CN104789699A - PCR primer for identifying PRV virulent virus and PRV gG and PRV gE gene deletion vaccine viruses - Google Patents

PCR primer for identifying PRV virulent virus and PRV gG and PRV gE gene deletion vaccine viruses Download PDF

Info

Publication number
CN104789699A
CN104789699A CN201510154094.2A CN201510154094A CN104789699A CN 104789699 A CN104789699 A CN 104789699A CN 201510154094 A CN201510154094 A CN 201510154094A CN 104789699 A CN104789699 A CN 104789699A
Authority
CN
China
Prior art keywords
prv
gene
poison
pcr
primer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201510154094.2A
Other languages
Chinese (zh)
Inventor
吴志明
闫若潜
赵明军
赵雪丽
刘梅芬
郑岩
刘慧�
程果
王东方
刘光辉
程俊贞
韩楠
项黎黎
郭育培
谢霞
梁松林
宋松林
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
HENAN PROVINCIAL CENTER FOR ANIMAL DISEASE CONTROL AND PREVENTION
Original Assignee
HENAN PROVINCIAL CENTER FOR ANIMAL DISEASE CONTROL AND PREVENTION
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by HENAN PROVINCIAL CENTER FOR ANIMAL DISEASE CONTROL AND PREVENTION filed Critical HENAN PROVINCIAL CENTER FOR ANIMAL DISEASE CONTROL AND PREVENTION
Priority to CN201510154094.2A priority Critical patent/CN104789699A/en
Publication of CN104789699A publication Critical patent/CN104789699A/en
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/705—Specific hybridization probes for herpetoviridae, e.g. herpes simplex, varicella zoster
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention provides a PCR primer for identifying a PRV (Pseudorabies Virus) virulent virus and PRV gG and PRV gE gene deletion vaccine viruses. The primer comprises P1, P2, P3, P4, P5 and P6. The PRV virulent virus, the PRV gG gene deletion vaccine virus and the PRV gE gene deletion vaccine virus can be quickly identified and detected at the same time by one reaction based on triple PCR detection established by the primer, detection can be completed within 3h, the primer has the advantages of high quickness, specificity, sensitivity, high flux and the like, and the requirement for large-batch and quick identification and detection of the PRV virulent virus, the PRV gG gene deletion vaccine virus and the PRV gE gene deletion vaccine virus can be met.

Description

Differentiate the PCR primer of the strong poison of PRV and PRV gG, PRV gE gene-deleted vaccine poison
Technical field
The invention belongs to animal epidemic quarantine detection technique field, concrete, relate to and differentiate the strong poison of PRV and PRV gG gene-deleted vaccine is malicious, the PCR primer of PRV gE gene-deleted vaccine poison.
Background technology
Pseudo-rabies (Pseudorabies, PR) is acute, the high degree in contact sexually transmitted disease of the boar extensively occurred in world wide.This disease transmission is strong, the polyinfection of easy secondary bacterial and other viruses or secondary infection, mainly with the acute death of newborn piglet and 4 week age above piglet there is nervous symptoms, pregnant sow with breeding difficulty, miscarriage, stillborn foetus and respiratory symptom for feature.In recent years, especially 2012, PR occurred in succession in China, popular inside the province more than 23, occurred high incidence and mortality ratio, the piglet sudden onset within newborn and 4 week age, and mortality ratio can up to the new characteristics of incidence of 100% grade.This disease, without special effect medicine therapeutic, becomes one of main epidemic disease of harm pig industry.
PR cause of disease is Pseudorabies virus (Pseudorabies virus, PRV), and belong to the herpesvirus suis I type of herpetoviridae Alphaherpesvirinae Varicellavirus, full-length genome is 150kbp.In PRV, 11 kinds of glycoprotein are found at present, respectively called after gB, gC, gD, gE, gG, gH, gJ, gK, gL, gM and gN.Wherein the gD albumen of gD genes encoding and gI albumen form complex body, and participating in the adsorption process of virus, is important immunogenic protein.GG gene is the nonessential glycoprotein gene of a secretion property in PRV.The gE albumen of gE genes encoding is a kind of glycoprotein promoting cytogamy, the diffusion of mediate retroviral between cell to cell.Affecting the tissue tropisms of PRV in pig body, is necessary through gasserian ganglion intrusion central nervous tissue.
At present, the widest PRV gene-deleted vaccine is used to be gE gene-deleted vaccine both at home and abroad.The U.S. uses gE, gG and gC gene-deleted vaccine.But use gG gene-deleted vaccine in Japan, that China produces take Strain Ea as the T K that parent's strain builds more -/ gG -/ LacZ +gene-deleted vaccine high security, immune effect is better than same kind of products at abroad, at home also widespread use.
Existing existing PRV diagnostic method is mainly for the serology ELISA diagnostic method that totivirus antibody or gE antibody lack, there is the shortcomings such as complex operation, technical requirements and Test Condition Requirements are high, the test period is long in these methods, be difficult to meet quick, a large amount of needs differentiating to detect, current PCR detection method, for the judgement of the term single gene of PRV, cannot meet the needs differentiating fast to detect the strong poison of PRV and PRV gG and PRV gE gene-deleted vaccine poison.Therefore set up a kind of quick, efficient, special, responsive detection method, thus can differentiate fast to detect to the strong poison of PRV, PRVgG and PRV gE gene-deleted vaccine poison simultaneously, significant for making a definite diagnosis fast of PR.
Summary of the invention
The object of this invention is to provide a kind of triple PCR detection primer can differentiating to detect the strong poison of PRV, PRV gG gene-deleted vaccine poison and PRV gE gene-deleted vaccine poison fast.
Another object of the present invention is to provide the application of above-mentioned primer in the strong poison of PCR discriminating PRV and PRVgG, PRV gE gene-deleted vaccine poison.
In order to realize the object of the invention, the present inventor is PRV gD, gG, gE gene order in a large amount of comparison GenBank, choose PRV gD, gG, gE gene high conservative and the sequence with type specificity gene is template, design and synthesis PRV gD, gG, gE gene-specific primer pair, respectively called after PRV gD-F (P1), PRV gD-R (P2), PRV gG-F (P3), PRV gG-R (P4), PRV gE-F (P5) and PRV gE-R (P6); Thus, provide and a kind ofly differentiate the strong poison of PRV and PRV gG gene-deleted vaccine is malicious, the PCR primer of PRV gE gene-deleted vaccine poison, concrete:
P1:3 '-GGAGGACGAG CTGGG GCTGC-5 ' (shown in SEQ ID NO:1 sequence);
P2:3 '-CCACGCCCCG CTTGA AGCTG-5 ' (shown in SEQ ID NO:2 sequence);
P3:3 '-ACACCCGCAC GCGCATCGAC-5 ' (shown in SEQ ID NO:3 sequence);
P4:3 '-TCTCGGCGGC GGTCACGTTC-5 ' (shown in SEQ ID NO:4 sequence);
P5:3 '-GTCACCGAGG TCCCGAGTCC (shown in SEQ ID NO:5 sequence);
P6:3 '-GCGTGTAGAG GCCCGTGTCG (shown in SEQ ID NO:6 sequence).
Wherein, P1 and P2 is for the PRV gD fragment that increases, and fragment length is 212bp.
P3 and P4 is for the PRV gG fragment that increases, and fragment length is 315bp.
P5 and P6 is for the PRV gE fragment that increases, and fragment length is 472bp.
The present invention also provides containing above-mentioned primer for differentiating the test kit of the strong poison of PRV and PRV gG, PRVgE gene-deleted vaccine poison.
Wherein, can also contain positive reference substance and negative controls in test kit provided by the present invention, positive reference substance can be the PRV cell culture of deactivation, PRV gE gene-deleted vaccine poison and PRV gG gene-deleted vaccine poison.
The present invention further provides above-mentioned primer or test kit are differentiated in the strong poison of PRV and PRV gG, PRV gE gene-deleted vaccine poison application at PCR.
Optionally, described application comprises the following steps:
1) testing sample STb gene is extracted;
2) with the testing sample STb gene extracted for template, with P1, P2, P3, P4, P5, P6 for amplimer, carry out pcr amplification reaction obtain amplified production;
3) agarose gel electrophoresis detection is carried out to PCR primer.
Optionally, described testing sample can be lymphoglandula, cerebral tissue or gasserian ganglion.
Wherein, the present invention has no particular limits the method extracting described testing sample STb gene, and the Total DNA extraction method of this area routine can be adopted to carry out.
Optionally, the reaction system of carrying out pcr amplification reaction use is counted with 25 μ L: the final concentration of P1, P2, P3, P4, P5, P6 is respectively 0.4 μm of ol/L, 2 × GC Buffer I 12.5 μ L, 5U/ μ L Ex Taq enzyme 0.25 μ L, PRV (Pseudorabies virus) DNA 2 μ L, mends to 25 μ L with deionized water.
Optionally, pcr amplification reaction condition is: after 94 DEG C of 5min, 94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 30s, 35 circulations; 72 DEG C of 10min.
Qualification result judges: negative control does not amplify band, and positive control amplifies 212bp, 315bp and 472bp band respectively, and for test is set up, otherwise test is false; There is 212bp, 315bp and 472bp in sample P CR product simultaneously, show that this sample is PRV wild virus infection sample; There is 212bp, 315bp band in sample simultaneously, show that this sample is that gE/PRV deletion of vaccine strain is positive; There is 212bp, 472bp band in sample simultaneously, show that this sample is that gG/PRV deletion of vaccine strain is positive; Only there is 212bp band in sample, show that this sample is that the two deletion of vaccine strain of gG/PRV, gE/PRV is positive; Do not occur in sample that any band is judged to PRV and infects negative.
PRV gD/gG/gE triple PCR quick detection reagent of the present invention and discriminating detection method can realize a reaction and differentiate detection fast to the strong poison of PRV, PRV gG gene-deleted vaccine poison and PRV gE gene-deleted vaccine poison simultaneously, can complete in 3h, there is the advantages such as quick, special, responsive, high-throughput, requirement in enormous quantities, to differentiate to detect the strong poison of PRV and PRV gG gene-deleted vaccine poison, PRV gE gene-deleted vaccine poison fast can be met.
Accompanying drawing explanation
Fig. 1 is PRV gD/gG/gE triple PCR product electrophorogram.
In figure, M.100bp gradient DNA molecular amount mark, P1.PRV positive control, P2.gG gene-deleted vaccine poison positive control, P3.gE gene-deleted vaccine poison positive control, N. negative control.
Fig. 2 is the sensitivity test of PRV gD/gG/gE triple PCR
In figure, M.100bp gradient DNA molecular amount mark, 1.PRV gD/gG/gE gene masculine plasmid (1.0 × 10 7copy/μ L), 2.PRV gD/gG/gE gene masculine plasmid (1.0 × 10 6copy/μ L), 3.PRV gD/gG/gE gene masculine plasmid (1.0 × 10 5copy/μ L), 4.PRVgD/gG/gE gene masculine plasmid (1.0 × 10 4copy/μ L), 5.PRV gD/gG/gE gene masculine plasmid (1.0 × 10 3copy/μ L), 6.PRV gD/gG/gE gene (1.0 × 10 2copy/μ L), 7.PRV gD/gG/gE gene plasmid (1.0 × 10 1copy/μ L), 8.PRV gD/gG/gE gene masculine plasmid (1.0 × 10 0copy/μ L).
Fig. 3 is PRV gD/gG/gE triple PCR specific test
In figure, M.100bp gradient DNA molecular amount mark, P1.PRV positive control, P2.gG gene-deleted vaccine poison positive control, P3.gE gene-deleted vaccine poison positive control, N. negative control, 1.LP-PRRSV, 2.CSFV, 3.PPV, 4.JEV, 5.SS2.
Fig. 4 is PRV gD/gG/gE triple PCR application test
In figure, M.100bp gradient DNA molecular amount mark, P1.PRV positive control, P2.gG gene-deleted vaccine poison positive control, P3.gE gene-deleted vaccine poison positive control, N. negative control.Wherein P1,4-6,9,12,14,16,20-22,24,27,31 is that PRV gD/gG/gE gene test is positive, P2,2,7,17,23,32 is that PRV gD/gE gene (the weak poison of gG genetically deficient) detects positive, P3, and 11,13,19,26,29,33,35 is that PRV gD/gG gene (the weak poison of gE genetically deficient) detects the positive.
Fig. 5 is that PRV gD PCR method is to 35 parts of doubtful sample agarose gel electrophoresis detected result figure;
In figure, M.DL 2000Marker gradient DNA molecular amount marks, P1.PRV positive control, P2.gG gene-deleted vaccine poison positive control, P3.gE gene-deleted vaccine poison positive control, N. negative control, wherein P1, P2, P3,3-6,9,11-14,16,19-22,24-27,29,32,33,35 be that PRV gD gene test is positive, all the other samples be feminine gender.
Fig. 6 is that PRV gG PCR method is to 35 parts of doubtful sample agarose gel electrophoresis detected result figure;
In figure, M.100bp gradient DNA molecular amount mark, P1.PRV positive control, P2.gG gene-deleted vaccine poison positive control, P3.gE gene-deleted vaccine poison positive control, N. negative control, wherein P1, P2, P3,3-6,9,11-14,16,19-22,24-27,29,31,33,35 be that PRV gG gene test is positive, all the other samples be feminine gender.
Fig. 7 is that PRV gE PCR method is to 35 parts of doubtful sample agarose gel electrophoresis detected result figure;
In figure, M.100bp gradient DNA molecular amount mark, P1.PRV positive control, P2.gG gene-deleted vaccine poison positive control, P3.gE gene-deleted vaccine poison positive control, N. negative control, wherein P1, P2,3-8,10,13,15,17-18,21-25,28,32-33 is that PRV gE gene test is positive, all the other samples be feminine gender.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is experiment condition all conveniently, as Sambrook equimolecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, Molecular cloning:a laboratorymanual, 2001) condition of, or according to manufacturer's specification sheets advising.
The foundation of the triple PCR detection system of the strong poison of embodiment 1PRV, PRV gG gene-deleted vaccine poison and PRV gE gene-deleted vaccine poison
1. materials and methods
1.1 material
1.1.1 strain
PRV strain isolated is by animal epidemic prevention and control centralab of Henan Province isolation identification; PRVgE gene-deleted vaccine poison, PRVgG gene-deleted vaccine is preserved by animal epidemic prevention and control centralab of Henan Province with other contrast viruses or bacterial strain.
1.1.2 instrument and reagent
PCR amplification instrument, German Biometra Products; Labworks image acquisition and analysis software, U.S. Alpha Innotech Products; Thermostatic water bath vibrator (HZQ-Q), Harbin Dong Lian electronic technology development corporation, Ltd. product; Table-type high-speed refrigerated centrifuge, U.S. Heraeus Products; Ex Taq archaeal dna polymerase, dNTPs, DNA recovery test kit etc. are all purchased from precious biotechnology (Dalian) company limited, and pGEM-T Easy carrier, JM109 competent cell are purchased from Promega company.
1.2 method
1.2.1 design of primers and synthesis
PRV gD, gG, gE gene order in comparison GenBank, choosing PRV gD, gG, gE gene high conservative and having type specificity gene order is template, design PRV gD, gG, gE gene-specific primer pair, called after PRV gD-F (P1), PRV gD-R (P2), CDV-R (P2), PRV gG-F (P3), PRV gG-R (P4), PRV gE-F (P5), PRV gE-R (P6) respectively, synthesized by precious biotechnology (Dalian) company limited, the primer sequence for the amplification of PRVgD/gG/gE gene triple PCR is as follows:
P1：3’-GGAGGACGAG CTGGG GCTGC-5’
P2：3’-CCACGCCCCG CTTGA AGCTG-5’
P3：3’-ACACCCGCAC GCGCATCGAC-5’
P4：3’-TCTCGGCGGC GGTCACGTTC-5’
P5：3’-GTCACCGAGG TCCCGAGTCC-5’
P6：3’-GCGTGTAGAG GCCCGTGTCG-5’。
1.2.2 the preparation of STb gene
With the PRV cell (PK of deactivation 15cell) culture, PRV gE gene-deleted vaccine poison, PRV gG gene-deleted vaccine poison is positive control, normal PK 15cell is negative control, STb gene is extracted with the gasserian ganglion containing PRV or cerebral tissue measuring samples, concrete operations are as follows: the PRV cell culture getting deactivation respectively, PRV gE gene-deleted vaccine poison, PRVgG gene-deleted vaccine poison, negative control and measuring samples (getting supernatant after appropriate PBS grinds as first added during for organizing) each 100 μ L are in 1.5ml centrifuge tube, add 500 μ L Digestive systems (containing final concentration 10mM Tris-HCl (pH 8.0), final concentration 25mM EDTA (pH 8.0), final concentration 0.5%SDS (w/v), final concentration 100mM NaCl) and the mixing of 10 μ L Proteinase Ks (final concentration 20mg/mL), put 55 DEG C of water-bath 30min ~ 1h, add 500 μ L Tris balance phenol/chloroform/primary isoamyl alcohol (volume ratio is 25:24:1) mixed solution extractings, centrifugal rear Aspirate supernatant adds the isopropanol precipitating DNA of equal-volume through-20 DEG C of precoolings, precipitate by mass percent 75% washing with alcohol, dry, finally add 20 μ L TE buffer solution precipitations ,-20 DEG C frozen for subsequent use.
1.2.3PRV the preparation of standard substance
Positive amplification product containing PRV type strain gD, gG, gE gene is cloned in pGEM-T Easy carrier respectively, screens positive recombinant plasmid and send precious biotechnology (Dalian) company limited to adopt T7 and SP6 primer to check order.Its OD of PRV type strain gD, gG, gE gene recombination positive plasmid application spectrophotometric determination checking order correct 260and OD 280value and OD 260/ OD 280value, repeats 5 times, pGEM-T/PRV gD and pGEM-T/PRV gG, pGEM-T/PRV gE plasmid standard OD altogether 260mean value is respectively 6.745,6.537 and 6.836, OD 280mean value is respectively 1.932,1.996 and 1.941, OD 260/ OD 280mean value is respectively 1.892,1.8882 and 1.905; With reference to plasmid DNA copies number calculating method, the concentration of calculating pGEM-T/PRV gD and pGEM-T/PRV gG, pGEM-T/PRV gE plasmid DNA solution is respectively 9.58 × 10 10copy/μ L, 9.42 × 10 10copy/μ L and 9.65 × 10 10copy/μ L, quantitatively and be diluted to 1.0 × 10 0~ 1.0 × 10 10copy/μ L ,-20 DEG C save backup.
1.2.4PRV the optimization of gD/gG/gE gene triple PCR reaction conditions
The pGEM-T/PRV gD of step 1.2.3 gained, pGEM-T/PRV gG and PRVgE recombinant plasmid are diluted to final concentration 1.0 × 10 respectively 5copy/μ L is as detection template, P1/P2, P3/P4, P5/P6 primer pair dilutes for final concentration 0.05,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8 and 1.0 μm of ol/L respectively, application PCR instrument (German Biometra company) adopts matrix method to screen P1/P2, P3/P4, P5/P6 combination of primers of different concns, obtains for the best primer concentration of PRV gD/gG/gE triple PCR and optimum reaction condition.
25 μ L reaction systems, wherein: each 0.4 μm of ol/L, the 2.5mmol/L dNTPs 4 μ L of P1/P2, P3/P4, P5/P6 final concentration, 2 × GC Buffer I 12.5 μ L, 5U/ μ L Ex Taq enzyme 0.25 μ L, PRV (Pseudorabies virus) DNA 2 μ L, mend to 25 μ L with deionized water; Reaction conditions is: after 94 DEG C of 5min, 94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 30s, 35 circulations; 72 DEG C of 10min.Pcr amplification product carries out electroresis appraisal, the results are shown in Figure 1.In figure, M.100bp gradient DNA molecular amount mark, P1.PRV positive control, P2.gG gene-deleted vaccine poison positive control, P3.gE gene-deleted vaccine poison positive control, N. negative control.
1.2.5 sensitivity test
The pGEM-T/PRV gD of step 1.2.3 gained, pGEM-T/PRV gG and PRVgE recombinant plasmid are made 10 times of serial dilutions respectively, and two kinds of plasmid 1:1 ratio mixing, often kind of plasmid final concentration is adjusted to 1.0 × 10 7~ 1.0 × 10 0copy/μ L, totally 8 extent of dilution, the PCR reaction conditions optimized by step 1.2.4 carries out the sensitivity test of PRV gD/gG/gE triple PCR.
PRV gD/gG/gE triple PCR is 100 copy/μ L to pGEM-T/PRV gD, pGEM-T/PRV gG and PRV gE tri-kinds of plasmid minimum detectabilities, occur that size is the object band of 212bp, 315bp and 472bp, showing that set up PRV gD/gG/gE triple PCR detection method can detect bottom line is 100 copy/μ L.(the results are shown in Figure 2)
1.2.6 specific test
By method described in step 1.2.2, RNA is extracted to Pestivirus suis (CSFV), porcine reproductive and respiratory syndrome virus classical strains (LP-PRRSV), pig Japanese B encephalitis virus (JEV) etc. and carry out reverse transcription, the then extracting directly DNA such as pig parvoviral (PPV), streptococcus suis 2-type (SS2), set the PRV cell culture of deactivation, PRV gG gene-deleted vaccine poison, PRV gE gene-deleted vaccine malicious as positive control is positive control, normal PK simultaneously 15cell is negative control, and the PCR reaction conditions optimized by step 1.2.4 carries out pcr amplification to verify the specificity of the method.
Adopt the PRV gD/gG/gE triple PCR detection method set up to carry out amplification to PRV and can obtain the object fragment that size is 212bp, 315bp and 472bp, amplification is carried out to PRVgE gene-deleted vaccine poison and can obtain the object fragment that size is 212bp, 315bp, amplification is carried out to PRVgG gene-deleted vaccine poison and can obtain the object fragment that size is 212bp, 472bp; But normal PK15 cell culture fluid, pig parvoviral, PRRS virus, Pestivirus suis, pig Japanese B encephalitis virus, streptococcus suis 2-type all fail to amplify any band, and the result is shown in Fig. 3, in figure, M.100bp gradient DNA molecular amount mark, P1.PRV positive control, P2.gG gene-deleted vaccine poison positive control, P3.gE gene-deleted vaccine poison positive control, N. negative control, 1.LP-PRRSV, 2.CSFV, 3.PPV, 4.JEV, 5.SS2.
1.2.7 stability and replica test
Respectively by pGEM-T/PRV gD, the pGEM-T/PRV gG of 10 of 4 concentration times of serial dilutions and PRV gE restructuring equivalent plasmid mixture (often kind of plasmid final concentration 1.0 × 10 3~ 1.0 × 10 0) according to step 1,2, the 4 PCR reaction conditionss optimized carry out pcr amplification, each series setting 3 repetitions, to verify stability and the repeatability of the method.
Replica test result shows, concentration is 10 7-10 2repeat to be the positive for 3 times of copy/μ L 6 concentration, concentration is 1.0 × 10 1copy/μ L and 1.0 × 10 0copy/μ L 3 revision test results are feminine gender.Illustrate that the PRV gD/gG/gE triple PCR detection method set up has good stability and repeatability.
1.2.8 application test
According to the PRV gD/gG/gE triple PCR set up and PRV gD, PRV gG, PRVgE term single gene PCR detection method (with reference to GB/T 18641-2002), preparation detection reagent, with through deactivation PRV cell culture, PRVgE gene-deleted vaccine poison, PRVgG gene-deleted vaccine poison for positive control, normal PK 15cell is negative control, and (sample number into spectrum is: 1 ~ 35 has carried out applying detection, and detected result is shown in Fig. 4-7, compares analysis to the detected result of these two kinds of methods to infect sample to 35 parts of clinical doubtful PRV.
Result shows, the method adopting the present invention to set up carries out triple RT-PCR amplification to 35 parts of clinical signs of suspected samples, the triple positive of result PRV gD/gG/gE is 14 parts, PRV gD/gG double (gE genetically deficient) positive is 7 parts, PRV gD/gE double (gG genetically deficient) positive is 5 parts, and 9 increment product are negative.
PRV gD pcr amplification positive is adopted to be 26 parts, PRV gG pcr amplification positive is adopted to be 21 parts, PRV gE pcr amplification positive is adopted to be 19 parts, show that PRV gD/gG/gE triple PCR method that the present invention sets up is than equally high with the susceptibility of single PCR method, this PCR method detected result and PRV Virus Isolation and PRV gD, gG, gE gene sequencing result coincidence rate are 100%.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.

Claims (6)

1. differentiate the PCR primer of the strong poison of PRV and PRV gG, PRV gE gene-deleted vaccine poison, it is characterized in that, described primer comprises:
P1：3’-GGAGGACGAG CTGGG GCTGC-5’
P2：3’-CCACGCCCCG CTTGA AGCTG-5’
P3：3’-ACACCCGCAC GCGCATCGAC-5’
P4：3’-TCTCGGCGGC GGTCACGTTC-5’
P5：3’-GTCACCGAGG TCCCGAGTCC-5’
P6：3’-GCGTGTAGAG GCCCGTGTCG-5’。
2. the test kit for differentiating the strong poison of PRV and PRV gG, PRVgE gene-deleted vaccine poison containing primer described in claim 1.
3. test kit described in primer described in claim 1 or claim 2 differentiates the application in the strong poison of PRV and PRV gG, PRV gE gene-deleted vaccine poison at PCR.
4. application according to claim 3, is characterized in that, comprises the following steps:
1) testing sample STb gene is extracted;
2) with the testing sample STb gene extracted for template, with P1, P2, P3, P4, P5, P6 for amplimer, carry out pcr amplification reaction obtain amplified production;
3) agarose gel electrophoresis detection is carried out to PCR primer.
5. application according to claim 4, it is characterized in that, the reaction system of carrying out pcr amplification reaction use is counted with 25 μ L: the final concentration of P1, P2, P3, P4, P5, P6 is respectively 0.4 μm of ol/L, 2 × GC Buffer I 12.5 μ L, 5U/ μ L Ex Taq enzyme 0.25 μ L, PRV (Pseudorabies virus) DNA 2 μ L, mends to 25 μ L with deionized water.
6. the application according to claim 4 or 5, is characterized in that, pcr amplification reaction condition is: after 94 DEG C of 5min, 94 DEG C of 30s, 63 DEG C of 30s, 72 DEG C of 30s, 35 circulations; 72 DEG C of 10min.
CN201510154094.2A 2015-04-02 2015-04-02 PCR primer for identifying PRV virulent virus and PRV gG and PRV gE gene deletion vaccine viruses Pending CN104789699A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201510154094.2A CN104789699A (en) 2015-04-02 2015-04-02 PCR primer for identifying PRV virulent virus and PRV gG and PRV gE gene deletion vaccine viruses

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201510154094.2A CN104789699A (en) 2015-04-02 2015-04-02 PCR primer for identifying PRV virulent virus and PRV gG and PRV gE gene deletion vaccine viruses

Publications (1)

Publication Number Publication Date
CN104789699A true CN104789699A (en) 2015-07-22

Family

ID=53554888

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201510154094.2A Pending CN104789699A (en) 2015-04-02 2015-04-02 PCR primer for identifying PRV virulent virus and PRV gG and PRV gE gene deletion vaccine viruses

Country Status (1)

Country Link
CN (1) CN104789699A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101831506A (en) * 2010-04-14 2010-09-15 中国农业科学院哈尔滨兽医研究所 Kit for authenticating deletion of vaccine strain and wild strain of PRVgE
CN103224995A (en) * 2013-04-10 2013-07-31 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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101831506A (en) * 2010-04-14 2010-09-15 中国农业科学院哈尔滨兽医研究所 Kit for authenticating deletion of vaccine strain and wild strain of PRVgE
CN103224995A (en) * 2013-04-10 2013-07-31 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

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
赵雪丽等: "鉴别猪伪狂犬病毒gE基因缺失疫苗和野毒感染的二重PCR诊断方法的建立和应用", 《中国畜牧兽医学会家畜传染病学分会第八届全国全员代表大会暨第十五次学术研讨会论文集》 *
马兴杰: "鉴别猪伪狂犬病毒gE/gB/gG的纳米PCR及荧光定量PCR方法的建立及评价", 《中国优秀硕士学位论文全文数据库 农业科技辑》 *

Similar Documents

Publication Publication Date Title
CN104561374A (en) Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain
CN102230029B (en) Ternary RT-PCR (Reverse Transcription-Polymerase Chain Reaction) detection reagent and method of type O, type A and type Asial foot and mouth disease viruses
CN103757139B (en) Canine distemper virus and canine parvovirus duplex TaqMan-MGB fluorescent quantitative PCR (polymerase chain reaction) detection kit and detection method thereof
CN105483291B (en) Pig Delta coronavirus, Porcine epidemic diarrhea virus, transmissible gastro-enteritis virus multiple RT-PCR detection primer
CN110184390A (en) For identifying the double FQ-PCR detection kit of African swine fever and wild strains of classical swine fever virus
CN105567871A (en) RT-RPA (reverse transcription recombinase polymerase amplification) detection kit for fast detecting high-pathogenicity porcine reproductive and respiratory syndrome virus and application thereof
CN107326100A (en) Aftosa and the double real-time fluorescence quantitative PCR detection kit of Sai Nika paddy virus
CN104561368B (en) Six boar virus multiple real-time fluorescence quantitative PCR quick diagnosis reagent kits
Tao et al. Cocirculation of two transmission lineages of echovirus 6 in Jinan, China, as revealed by environmental surveillance and sequence analysis
CN103627816B (en) A kind of multiple RT-PCR detection kit of porcine reproductive and respiratory syndrome virus and application thereof
CN108866243B (en) Porcine enterocoronavirus 4-fold fluorescent quantitative PCR detection kit
CN105525040A (en) Real-time fluorescent RPA kit and test strip RPA kit for rapid detection of porcine type 2 circovirus and application of real-time fluorescent RPA kit and test strip RPA kit
CN103725793B (en) The multiple fluorescence quantitative RT-PCR amplifing reagent of PRRSV
CN107236824A (en) A kind of fluorescent quantitation RT PCR kits and application for porcine reproductive and respiratory syndrome virus specific detection
Wang et al. An overview of nucleic acid testing for the novel coronavirus SARS-CoV-2
CN102212617B (en) Primer pair, probe and kit for detecting classical swine fever virus wild strain
CN105907890A (en) Primers, probe and method for rapidly distinguishing HP-PRRS (High pathogenic porcine reproductive and respiratory syndrome) vaccine strain GDr180 from HP-PRRS wild strain
CN105886663A (en) Locked nucleic acid sensitivity-enhanced fluorescent quantitative PCR (polymerase chain reaction) detection reagent kit for wild strains of porcine pseudorabies viruses
Gao et al. Establishment of a dual real-time PCR assay for the identification of African swine fever virus genotypes I and II in China
Cao et al. A duplex fluorescent quantitative PCR assay to distinguish the genotype I and II strains of African swine fever virus in Chinese epidemic strains
CN103725794A (en) Fluorescent quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) primers, probes and method for detecting PRRSV (porcine reproductive and respiratory syndrome virus)
CN105112558B (en) The triple real time fluorescent quantitative RT PCR detection kits of the type of foot and mouth disease virus O, A and Asia I
CN105385683B (en) A kind of multiple RT PCR detection kits of Porcine epidemic diarrhea virus
CN105779650A (en) Triple fluorescent quantitative PCR primer, probe and kit for identifying pseudorabies virus strains
CN106119421B (en) QYYZ plants of pig blue-ear disease of fluorogenic quantitative detection primer, probe and kit

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
EXSB Decision made by sipo to initiate substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20150722