CN101831506B - Kit for authenticating deletion of vaccine strain and wild strain of PRVgE - Google Patents
Kit for authenticating deletion of vaccine strain and wild strain of PRVgE Download PDFInfo
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- CN101831506B CN101831506B CN2010101463534A CN201010146353A CN101831506B CN 101831506 B CN101831506 B CN 101831506B CN 2010101463534 A CN2010101463534 A CN 2010101463534A CN 201010146353 A CN201010146353 A CN 201010146353A CN 101831506 B CN101831506 B CN 101831506B
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Abstract
The invention discloses a loop-mediated isothermal amplification kit for rapid testing and authenticating the deletion of vaccine strain and wild strain of pseudorabies virus (PRV) gE. The kit comprises the following components: mixed buffer solution of isothermal reaction, DNA polymerase, distilled water, a primer mixture 1 and a primer mixture 2, wherein the primer mixture 1 comprises 3 pairs of primers and the primer mixture 2 comprises 3 pairs of primers. The invention adopts the loop-mediated isothermal amplification (LAMP) technology, has strong specificity, higher sensitivity than the PCR measurement method, does not need an expensive PCR instrument, only needs a common metal bath or water bath tank, can use the gel electrophoresis method to observe the measurement result, can use the centrifugation method to observe the precipitation, is simple and rapid, can be used for the authentication detection of the wild strain of the PRV, and also can be used for authenticating the deletion of vaccine strain and wild strain of PRV gE and is particularly suitable for the detection of PRV of a base-level veterinarian detection organization and the scaled hoggery.
Description
Technical field
The invention belongs to the test kit of differentiating PRV, relate in particular to a kind of rapid detection and the loop-mediated isothermal amplification kit of differentiating PRV gE deletion of vaccine strain and street strain, belong to the detection range of PRV.
Background technology
PRV can cause multiple domestic animal and wildlife with heating, very itch (except the pig) and encephalomyelitis is the disease of cardinal symptom.Pig is reservoir host and the contagium of PRV, mainly causes Gestation period sow miscarriage, stillbirth, mummy tire; Mostly newborn piglet is the acute fatal process, has tangible nervous symptoms, and mortality ratio is almost 100%; Adult pig is latent infection more.Immunization is the main policies of preventing and treating pseudoabies, and it is less toxic vaccine with Bartha-K61 strain preparation that China uses many at present.The appearance of back clinical symptom though the PRV less toxic vaccine can prevent infections effectively, inoculation pig still possibly infected by strong poison, infect can form hide and subsequently the virus of latent infection can be activated and cause and disseminate.Therefore, it is important to set up the antigen detection method ten minutes of the early stage poison of sldh gene deletion attenuated vaccine fast and wild virus infection of PRV.
The antigen detection method of PRV is a lot, like virus separation, quantitative fluorescent PCR, colloidal gold antigen test strip.But virus is separated time and effort consuming, and the fluorescence quantitative PCR detection cost is high and need could accomplish by the auxiliary of some special test apparatuses, though the quick susceptibility of colloidal gold antigen test strip is not high.
Summary of the invention
The purpose of this invention is to provide a kind of can rapid detection and the loop-mediated isothermal amplification kit of differentiating PRV gE deletion of vaccine strain and street strain; Need could accomplish detection by the auxiliary of some specific apparatus with what solve that prior art exists, less stable, troublesome poeration, efficient are low, the problem of need cryopreservation, accumulating inconvenience.
The objective of the invention is to realize through following technical scheme:
A kind of rapid detection and the loop-mediated isothermal amplification kit of differentiating PRV gE deletion of vaccine strain and street strain, its moity comprises: isothermal reaction cocktail buffer, archaeal dna polymerase, zero(ppm) water, primer mixture 1 and primer mixture 2; Wherein, Described primer mixture 1 by 3 pairs of primers according to etc. mol ratio form; The sequence of the 1st pair of primer is respectively shown in SEQ ID NO:1 and the SEQ ID NO:2; The sequence of the 2nd pair of primer is respectively shown in SEQ ID NO:3 and the SEQ ID NO:4, and the sequence of the 3rd pair of primer is respectively shown in SEQ ID NO:5 and the SEQ ID NO:6; Described primer mixture 2 by 3 pairs of primers according to etc. mol ratio form; The sequence of the 1st pair of primer is respectively shown in SEQ ID NO:7 and the SEQ ID NO:8; The sequence of the 2nd pair of primer is respectively shown in SEQ ID NO:9 and the SEQ ID NO:10, and the sequence of the 3rd pair of primer is respectively shown in SEQ I D NO:11 and the SEQ ID NO:12.
Wherein, the isothermal reaction cocktail buffer that described isothermal reaction cocktail buffer is 2 * U comprises following component: 40mM Tris-HCl, 20mM KCl, 20mM (NH
4)
2SO
4, 0.2% TritonX-100,5mM dNTP, 5M trimethyl-glycine; In order to reach better effect, can also contain other compositions such as stablizer and toughener in the described isothermal reaction cocktail buffer.
Described archaeal dna polymerase is preferably the Bst archaeal dna polymerase.
In order to reach better detection effect, test kit of the present invention can also contain the positive control PRV-PMD18T-gE plasmid and the PRVgE deletion of vaccine strain positive control plasmid PRV-PMD18T-gG plasmid of the wild poison of PRV; The construction process of these two plasmids is following: the primer of at first synthetic PRV-GE gene and PRV-GG gene, and primer sequence is following:
GE-FP：5’-CGGCTTCGACGTCTGGTT-3’；
GE-RP：5’-GCGGTCACGCCATAGTTG-3；
gG-FP：5’-CGATGAAGTGGGCAACGTGG?ATCCTC-3’；
gG-RP 5’-CGTCAGGCGGAGGCCACGTGGCGGTA-3’；
With the wild malicious PRV-JX strain of PRV (can buy from market, for example: Chinese veterinary microorganism culture presevation administrative center) and malicious PRV-Bartha strain a little less than the PRV (can buy from market, for example: Chinese veterinary microorganism culture presevation administrative center) DNA is a template; Pcr amplification purpose fragment, glue reclaims the purpose fragment respectively then, is cloned on the PMD18T carrier; Transform DH5 α competent cell then; Screen positive bacterium colony, shake the bacterium amplification, plasmid extraction kit extracts the purpose plasmid.The plasmid that makes up is prone to preserve, and does not have infectivity, does not have the danger of the poison that looses, and the DNA that can substitute totivirus is as positive template;
Preferably, the volume of each moity of detection kit of the present invention and preservation condition are following:
| Numbering | Test kit is formed | Volume (20T/40T) | |
| 1 | 2×U-LAMP?Mix | 750μL/1500μL | -20 |
| 2 | The Bst archaeal dna polymerase | 60μL/120μL | -20 |
| 3 | Primer mixture 1 (10 *, detect the wild malicious tailored version primer of PRV) | 360μL/720μL | -20 |
| 4 | Primer mixture 2 (detecting PRVgE deletion of vaccine strain and street strain's universal primer) | 360μL/720μL | -20 |
| 5 | Template 1 (PRV-gE plasmid) | 60μL | -20℃ |
| 6 | Template 2 (PRV-gG plasmid) | 60μL | -20℃ |
| 7 | Free nucleic acid ddH 2O | 1000μL | -20℃ |
| 8 | Reaction tubes (100) pipe arrangement frame | 200μL | -20℃ |
Further preferred; The volume of each component is in the test kit of the present invention: the volume of isothermal reaction cocktail buffer is more than or equal to 25 μ L; The volume of BstDNA polysaccharase is more than or equal to 2 μ L; The volume of zero(ppm) water is more than or equal to 9.0 μ L, and the volume of primer mixture 1 is more than or equal to 6 μ L, and the volume of primer mixture 2 is more than or equal to 6 μ L.
The detection method of test kit of the present invention comprises:
(1) DNA of extraction tissue sample to be detected;
(2) at first carry out wild malicious detection:
Each set of dispense of detection architecture is such as following:
2×U-LAMP?Mix 12.5μL
Primer mixture 1 (10 *, detect the wild malicious tailored version primer of PRV) 6 μ L
PRV-PMD18T-gE plasmid or viral DNA to be checked 1 μ L
Bst archaeal dna polymerase 1 μ L
Free nucleic acid ddH
2O 4.5 μ L
Each sample to be checked all need be established the positive control (the positive contrast of PRV-PMD18T-gE plasmid) of wild poison and not add the negative water contrast of template; Repeatedly use, can establish positive control and negative control first, can not establish positive control later on, but negative control can not omit.
With the said components mixing, place 65 ℃ of constant water bath box to place 80 ℃ of 10 minutes deactivation Bst archaeal dna polymerases 60 minutes; Amplified production detects on Ultraviolet Detector through 2% agarose gel electrophoresis or directly with amplified production centrifugal (5000 left the heart 10 minutes), precipitates at the bottom of the observation tube; What be positive is street strain, the attenuated vaccine strain that is that is negative and reacts;
(3) will pass through detection is not the detection that the sample of street strain carries out the weak poison of PRVgE deletion of vaccine strain:
Each set of dispense of detection architecture is such as following:
25 each set of dispense of μ L versatility primer detection architecture are such as following:
2×U-LAMP?Mix 12.5μL
Primer mixture 2 (10 *, detect PRVgE
-Deletion of vaccine strain and street strain's universal primer)
6μL
Template 2 (PRV-gG plasmid) or viral DNA to be checked 1 μ L
Bst archaeal dna polymerase 1 μ L
Free nucleic acid ddH
2O 4.5 μ L
With the said components mixing, place 65 ℃ of constant water bath box to place 80 ℃ of 10 minutes deactivation Bst archaeal dna polymerases 60 minutes; Amplified production detects on Ultraviolet Detector through 2% agarose gel electrophoresis or directly with amplified production centrifugal (5000 left the heart 10 minutes), precipitates at the bottom of the observation tube; That be positive is PRVgE
-Deletion of vaccine strain;
(4) result judges
4.1 agarose gel electrophoresis: amplified production detects on Ultraviolet Detector through 2% agarose gel electrophoresis, and positive reaction can be observed the stepped band of LAMP characteristic, and negative reaction does not then have.
4.2 the centrifuging deposition is observed: amplified production 5000 left the heart after 10 minutes, positive reaction has macroscopic white precipitate in the pipe bottom, and negative reaction does not then have.
The present invention has at first designed primer according to PRV with reference to strain NC_006151 strain gE gene and gG gene conservative region sequence, and gE gene conservative gene sequence is that the wild poison of PRV is common, and the gE deletion of vaccine strain does not then have.The present invention adopts LAMP technology, high specificity, and higher sensitivity is arranged than PCR detection method; But do not need expensive PCR appearance, only need common metal bath or water bath to get final product, and the available gel electrophoresis method of result is observed; Also can observe deposition through centrifugation method; Simple and quick, both can be used for the detection of the wild poison of PRV, also can be used for (PRV) gE
-The discriminating of deletion of vaccine strain and street strain detects, and is particularly suitable for the detection for PRV of animal doctor feeler mechanism of basic unit and large scale of pig farm field.
The present invention uses the LAMP technology; Each boar according to using within Chinese territory all lacks this characteristic of gE gene with PRV genetically engineered attenuated vaccine strain; Successfully set up a kind of practicality fast cheap the strong and weak poison of PRV differentiate detection method, the early stage quick diagnosis that infects for PRV provides strong diagnostic tool.
Description of drawings
Fig. 1 uses the detected result of test kit of the present invention (PRV-GE-LAMP detection method) to two strain attenuated vaccine strains and two strain street strains; 1.PRV-GE-LAMP detect PRV-JX street strain; 2.PRV-GE-LAMP detect PRV-HLJ street strain; 3.PRV-GE-LAMP detect the PRV-Bartha attenuated vaccine strain; 3.PRV-GE-LAMP detect PRV-Plus strain attenuated vaccine strain; 5..PRV-GE-LAMP negative control.
Fig. 2 uses the detected result of test kit of the present invention (PRV-GG-LAMP detection method) to two strain attenuated vaccine strains and two strain street strains; 1.PRV-GG-LAMP detect PRV-JX street strain; 2.PRV-GG-LAMP detect PRV-HLJ street strain; 3.PRV-GG-LAMP detect the PRV-Bartha attenuated vaccine strain; 3.PRV-GG-LAMP detect PRV-Plus strain attenuated vaccine strain; 5..PRV-GG-LAMP negative control.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
Test materials
Bs tDNA polysaccharase is available from Niu Yinglun biotechnology (Beijing) ltd;
Test Example 1 test kit of the present invention detects porcine pseudorabies virus 2 strain gE
-The application test of deletion of vaccine strain and 2 strain street strains
Use test kit of the present invention to using maximum PRVgE within Chinese territory
-Deletion of vaccine strain Bartha strain and Plus strain and the two strain terrain JX of isolating street strain strains and HLJ strain detect.
Detection method is following:
1, the preparation of PRV-LAMP amplification reaction solution: the various components beyond dissolving is dezymotized on ice, mixing.Each detection of carrying out wild poison earlier can be carried out PRVgE if not wild poison
-The detection of the weak poison of deletion of vaccine strain.
Wild malicious each set of dispense of tailored version primer detection architecture of 25 μ L is such as following:
2×U-LAMP?Mix 12.5μL
Primer mixture 1 (10 *, detect the wild malicious tailored version primer of PRV) 6 μ L
Template 1 (PRV-gE plasmid) or viral DNA to be checked 1 μ L
Bst archaeal dna polymerase 1 μ L
Free nucleic acid ddH
2O 4.5 μ L
The positive control that each sample to be checked all need be established wild poison contrasts with the negative water that does not add template, whether detects wild virus infection, and primer is with primer mixture 1, if template concentrations is lower, can add the usage quantity of large form, reduces the usage quantity of water simultaneously.
25 each set of dispense of μ L versatility primer detection architecture are such as following:
2×U-LAMP?Mix 12.5μL
Primer mixture 2 (10 *, detect PRVgE
-Deletion of vaccine strain and street strain's universal primer))
6μL
Template 2 (PRV-gG plasmid) or viral DNA to be checked 1 μ L
Bst archaeal dna polymerase 1 μ L
Free nucleic acid ddH
2O 4.5 μ L
Each sample to be checked all need be established positive control and not add the negative water contrast of template, and whether detect is PRVgE
-The deletion attenuated vaccine strain with primer mixture 2, if template concentrations is lower, can add the usage quantity of large form, reduces the usage quantity of water simultaneously.
2, isothermal duplication condition: mixing places 65 ℃ of constant water bath box to place 80 ℃ of 10 minutes deactivation Bst archaeal dna polymerases 60 minutes.Amplified production detects on Ultraviolet Detector through 2% agarose gel electrophoresis or directly with amplified production centrifugal (5000 left the heart 10 minutes), precipitates at the bottom of the observation tube.
3, the result judges
3.1 agarose gel electrophoresis: amplified production detects on Ultraviolet Detector through 2% agarose gel electrophoresis, and positive reaction can be observed the stepped band of LAMP characteristic.Negative reaction does not then have.
3.2 the centrifuging deposition is observed: amplified production 5000 left the heart after 10 minutes, positive reaction has macroscopic white precipitate in the pipe bottom, and negative reaction does not then have.
Preliminary Applications result: use test kit of the present invention to using maximum PRVgE within Chinese territory
-Deletion of vaccine strain Bartha strain and Plus strain and the isolating street strain of two strain terrains (PRV-JX, PRV-HLJ) carried out detecting (above-mentioned strain to be detected available from Chinese veterinary microorganism culture presevation administrative center):
Detected result shows, uses test kit of the present invention (PRV-GE-LAMP detection method) and can detect malicious PRV-JX strain in open country and wild malicious PRV-HLJ strain, the distinctive stepped band of LAMP occurs, and the result is positive; And PRVgE
-Vaccine strain PRV-Bartha strain and PRV-Plus strain can not detect, and do not have stepped purpose band, and reaction result is negative.Detected result proof PRV-JX strain and PRV-HLJ strain are the wild poison of PRV.
Use test kit of the present invention (PRV-GG-LAMP detection method) and can detect, to PRVgE malicious PRV-JX strain in open country and wild malicious PRV-HLJ strain
-Vaccine strain PRV-Bartha strain and PRV-Plus strain can detect equally, the distinctive stepped band of LAMP occurs, and the result is positive.Can judge that in conjunction with the detected result of PRV-GE-LAMP PRV-Bartha strain and PRV-Plus strain are PRV GE gene-deleted vaccine poison.
Test-results proof PRV-JX, PRV-HLJ is a street strain, and PRV-Bartha strain and PRV-Plus strain are PRVgE
-Attenuated vaccine strain; Checking PRV-GE-LAMP detection method is the method for detecting specificity of the wild poison of PRV; And the PRV-GG-LAMP detection method is the universal test method of PRV open country poison and the strain of GE deletion attenuated vaccine, can judge accurately that the wild poison of PRV still is a GE deletion of vaccine poison but unite use through the two.
Claims (6)
1. rapid detection and the loop-mediated isothermal amplification kit of differentiating PRV gE deletion of vaccine strain and street strain, its moity comprises: isothermal reaction cocktail buffer, archaeal dna polymerase, zero(ppm) water, primer mixture 1 and primer mixture 2; It is characterized in that: described primer mixture 1 is made up of 3 pairs of primers; Wherein, The sequence of the 1st pair of primer is respectively shown in SEQ ID NO:1 and the SEQ ID NO:2; The sequence of the 2nd pair of primer is respectively shown in SEQ ID NO:3 and the SEQ ID NO:4, and the sequence of the 3rd pair of primer is respectively shown in SEQ ID NO:5 and the SEQ ID NO:6; Described primer mixture 2 is made up of 3 pairs of primers; Wherein, The sequence of the 1st pair of primer is respectively shown in SEQ ID NO:7 and the SEQ ID NO:8; The sequence of the 2nd pair of primer is respectively shown in SEQ ID NO:9 and the SEQ ID NO:10, and the sequence of the 3rd pair of primer is respectively shown in SEQ ID NO:11 and the SEQ ID NO:12.
2. according to the described loop-mediated isothermal amplification kit of claim 1, it is characterized in that: in the described primer mixture 1 the 3 pairs of primers according to etc. mol ratio form, in the described primer mixture 2 the 3 pairs of primers according to etc. mol ratio form.
3. according to the described loop-mediated isothermal amplification kit of claim 1, it is characterized in that: the isothermal reaction cocktail buffer that described isothermal reaction cocktail buffer is 2 * U, it comprises following component: 40mMTris-HCl, 20mM KCl, 20mM (NH4)
2SO
4, 0.2%TritonX-100,5mM dNTP and 5M trimethyl-glycine.
4. according to the described loop-mediated isothermal amplification kit of claim 1, it is characterized in that: described archaeal dna polymerase is the Bst archaeal dna polymerase.
5. according to the described loop-mediated isothermal amplification kit of claim 1, it is characterized in that: the positive control PRV-PMD18T-gE plasmid or the PRV gE deletion of vaccine strain positive control PRV-PMD18T-gG plasmid that also contain the wild poison of pseudorabies virus.
6. according to the described loop-mediated isothermal amplification kit of claim 1; It is characterized in that; The volume of each moity is: the volume of isothermal reaction cocktail buffer is more than or equal to 25 μ L, and the volume of archaeal dna polymerase is more than or equal to 2 μ L, and the volume of zero(ppm) water is more than or equal to 9.0 μ L; The volume of primer mixture 1 is more than or equal to 6 μ L, and the volume of primer mixture 2 is more than or equal to 6 μ L.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101463534A CN101831506B (en) | 2010-04-14 | 2010-04-14 | Kit for authenticating deletion of vaccine strain and wild strain of PRVgE |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101463534A CN101831506B (en) | 2010-04-14 | 2010-04-14 | Kit for authenticating deletion of vaccine strain and wild strain of PRVgE |
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| Publication Number | Publication Date |
|---|---|
| CN101831506A CN101831506A (en) | 2010-09-15 |
| CN101831506B true CN101831506B (en) | 2012-05-30 |
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103224995B (en) * | 2013-04-10 | 2015-07-15 | 中国农业科学院哈尔滨兽医研究所 | Nanometer PCR detection kit for rapidly identifying and diagnosing virulent virus and attenuated virus of porcine pseudorabies virus, and applications thereof |
| CN104726611A (en) * | 2013-12-19 | 2015-06-24 | 普莱柯生物工程股份有限公司 | Method, primer, and kit used for detecting porcine pseudorabies virus field strain |
| CN105368791A (en) * | 2014-02-21 | 2016-03-02 | 普莱柯生物工程股份有限公司 | Porcine pseudorabies virus gene-deleted strain, vaccine composition and their preparation method and application |
| CN104789699A (en) * | 2015-04-02 | 2015-07-22 | 河南省动物疫病预防控制中心 | PCR primer for identifying PRV virulent virus and PRV gG and PRV gE gene deletion vaccine viruses |
| CN105112565A (en) * | 2015-09-07 | 2015-12-02 | 山东省农业科学院畜牧兽医研究所 | LAMP detection method for detecting pseudorabies virus GE gene |
| CN107828917A (en) * | 2017-11-30 | 2018-03-23 | 山东新希望六和集团有限公司 | For detecting the wild malicious primer and probe of PRV, PCR kit for fluorescence quantitative and method, application |
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