CN103695562A - Detection method and detection kit for PRV (pseudorabies virus) virulence gene mutation - Google Patents
Detection method and detection kit for PRV (pseudorabies virus) virulence gene mutation Download PDFInfo
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- CN103695562A CN103695562A CN201310652245.8A CN201310652245A CN103695562A CN 103695562 A CN103695562 A CN 103695562A CN 201310652245 A CN201310652245 A CN 201310652245A CN 103695562 A CN103695562 A CN 103695562A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a primer group for detecting the PRV (pseudorabies virus) virulence genes gE, gC, gD and TK, and also discloses an application of the primer group, a kit comprising the primer group and a method for performing PCR amplification of the PRV virulence genes by use of the primer group. The primer group can be used for exclusively identifying whether the PRV virulence gene mutates; and with high specificity and sensitivity, the primer group can guarantee the accuracy of the detection result and is of great significance to guide practice and production.
Description
Technical field
The present invention relates to field of virus detection, be specifically related to detection method and the detection kit of PRV virulence gene sudden change.
Background technology
Pseudoabies (Pseudorabies, PR has another name called Aujeszky's disease, AD) be a kind of acute infectious disease of the multiple domestic animal, wildlife, companion animals and the laboratory animal that are caused by pseudorabies virus (Pseudorabies Virus, PRV).Under field conditions (factors), this disease is most commonly in pig, ox, sheep, dog, cat and muroid.Pseudorabies virus, except to pig, is all the lethality course of disease to all susceptible animals.To pig main manifestations, be nervous symptoms, salivation, expiratory dyspnea, breeding difficulty, cessation of growth cessation, weightlessness and high mortality.The clinical manifestation of sucking piglets infection pseudoabies is typical case, serious in consistent the most, all occurs nervous symptoms, paralysis, exhaustion death, and mortality ratio is almost up to 100%; Mortality ratio declines gradually with age, and becoming pig is only 2%, does not fall ill, and be stealthy process but generally become pig to infect, and rarely seenly sneezes, the light symptoms such as cough, fervescence; Pregnant sow infects Pseudorabies virus can cause miscarriage, stillborn foetus, the weak son of product and mummified fetus; To boar, can cause vaginalitis.This disease has the infectivity of height, once in swinery, find, will be very soon by air with contact through respiratory infectious to other pigs and pig farm, recent research shows that pseudorabies virus can also be by milk and genital tract infection.
Different Pseudorabies virus has different virulence, according to the difference of its virulence, can be divided into: virulent strain, and common street strain, attenuation is thorough strain, the complete low virulent strain of attenuation.Clinical symptom that the strain of different virulence causes is different, and the clinical symptom that virulent strain causes is: except common sympton, can also cause that skin very itches.The clinical symptom that common street strain causes is mainly: breeding difficulty etc.The halfway strain of attenuation mainly causes: grice diarrhoea, the slight breeding difficulty of sow.The complete low virulent strain of attenuation is not pathogenic, and safe strain, so used as vaccine.The difference of the virulence of PRV is determined by its virulence gene, both at home and abroad the gene studies of pseudo-rabies is shown at present, and the major decision gene that affects pseudo-rabies virulence concentrates on gE, gC, gD, TK, gB, these genes of RR.Through the literature search of prior art is found, Chinese invention patent application is not about detecting the test kit of these four virulence genes of PRV at present, but in recent years, PRV presents certain popularity, in order better to monitor the variation of virulence gene, to analyze the virulence of PRV, change, develop a kind of test kit that can the several virulence genes sudden changes of rapid detection and be necessary very much.
Summary of the invention
Therefore the present invention is intended to design one group of primer, amplification for these genes, then PCR product is carried out to sequencing, according to result compare of analysis, go out the difference between different strains, and conjugated antigen forecasting software is analyzed, whether can analyze virus virulence and antigenicity changes, this can be used for Molecule Epidemiology Investigation by Guiding Practice, instruct vaccine potency check, according to the virulence variation tendency of the result judgement Pseudorabies virus of prediction, then the Immunization experimental result of combined vaccine instructs the efficacy test of vaccine.
Primer sets for detection of PRV virulence gene sudden change provided by the invention, comprises the primer sets of increase respectively PRV virulence gene gE, gC, gD and TK,
Wherein, the primer sets of amplification gE comprises the primer sets of aligning primer shown in aligning primer shown in the primer sets of aligning primer shown in aligning primer shown in SEQ ID No.1 and SEQ ID No.2, the primer sets with aligning primer shown in aligning primer shown in SEQ ID No.3 and SEQ ID No.4 and SEQ ID No.5 and SEQ ID No.6;
Wherein, the primer sets of amplification gC comprises the primer sets of aligning primer shown in aligning primer shown in the primer sets of aligning primer shown in aligning primer shown in SEQ ID No.7 and SEQ ID No.8 and SEQ ID No.9 and SEQ ID No.10;
Wherein, the increase primer sets of described gD comprises the primer sets of aligning primer shown in aligning primer shown in the primer sets of aligning primer shown in aligning primer shown in SEQ ID No.11 and SEQ ID No.12 and SEQ ID No.13 and SEQ ID No.14;
Wherein, the increase primer sets of described TK is the primer sets of aligning primer shown in aligning primer shown in SEQ ID No.15 and SEQ ID No.16.
The application of primer sets described in the present invention also provides in detecting the sudden change of PRV virulence gene.
PRV virulence gene sudden change detection reagent or the test kit of the primer sets described in the present invention also provides.
The present invention also provides the method for a kind of PRV of detection virulence gene sudden change, and it is template for take the DNA of PRV, by described primer sets, carries out pcr amplification, after each PCR product is checked order respectively, carries out sequence alignment.
Wherein, use 0.5～1.5 primer of μ l and the DNA profiling of 50～100ng in the reaction system of every 25 μ l, reaction conditions is:
96～94 ℃ of denaturation 2min～5min;
96～94 ℃ of sex change 30s～40s;
60～64 ℃ of annealing 30s～60s;
72 ℃ are extended 30s～60s;
30-40 circulation;
72 ℃ are extended 10min.
The present invention is according to the gE in PRV in GeneBank, gC, gD, TK gene order has been carried out specific design of primers, so the PCR product increasing can identify gE, gC, gD, whether tetra-virulence genes of TK undergo mutation, and can guarantee accuracy and the uniqueness of the sequencing result of increased sequence.
Primer sets provided by the invention and test kit can be for detection of gE, gC, gD, whether these four virulence genes of TK undergo mutation, be used for Molecule Epidemiology Investigation and instruct PRV virulence variation tendency can instruct the evaluation of vaccine result of use simultaneously, therefore have important practical significance.
The different virulence gene PCR of the present invention can complete under identical reaction conditions and identical reaction system, the omnidistance time spending was less than two hours, amplified production can directly check order, and therefore when centralized detecting PRV sample, has fast and convenient feature.
Accompanying drawing explanation
Fig. 1 is the electrophoresis result of the PCR product of the different goal gene of amplification.
Fig. 2 is the sudden change position of the gC gene with Vector NTI software analysis.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
Embodiment 1
The gE that the NC_001651 sequence of announcing according to NCBI GenBank is announced, gD, gC, the sequence of TK, designs for goal gene gE, gD, gC, the PCR primer pair of TK amplification, the results are shown in Table 1.
Detect gE, gC, gD, TK transgenation step:
PRV infects the extracting genome DNA of sample (serum or tissue):
Use QIAamp DNA MiniKit (250) Cat.No.51306 to extract PRV genomic dna in blood or tissue, this DNA is template DNA used.
GE, gC, gD, the pcr amplification of tetra-genes of TK:
Each reaction system is 50 μ l.
PCR reaction system is as follows:
The reaction conditions of PCR circulation:
After finishing, reaction carries out 1%Agarose detected through gel electrophoresis.Detected result is as Fig. 1.The sample (PCR product, at least 40 μ l) of amplification positive findings send order-checking company (Invitrogen company) to check order.
Sequencing result is analyzed, with any biosoftware (Sequencher or DNAstar) that can open sequencing result, result is analyzed, whether the sequence of downloading in analysis and GenBank changes, this variation comprises: the variation of single base (amino acid of coding is changed), the disappearance of a plurality of bases, the insertion of a plurality of bases.If there is above variation, illustrate that the virulence gene of PRV exists sudden change.In conjunction with clinical prevailing disease situation and transgenation result, draw the molecular epidemiology result of PRV, with this result, carry out Guiding Practice work.
The sudden change result of the strain virus recording in the present invention is as Fig. 2, and this sports the sudden change of gC gene, relatively finds to exist the disappearance of six bases in place with other sequences.There is change in the virulence gene of the presentation of results PRV of this sudden change, this variation has important directive significance for the Molecule Epidemiology Investigation of PRV.
Embodiment 2
For checking a test kit for PRV virulence gene sudden change, comprise tetra-main virulence gene gE of PRV, gC, gD, the primer sets of TK, each described pcr amplification primer group is shown in table 1.Also comprise pcr amplification reagent, amplifing reagent comprises dNTP, LA TaqDNA polysaccharase, buffer etc.This test kit is stored in-20 ℃, reduces multigelation as far as possible.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Sequence table
Animal medicine research centre, <110> BeiJing DaBei farm Science Group Co., Ltd
BeiJing DaBei farm Science Group Co., Ltd
Fuzhou Dabeinong Bioisystech Co., Ltd
Detection method and the detection kit of the sudden change of <120> PRV virulence gene
<130>
<160> 16
<170> PatentIn version 3.3
<210> 1
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<212> DNA
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<400> 1
gtgggtggcg ttttatctcc gtccg 25
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<212> DNA
<213> artificial sequence
<400> 2
gcgtgtagag gcccgtgtcg ttgg 24
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<212> DNA
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tggtcgccgc acctgagcgt cct 23
<210> 4
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ggcgacgtgg ccgttgtggg tca 23
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cggacgagtc ggggctgtac gtg 23
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<400> 6
atttaagcgg ggcgggacat caa 23
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<212> DNA
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<400> 7
gccctgctcg tgcaggcgta cgt 23
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<211> 23
<212> DNA
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<400> 8
cctcggtgcc gttggcggac agc 23
<210> 9
<211> 28
<212> DNA
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atgctcgctc tgctggcgct ctacacgg 28
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cgggtcggac tcgctgtcgt ttattgattc g 31
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gtcccaggtt cccatacact cac 23
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gtaccagtag ttcaccacct ccc 23
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gcgtgaacat cctcaccgac ttc 23
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gaaacagcag cgtcccgtct atc 23
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gggtcaagga cgccttctta agcgtctcg 29
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gggcacggca aactttattg ggatgacata ca 32
Claims (5)
1. for detection of the primer sets of PRV virulence gene sudden change, it is characterized in that, comprise the primer sets of increase respectively PRV virulence gene gE, gC, gD and TK,
Wherein, the primer sets of amplification gE comprises the primer sets of aligning primer shown in aligning primer shown in the primer sets of aligning primer shown in aligning primer shown in SEQ ID No.1 and SEQ ID No.2, the primer sets with aligning primer shown in aligning primer shown in SEQ ID No.3 and SEQ ID No.4 and SEQ ID No.5 and SEQ ID No.6;
Wherein, the primer sets of amplification gC comprises the primer sets of aligning primer shown in aligning primer shown in the primer sets of aligning primer shown in aligning primer shown in SEQ ID No.7 and SEQ ID No.8 and SEQ ID No.9 and SEQ ID No.10;
Wherein, the increase primer sets of described gD comprises the primer sets of aligning primer shown in aligning primer shown in the primer sets of aligning primer shown in aligning primer shown in SEQ ID No.11 and SEQ ID No.12 and SEQ ID No.13 and SEQ ID No.14;
Wherein, the increase primer sets of described TK is the primer sets of aligning primer shown in aligning primer shown in SEQ ID No.15 and SEQ ID No.16.
2. the application of primer sets claimed in claim 1 in detecting the sudden change of PRV virulence gene.
3. the PRV virulence gene sudden change detection reagent or the test kit that contain primer sets claimed in claim 1.
4. detect a method for PRV virulence gene sudden change, it is template for take the DNA of PRV, by primer sets claimed in claim 1, carries out pcr amplification, after each PCR product is checked order respectively, carries out sequence alignment.
5. method as claimed in claim 4, is characterized in that, uses 0.5～1.5 primer of μ l and the DNA profiling of 50～100ng in the reaction system of every 25 μ l, and reaction conditions is:
96～94 ℃ of denaturation 2min～5min;
96～94 ℃ of sex change 30s～40s;
60～64 ℃ of annealing 30s～60s;
72 ℃ are extended 30s～60s;
30-40 circulation;
72 ℃ are extended 10min.
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| CN201310652245.8A CN103695562A (en) | 2013-12-05 | 2013-12-05 | Detection method and detection kit for PRV (pseudorabies virus) virulence gene mutation |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152586A (en) * | 2014-08-18 | 2014-11-19 | 咸阳职业技术学院 | Multiplex nested polymerase chain reaction diagnosis kit for identifying pseudorabies gE gene deleted vaccine strain and wild strain, and detection method thereof |
| CN109913585A (en) * | 2019-03-25 | 2019-06-21 | 新乡学院 | A method of PRV is detected using PCR-ELISA |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775443A (en) * | 2010-01-06 | 2010-07-14 | 天津市畜牧兽医研究所 | LAMP kit for detecting PRV and preparation method thereof |
| CN102994458A (en) * | 2012-11-26 | 2013-03-27 | 中国农业科学院哈尔滨兽医研究所 | Porcine pseudorabies virus virulent strain, and gene deletion vaccine strain thereof and applications thereof |
-
2013
- 2013-12-05 CN CN201310652245.8A patent/CN103695562A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101775443A (en) * | 2010-01-06 | 2010-07-14 | 天津市畜牧兽医研究所 | LAMP kit for detecting PRV and preparation method thereof |
| CN102994458A (en) * | 2012-11-26 | 2013-03-27 | 中国农业科学院哈尔滨兽医研究所 | Porcine pseudorabies virus virulent strain, and gene deletion vaccine strain thereof and applications thereof |
Non-Patent Citations (4)
| Title |
|---|
| 刘红亮: "伪狂犬病病毒河南株的分离鉴定与TK基因的克隆和序列分析", 《中国优秀硕士学位论文全文数据（电子期刊）农业科技辑》 * |
| 杨安钢等主编: "《生物化学与分子生物学实验技术》", 30 June 2008, 高等教育出版社 * |
| 王键义: "猪伪狂犬病病毒gC基因的克隆及生物信息学分析", 《万方》 * |
| 陈磊: "伪狂犬病病毒新疆株的分离鉴定及其 gD 和gE 基因的克隆与序列分析", 《中国优秀硕士学位论文全文数据（电子期刊）农业科技辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152586A (en) * | 2014-08-18 | 2014-11-19 | 咸阳职业技术学院 | Multiplex nested polymerase chain reaction diagnosis kit for identifying pseudorabies gE gene deleted vaccine strain and wild strain, and detection method thereof |
| CN104152586B (en) * | 2014-08-18 | 2016-01-27 | 咸阳职业技术学院 | Differentiate pseudo-rabies gE gene-deleted vaccine strain and street strain's compound nested polymerase chain reaction diagnostic kit and detection method |
| CN109913585A (en) * | 2019-03-25 | 2019-06-21 | 新乡学院 | A method of PRV is detected using PCR-ELISA |
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