucleotide sequences shown in SEQ ID NO:2, and without RNA enzyme aqua sterilisa 28 μ L;
Endonuclease reaction liquid: 0.1% (w/v) BSA(bovine serum albumin) 2 μ L, the endonuclease reaction damping fluid 2 μ L of 10 times of dilutions, restriction endonuclease
saciI 1 μ L, sterilizing distilled water 5 μ L;
Described PCR damping fluid is by Tris-HCl(trishydroxymethylaminomethane hydrochloride), KCl and MgCl
2mixed aqueous solution composition, pH value is 8.5, and wherein Tris-HCl concentration is 100mM, KCl concentration is 500nM, MgCl
2concentration is 15nM;
Described endonuclease reaction damping fluid is made up of the mixed aqueous solution of magnesium acetate, dithiothreitol (DTT) and Potassium ethanoate, and wherein the concentration of magnesium acetate is 100mM, and the concentration of dithiothreitol (DTT) is 5mM, and the concentration of Potassium ethanoate is 660mM.
The present invention also provides the method detecting pig blue-ear disease living vaccine JXA1-R strain with the test kit of above-mentioned detection pig blue-ear disease living vaccine JXA1-R strain, be use described One step RT-PCR reaction solution to increase after measuring samples is extracted RNA, RT-PCR response procedures is: 50 DEG C 30 minutes; 94 DEG C 2 minutes; 94 DEG C 45 seconds, 55 DEG C 45 seconds, 72 DEG C 1 minute, totally 40 circulations; 72 DEG C, extend 8 ~ 10 minutes; Getting part amplified production joins in endonuclease reaction liquid, reacts 1 hour at 37 DEG C; Termination reaction is got digestion products and is carried out agarose gel electrophoresis, and by observation electrophoretic band of taking pictures, the fragment if there is 487bp length is judged as in measuring samples containing pig blue-ear disease living vaccine JXA1-R strain.
Compared with prior art, the present invention has following beneficial effect:
Compared with fluorescence quantifying PCR method, the present invention mainly have employed regular-PCR method, and the endonuclease reaction eventually through special site is differentiated to detect wild poison and vaccine degree, and agents useful for same consumptive material relative price is cheap, and testing cost is lower.
Compared with sequence measurement, the present invention does not need to carry out cloning and sequencing to PCR primer again after PCR reaction, and only need application enzymatic cleavage methods to obtain result, efficiently easy and simple to handle, cost is low.
Accompanying drawing explanation
Fig. 1 is that the RT-PCR product of different virus strain is through restriction endonuclease
sacthe restriction enzyme digestion and electrophoresis figure of II, wherein " the moon " is negative control, 1 is high-pathogenicity blue ear disease JXA1 street strain, 2 is Bo Linge porcine reproductive and respiratory syndrome living vaccine (VR-2332 strain), 3 is porcine reproductive and respiratory syndrome strain SB strain, 4 is swine Fever Vaccine (swine fever prestige), 5 is transmissible gastro-enteritis virus KY strain, 6 is JXA1-R vaccine strain, M is the standard nucleic acid mark of the DL2000 that the precious biotech firm of TAKARA produces, and is 100bp, 250bp, 500bp, 750bp, 1000bp and 2000bp nucleic acid bands from bottom to up respectively.
Embodiment
Explain the present invention further below in conjunction with specific embodiment, to make those skilled in the art can understand the present invention better and can be implemented, but embodiment is not as a limitation of the invention.Except specified otherwise, be this area routine techniques means.
" N × " described in embodiment represents dilution N doubly, and wherein N is natural number.
Experiment material and reagent source: JXA1-R vaccine strain, swine Fever Vaccine (swine fever prestige) are purchased from Guangdong great Hua Nong animal health-care product stock company; Bo Linge porcine reproductive and respiratory syndrome living vaccine (VR-2332 strain) is purchased from Berlin, Guangzhou agricultural-food company limited; High-pathogenicity blue ear disease JXA1 street strain nucleic acid is presented by Guangdong great Hua Nong animal health-care product stock company; Transmissible gastro-enteritis virus KY strain is by this laboratory separation and Culture.Restriction endonuclease
saciI purchased from the precious biotech firm of TAKARA.
embodiment 1
Complete JXA1-R vaccine strain and differentiate that detection is mainly divided into two steps to realize.
The first step: vaccine and virus liquid RT-PCR
(1) process of vaccine and virus liquid and nucleic acid extraction:
For subsequent use after the dilution of vaccine 5mL sterilizing distilled water; For subsequent use after virus liquid sterilizing distilled water 5 times dilution.
The vaccine handled well and virus liquid body fluid viral DNA/RNA pillar method in a small amount extraction agent box (purchased from the precious biotech firm of TAKARA, also can use other method for extracting nucleic acid) extract nucleic acid, save backup.
(2) nucleic acid RT-PCR:
The amplimer of RT-PCR is as follows:
Upstream primer S66:5 '-TCTCCCAAAGATGATTCTCGAG-3 ' (SEQ ID NO:1);
Downstream primer S88:5 '-AATCACCCGGAGAATAACCAC-3 ' (SEQ ID NO:2).
One step RT-PCR reaction solution: the PCR damping fluid 5 μ L of 10 times of dilutions, the ThermoScript II 1 μ L of the archaeal dna polymerase 1 μ L of 5U/ μ L, 200U/ μ L, the RNA enzyme inhibitors 1 μ L of the dNTPs1 μ L of 10mM, 200U/ μ L, the MgCl of 25mM
28 μ L, each 1 μ L of primer S66 and S88 of 20 μMs, the RNA 3 μ L of extracting, and without RNA enzyme aqua sterilisa 28 μ L.Wherein PCR damping fluid is by Tris-HCl, KCl and MgCl
2mixed aqueous solution composition, pH value is 8.5, and wherein Tris-HCl concentration is 100mM, KCl concentration is 500nM, MgCl
2concentration is 15nM.
Or press the One step RT-PCR test kit (PrimeScript that the precious biotech firm of TAKARA produces
(R)one Step RT-PCR Kit Ver.2, catalog number (Cat.No.) is DRR055A) operation instruction prepares, reaction system 25 μ L, be specially: PrimeScript 1 Step Enzyme Mix 1 μ L, 2 × 1 Step Buffer 12.5 μ L, concentration is the upstream primer S66 1 μ L of 10 μMs, and concentration is the downstream primer S88 1 μ L of 10 μMs, the RNA 3 μ L of extracting, RNase Free dH
2o 6.5 μ L.
Above reaction system prepares to be placed in PCR instrument carries out RT-PCR reaction, and response procedures is: 50 DEG C of 30min reverse transcriptions; The deactivation of 94 DEG C of 2min ThermoScript II; 94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 60s, totally 40 circulations; 72 DEG C extend 8 ~ 10min; Last 8 DEG C of coolings, reaction terminates.
Second step: the enzyme of PCR primer cuts qualification
The preparation of endonuclease reaction system: the PCR primer 10 μ L that the first step obtains, 0.1% (w/v) BSA 2 μ L, 10 × damping fluid (is made up of the mixed aqueous solution of magnesium acetate, dithiothreitol (DTT) and Potassium ethanoate, wherein the concentration of magnesium acetate is 100mM, the concentration of dithiothreitol (DTT) is 5mM, the concentration of Potassium ethanoate is 660mM) 2 μ L, restriction endonuclease
saciI 1 μ L, sterilizing distilled water 5 μ L.
Mix gently after endonuclease reaction system prepares, be placed in 37 DEG C of water-bath (or constant incubator) effect 1h(1 hour).
After endonuclease reaction terminates, add 2 μ L 10 × loading buffer, mix with termination reaction, get 5 μ L endonuclease reaction products and carry out agarose gel electrophoresis, take pictures in gel imaging system after electrophoresis terminates.There is 487bp object band in result, is judged to be that JXA1-R vaccine strain is positive; There is 244bp band, be judged to be that (other strains have at ORF1a 3195 ~ 3682 nucleotide fragments non-JXA1-R vaccine strain
saciI restriction enzyme site), as Fig. 1.Because the corresponding amplified fragments of other strains ORF1a passes through
saciI enzyme cut after fragment length be 244bp, much smaller than 487bp, electrophoretic band is easy to make a distinction, and the gradient difference value of electrophoresis DNA molecular weight standard is usually at more than 100bp, therefore can be judged as roughly the sample that be containing JXA1-R vaccine strain of molecular weight at about 460bp ~ 510bp.
SEQUENCE LISTING
<110> Guangdong Wen'S Foodstuffs Group Co., Ltd.
<120> mono-kind detects method and the test kit of pig blue-ear disease living vaccine JXA1-R strain
<130>
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213> artificial sequence
<400> 1
tctcccaaag atgattctcg ag 22
<210> 2
<211> 21
<212> DNA
<213> artificial sequence
<400> 2
aatcacccgg agaataacca c 21
Claims (4)
1. one kind is detected the method for pig blue-ear disease living vaccine JXA1-R strain, it is characterized in that, adopt rflp analysis method, the primer of design and synthesis amplification pig blue-ear disease living vaccine JXA1-R strain ORF1a 3195 ~ 3682 nucleotide fragments, extract the RNA in measuring samples, by RT-PCR, by described primer amplification JXA1-R strain ORF1a 3195 ~ 3682 nucleotide fragments, amplified production and restriction endonuclease
saciI carries out endonuclease reaction, and digestion products occurs that after agarose gel electrophoresis the band of length 487bp is judged as in measuring samples containing pig blue-ear disease living vaccine JXA1-R strain;
Described RT-PCR is One step RT-PCR, and the nucleotide sequence of described primer is as shown in SEQ ID NO:1 ~ 2.
2. detect a test kit for pig blue-ear disease living vaccine JXA1-R strain, it is characterized in that, this test kit comprises the primer nucleotide sequences shown in SEQ ID NO:1 ~ 2 and restriction endonuclease
saciI.
3. the test kit of detection pig blue-ear disease living vaccine JXA1-R according to claim 2 strain, is characterized in that, this test kit is made up of One step RT-PCR reaction solution and endonuclease reaction liquid two group reagent, wherein:
One step RT-PCR reaction solution: the PCR damping fluid 5 μ L of 10 times of dilutions, the ThermoScript II 1 μ L of the archaeal dna polymerase 1 μ L of 5U/ μ L, 200U/ μ L, the RNA enzyme inhibitors 1 μ L of the dNTPs1 μ L of 10mM, 200U/ μ L, the MgCl of 25mM
28 μ L, the SEQ ID NO:1 of 20 μMs and each 1 μ L of the primer nucleotide sequences shown in SEQ ID NO:2, and without RNA enzyme aqua sterilisa 28 μ L;
Endonuclease reaction liquid: 0.1% (w/v) BSA 2 μ L, the endonuclease reaction damping fluid 2 μ L of 10 times of dilutions, restriction endonuclease
saciI 1 μ L, sterilizing distilled water 5 μ L;
Described PCR damping fluid is by Tris-HCl, KCl and MgCl
2mixed aqueous solution composition, pH value is 8.5, and wherein Tris-HCl concentration is 100mM, KCl concentration is 500nM, MgCl
2concentration is 15nM;
Described endonuclease reaction damping fluid is made up of the mixed aqueous solution of magnesium acetate, dithiothreitol (DTT) and Potassium ethanoate, and wherein the concentration of magnesium acetate is 100mM, and the concentration of dithiothreitol (DTT) is 5mM, and the concentration of Potassium ethanoate is 660mM.
4. the method for pig blue-ear disease living vaccine JXA1-R strain is detected with the test kit of detection pig blue-ear disease living vaccine JXA1-R according to claim 3 strain, it is characterized in that, measuring samples extracts after RNA and uses described One step RT-PCR reaction solution to increase, and RT-PCR response procedures is: 50 DEG C 30 minutes; 94 DEG C 2 minutes; 94 DEG C 45 seconds, 55 DEG C 45 seconds, 72 DEG C 1 minute, totally 40 circulations; 72 DEG C, extend 8 ~ 10 minutes; Getting part amplified production joins in endonuclease reaction liquid, reacts 1 hour at 37 DEG C; Termination reaction is got digestion products and is carried out agarose gel electrophoresis, and by observation electrophoretic band of taking pictures, the fragment if there is 487bp length is judged as in measuring samples containing pig blue-ear disease living vaccine JXA1-R strain.
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| CN114774468B (en) * | 2022-04-20 | 2022-12-20 | 温氏食品集团股份有限公司 | Allele molecular marker and anti-blue-ear-disease pig group construction method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101307305A (en) * | 2008-04-30 | 2008-11-19 | 中国动物疫病预防控制中心 | Low virulent strain of porcine reproductive and respiratory syndrome virus, immunogenicity immunogenicity material and vaccine |
| CN101376909A (en) * | 2007-08-31 | 2009-03-04 | 中国农业科学院特产研究所 | A segment of sequence and a restriction enzyme site for differentiating canine distemper viral vaccine strain and wild strain |
| CN101423836A (en) * | 2008-11-05 | 2009-05-06 | 中国农业科学院哈尔滨兽医研究所 | Porcine circovirus I type infectious clone and virus rescued thereby and application thereof |
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| CN101376909A (en) * | 2007-08-31 | 2009-03-04 | 中国农业科学院特产研究所 | A segment of sequence and a restriction enzyme site for differentiating canine distemper viral vaccine strain and wild strain |
| CN101307305A (en) * | 2008-04-30 | 2008-11-19 | 中国动物疫病预防控制中心 | Low virulent strain of porcine reproductive and respiratory syndrome virus, immunogenicity immunogenicity material and vaccine |
| CN101423836A (en) * | 2008-11-05 | 2009-05-06 | 中国农业科学院哈尔滨兽医研究所 | Porcine circovirus I type infectious clone and virus rescued thereby and application thereof |
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Address after: 527400 9 East Causeway North Road, Xinxing County new town, Yunfu, Guangdong Patentee after: Winson food group Limited by Share Ltd Address before: 527400 Wen's Group Headquarters, No. 9 Dongdi North Road, Xinxing Town, Yunfu City, Guangdong Province Patentee before: Guangdong Wens Foodstuff Group Co., Ltd. |