CN104561374A - Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain - Google Patents
Detection reagent and method for identifying porcine pseudorabies virus vaccine strain and wild strain Download PDFInfo
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Abstract
The invention provides a detection reagent and a method for identifying a porcine pseudorabies virus vaccine strains and a wild strain, and belongs to the field of animal pathogen detection. The detection reagent comprises two primer pairs as shown in SEQ ID NO.1-4 and probes as shown in SEQ ID NO.5-6. The invention further provides a method for identifying a porcine pseudorabies virus vaccine strain gD gene and a wild strain gE gene by using the detection reagent. The porcine pseudorabies virus vaccine strains and the wild strain can be simultaneously identified through dual fluorogenic quantitative PCR, high-flux detection on large-scale samples can be achieved, the detection reagent has the characteristics of rapidness, specificity, sensitivity, accuracy, simplicity and convenience, provides an effective tool for surveying porcine pseudorabies infection sources and tracing infection environments and disease sources, and has relatively good application value in prevention and control on porcine pseudorabies.
Description
Technical field
The present invention relates to animal epidemic inspection field, particularly relating to the bifluorescence quantification PCR primer for differentiating porcine pseudorabies virus vaccine strain and street strain and probe, the invention still further relates to the method and the detection reagent that utilize this primer and probe to carry out porcine pseudorabies virus vaccine strain gD gene and street strain gE gene test.
Background technology
Porcine pseudorabies is a kind ofly itching and acute infectious disease that encephalomyelitis is main clinic symptoms with heating, very of being caused by PRV (Pseudorabies virus) (Pseudorabies virus, PRV), can cause pregnant sow miscarriage, produce stillborn foetus and fetus mummification; Then cause nervous symptoms to newborn piglet, occur ataxia, paralysis, exhaustion is dead, mortality ratio 100%; Adult Pig is often inapparent infection, and the Adult Pig in inapparent infection of resistance to mistake is the major source of infection of this disease.Porcine pseudorabies is found in the U.S. the earliest, because of clinical manifestation and rabies similar, therefore claim pseudoabies.Before the sixties in 20th century, porcine pseudorabies does not cause significant damage to pig industry, but in 60 ~ seventies, the appearance of robbing strain causes the quantity that this disease is broken out on pig farm significantly to increase, and each age in days pig all can infect, symptom is also obviously aggravated, and this change extensively exists in the U.S. and West Europe.This disease in the world popular in rising trend, the popular of this disease is reported in existing more than 50 countries and regions in the world.China nineteen forty-seven Liu Yong is pure be separated to PRV from pig body since, existing 24 provinces, municipal report are popular.
PRV (Pseudorabies virus) belongs to herpetoviridae A type herpesviral subfamilies, herpesvirus suis I type.At present, PRV only finds One serotype, but different strain is variant in biological characteristics and virulence etc.PRV has pantropic, in the internal breeding of Various Tissues culturing cell, and can produce eosinophilic inclusion in obvious cytopathy and core, to rabbit kidney and porcine kidney cell particularly responsive.PRV genome is linear double-stranded DNA, and size is about 180kb, and GC% content is up to 74%.PRV genome is made up of 72 reading frames, codified 70 kinds of albumen, find at present and name have 11 kinds of glycoprotein, wherein gB, gC, gD, gE are relevant with viral virulence with the glycoprotein of gI genes encoding.The detection method of current existing PRV mainly contains virus purification discriminating, animal inoculation pvaccination test, immunological detection method, Serology test and molecular biology for detection.That detects for PCR is mainly gB, gE and gH gene, and wherein gE gene GC% content is that virus replication institute is necessary up to 74.16%, gH gene, and glycoprotein gH is comparatively conservative in simplexvirus.
Due to the vaccine that the widely used pseudorabies disease vaccine of current China is gE genetically deficient, this gene is one of Major Virulence Factors of PRV, but is not needed for virus replication, and gD is relevant with virus replication, is present in vaccine strain and street strain.Therefore, if a kind of method can differentiating gE gene and gD gene can be provided simultaneously, will contribute to differentiating fast to distinguish porcine pseudorabies street strain and gE gene-deleted strain (PRV vaccine strain), and contribute to effectively judging whether pig infects the PRV of street strain.At present, the deficiencies such as existing PRV detection method is low based on the method such as ELISA, PCR and virus purification ubiquity susceptibility, length consuming time, this is unfavorable for carrying out of these sick prevention and control very much.Therefore, if a kind of fluorescence PCR method can differentiating gE gene and gD gene can be provided simultaneously, to contribute to differentiating fast to distinguish porcine pseudorabies street strain and gE gene-deleted strain (PRV vaccine strain), contribute to effectively judging whether pig infects the PRV of street strain, significant to the prevention and control of this disease.
Summary of the invention
The object of the present invention is to provide for differentiate the Auele Specific Primer of porcine pseudorabies virus vaccine strain gD gene and street strain gE gene to and probe;
Another object of the present invention is the fluorescence quantifying PCR method and the detection reagent thereof that provide discriminating porcine pseudorabies virus vaccine strain and street strain.
For achieving the above object, the DNA sequence dna of the present invention to known porcine pseudorabies virus gD and gE gene specific gene is compared, filter out porcine pseudorabies virus gD and gE gene high conservative and there is type specificity gene order, 2 pairs of Auele Specific Primers that design detects gD gene and gE gene to 2 TaqMan MGB probes, called after PRV-F (P1) respectively, PRV-R (P2), PRV-F (P3), PRV-R (P4), PRV-MGB-FAM-probe (P5), PRV-MGB-VIC-probe (P6), 2 pairs of Auele Specific Primers pair, the nucleotide sequence of 2 TaqManMGB probes is respectively as shown in SEQ ID NO.5-6.
The invention provides above-mentioned 2 pairs of Auele Specific Primers and 2 TaqMan MGB probes are combined, can be used for bifluorescence quantifying PCR method and differentiate to detect porcine pseudorabies virus vaccine strain and street strain.
The invention provides a kind of detection reagent differentiating porcine pseudorabies virus vaccine strain and street strain, containing 2 pairs of Auele Specific Primers to 2 fluorescent probes; The right nucleotide sequence of the 2 pairs of Auele Specific Primers is as shown in SEQ ID NO.1 ~ 4, and the nucleotide sequence of 2 fluorescent probes is as shown in SEQID NO.5 ~ 6.
Detection reagent of the present invention also comprises: DNA cleavage liquid, fluorescent quantitation reaction solution, negative template, positive template, and described negative template is distilled water, and described positive template is porcine pseudorabies virus genomic dna.
Further, its 25 μ L total working system of detection reagent of the present invention is:
The working routine of detection reagent of the present invention is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 15sec, 60 DEG C of annealing 30sec, 40 circulations.
The present invention also provides a kind of detection reagent differentiating porcine pseudorabies virus vaccine strain and street strain.
The invention provides above-mentioned 2 pairs of Auele Specific Primers and in preparation, 2 fluorescent probes differentiate that the application in porcine pseudorabies toxic vaccine strain and street strain's detection reagent also belong to protection scope of the present invention.
The invention provides a kind of bifluorescence quantifying PCR method differentiating porcine pseudorabies virus vaccine strain gD gene and street strain gE gene, particularly, application 2 pairs of Auele Specific Primers of the present invention and 2 fluorescent probes, with PRV genomic dna for template, differentiated by fluorescence quantifying PCR method and realization.
Must set up negative control and positive control when aforesaid method detects sample at every turn, the judging criterion of two kinds of contrast establishments is as follows in the detection:
Under " FAM/NONE " and " VIC/NONE " double-tagging pattern, there are two specific amplification curve Ct value≤29.0 in positive control; Negative control should without specific amplification curve and Ct value > 32.0 or nothing.Be discontented with as negative control and positive control result and be enough to upper condition, it is invalid that this time experiment is considered as.
Sample results judging criterion is as follows:
During Ct value≤29.0, sample result is positive;
The sample result of Ct value >=32.0 is negative;
When Ct value is between 29.0 ~ 32.0, and occur that the sample of specific amplification curve needs repetition, result is that positive is judged to be the positive, and result is that negative patient is judged to be feminine gender; If revision test Ct value is still judged to be positive amplification lower than 32, be judged to be negative amplification more than 32.If detect in sample, gE and the gD Gene Double of PRV is positive, is judged to street strain.
The invention provides the Auele Specific Primer for differentiating porcine pseudorabies virus vaccine strain gD gene and street strain gE gene and probe, detect gD gene for PRV gE gene-deleted vaccine and examine and do not measure gE gene, establish the bifluorescence quantifying PCR method differentiating porcine pseudorabies virus vaccine strain gD gene and street strain gE gene, have very high detection sensitivity, the lowest detection detecting porcine pseudorabies virus vaccine strain gD and street strain gE gene is limited to 1.0 × 10
1copy/μ L; The method also has the specificity of 100%, and it is reproducible, can be used as the means detecting clinical samples, improves the positive rate differentiating porcine pseudorabies virus vaccine virus and street strain, simple to operate, can complete, greatly shorten sense cycle in 1h ~ 2h.Detection reagent of the present invention will contribute to differentiating fast to distinguish PRV street strain and gE gene-deleted strain (porcine pseudorabies vaccine strain), contribute to effectively judging whether pig infects the PRV of street strain, for porcine pseudorabies investigation of infectious agent, communication environments, cause of disease are reviewed and provided effective tool, significant to the prevention and control of this disease.
Accompanying drawing explanation
Fig. 1 is the double FQ-PCR amplification curve of PRV gD/gE gene, and wherein, 1 is VIC probe (P6 probe) gD gene amplification curve, and 2 is FAM probe (P5 probe) gE gene amplification curve, and 3,4 is negative control.
Fig. 2 is that the double FQ-PCR of PRV gD/gE gene detects gD sensitizer gene perception amplification curve; 1 ~ 7 is plasmid concentration 1.0 × 10 corresponding respectively
6, 1.0 × 10
5, 1.0 × 10
4, 1.0 × 10
3, 1.0 × 10
2, 1.0 × 10
1, 1.0 × 10
0copy/μ L; 8,9 is negative control.
Fig. 3 is that the double FQ-PCR of PRV gD/gE gene detects gD gene typical curve.
Fig. 4 is that the double FQ-PCR of PRV gD/gE gene detects gE sensitizer gene perception amplification curve: 1 ~ 6 is plasmid concentration 1.0 × 10 corresponding respectively
5, 1.0 × 10
4, 1.0 × 10
3, 1.0 × 10
2, 1.0 × 10
1, 1.0 × 10
0copies/ μ L, 7,8 is negative control.
Fig. 5 is that the double FQ-PCR of PRV gD/gE gene detects gE gene typical curve.
Fig. 6 is PRV gD/gE gene double FQ-PCR system single detection gD gene-specific amplification curve; 1:gD gene VIC passage amplification curve; 2:gE gene FAM passage amplification curve, 3,4: negative control.
Fig. 7 is the single detection of a PRV gD/gE gene double FQ-PCR system gE gene-specific amplification curve; 1:gE gene FAM amplification curve, 2:gD gene VIC amplification curve, 3,4: negative control.
Fig. 8 is that the double FQ-PCR of PRV gD/gE gene detects other virus-specific amplification curves; 1:gD gene amplification curve, 2:gE gene amplification curve, 3,4:CSFV cDNA, 5,6:PRRSV cDNA, 7,8:PCV-2DNA, 9,10:PPV DNA, 11,12: negative control.
Fig. 9 is the double FQ-PCR method repeatability of PRV gD/gE gene and stability test result figure; The plasmid concentration that D1-D3, D4-D6, D7-D9 are corresponding is respectively 1.0 × 10
5, 1.0 × 10
4, 1.0 × 10
3the gD gene amplification curve of copy/μ L, P1-P3, P4-P6, P7-P9, plasmid concentration corresponding is respectively 1.0 × 10
5, 1.0 × 10
4, 1.0 × 10
3the gE gene amplification curve of copy/μ L, D10, P10 are negative control.
Figure 10 is that PRV gD/gE gene double FQ-PCR method is to 47 parts of doubtful sample detection result figure; D+ is gD gene standard amplification curve, P+ is gE gene standard amplification curve, D (P) 2, D (P) 8-D (P) 9, D (P) 10, D (P) 13D (P) 17, D (P) 33-D (P) 39, D (P) 40--D (P) 41, D (P) 47 is for being the PRV gD/gE gene test positive, all the other samples are negative, and N is negative control.
Figure 11 is that PRV gD/gE gene RT-PCR method is to 48 parts of doubtful sample agarose gel electrophoresis detected result figure; P1 is the contrast of gD gene masculine, and P2 is the contrast of gE gene masculine, and P3 is gD/gE Gene Double positive control, and N is negative control, and 8,21,24,26,33 ,-37,39,41,47 is that gD/gE gene test is two positive, and all the other samples are negative.
Embodiment
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art.
The PRV (Pseudorabies virus) strain isolated of the embodiment of the present invention is separated by animal epidemic prevention and control centralab of Henan Province to be differentiated; Other contrast viruses or bacterial strain are preserved by animal epidemic prevention and control centralab of Henan Province.
The instrument used in embodiment: fluorescent PCR instrument, American AB I Products, model ABI7000; 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 detection reagent etc. are all purchased from precious biotechnology (Dalian) company limited, and pGEM-T Easy carrier, JM109 competent cell are purchased from Promega company.
Embodiment 1 differentiates the Auele Specific Primer of porcine pseudorabies virus vaccine strain gD gene and street strain gE gene and the design of probe
When the present invention designs primer and probe, note the catastrophe point avoiding sequence, through repeated screening and checking, PRV gD gene and PRV gE gene order in a large amount of comparison GenBank, choosing PRV gD gene and PRV gE gene high conservative and having type specificity gene order is template, design gD/gE Auele Specific Primer to TaqMan MGB probe, called after PRV-F (P1) respectively, PRV-R (P2), PRV-F (P3), PRV-R (P4), PRV-MGB-FAM-probe (Probe-P5), PRV-MGB-VIC-probe (Probe-P6), synthesized by precious biotechnology (Dalian) company limited, for gD/gE double TaqMan MGBFQ-PCR increase primer and probe sequence as follows:
Primer P1:GGTTCAACGAGGGCCAGTA (SEQ ID NO.1)
Primer P2:ATGAAGTCGGTGAGGATGTTCA (SEQ ID NO.2)
Primer P3:CGGCTTCGACGTCTGGTT (SEQ ID NO.3)
Primer P4:GCCATAGTTGGGYCCATTCG (SEQ ID NO.4)
Probe PROBE-P5:5'-FAM-CTGGTGTYCGTCGACG-MGB-3'(SEQ ID NO.5) (above-mentioned Y representative annexs base C or T)
Probe PROBE-P6:5'-VIC-CCGGAGAAACCGGAAG-MGB-3'(SEQ ID NO.6)
Embodiment 2 differentiates the foundation of the duplex fluorescent PCR detection method of porcine pseudorabies virus vaccine strain gD gene and street strain gE gene
1, the preparation of template ribonucleic acid: adopt Trizol method to extract total serum IgE.Concrete operations are as follows: get each 200 μ L of measuring samples respectively in 1.5ml centrifuge tube, add the Trizol of 600 μ L again in vortice concussion 2 ~ 3min, add 200 μ L chloroforms, after centrifugal, get supernatant and proceed in another 1.5ml centrifuge tube, add 200 μ L isopropanol precipitatings, mass percent 75% washing with alcohol precipitates, dry, finally uses 20 μ L DEPC (tetra-sodium diethyl ester) water dissolution precipitations, get 10 μ L for reverse transcription, all the other-20 DEG C preservations.
2, the preparation of template DNA: with the pig PK-15 cell culture of PRV for positive control, normal pig PK-15 cell is negative control, with the nose swab containing PRV, tonsilla, the measuring samples such as lung extract STb gene, concrete operations are as follows: the PRV cell culture getting deactivation respectively, negative control and each 100 μ L of measuring samples are in 1.5ml centrifuge tube, add 500 μ L Digestive systems: containing final concentration 10mM Tris-HCl, pH 8.0, final concentration 25mMEDTA, pH 8.0, weightmeasurement ratio final concentration 0.5%SDS, final concentration 100mM NaCl, then with 10 μ L final concentration 20mg/mL Proteinase K mixings, put 55 DEG C of water-bath 30min ~ 1h, add 500 μ L Tris balance phenol/chloroform/primary isoamyl alcohol, volume ratio is the extracting of 25:24:1 mixed solution, 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, drying, finally add 20 μ L TE buffer solution precipitations ,-20 DEG C frozen for subsequent use.
3, reverse transcription
Often pipe reverse transcription reaction system is containing following composition: 5 × M-MLV damping fluid 4 μ L; 2.5mmol/L dNTPs (triphosphate deoxy-nucleotide) 4 μ L; M-MLV ThermoScript II 0.5 μ L; RNA enzyme inhibitors 0.5 μ L; Random primer 1 μ L; Cumulative volume 10 μ L.In every pipe reverse transcription reaction system, add the RNA 10 μ L obtained by step 2.1,37 DEG C of water-bath 1h or be placed in PCR instrument 37 DEG C reaction 1h, after reaction terminates, 70 DEG C, 15min deactivation ThermoScript II, be directly used in pcr amplification below or-20 DEG C frozen for subsequent use.
4, the preparation of PRV standard substance
Positive amplification product containing PRV type strain gD gene and PRV type strain 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 restructuring positive plasmid application spectrophotometric determination checking order correct
260and OD
280value and OD
260/ OD
280value, repeats 5 times: pGEM-T/gD and pGEM-T/gE plasmid standard OD altogether
260mean value is respectively 3.501 and 3.681, OD
280mean value is respectively 1.924 and 1.966, OD
260/ OD
280mean value is respectively 1.819 and 1.872; With reference to plasmid DNA copies number calculating method, the concentration calculating pGEM-T/CDV and pGEM-T/CPV plasmid DNA solution is respectively 8.38 × 10
10copy/μ L and 8.71 × 10
10copy/μ L, quantitatively and be diluted to 1.0 × 10
0~ 1.0 × 10
10copy/μ L ,-20 DEG C save backup.
5, the optimization of the double FQ-PCR reaction conditions of PRV
PGEM-T/gD and the pGEM-T/gE recombinant plasmid of step 1.2.4 gained is diluted to final concentration 1.0 × 10 respectively
5copy/μ L is as detection template, P1/P2, P3/P4 primer pair dilutes for final concentration 0.05 respectively, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 1.0 μm of ol/L, Probe-P5, it is 0.05 that Probe-P6 probe is diluted to final concentration respectively, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 and 1.0 μm of ol/L, application fluorescent PCR instrument (American AB I company, model: ABI7000) adopt matrix method to screen primer and the probe combinations of different concns, screen the best primer concentration of double FQ-PCR for gD/gE gene, concentration and probe concentration and optimum reaction condition:
25 μ L reaction systems, the each 0.4 μm of ol/L of gD and gE upstream and downstream primer P1/P2, P3/P4 final concentration, Probe-P5 final concentration is 0.6 μm of ol/L, Probe-P6 final concentration is 0.7 μm of ol/L, 2.5mmol/L dNTPs 2 μ L, 10 × Ex Taq damping fluid 2.5 μ L, 5U/ μ L Ex Taq enzyme 0.25 μ L, 1.0 × 10
5the each 2 μ L of plasmid template of copy/μ L, supply deionized water to final volume 25 μ L; The response procedures optimized is: 94 DEG C, after 5min, and 94 DEG C of 15s, 60 DEG C of 30s, 40 circulations.
Result judges: threshold setting is as the criterion with the vertex of threshold line just above normal negative controls amplification curve, and different instrument can adjust according to instrument noise situation.The detected result of negative control should without specific amplification curve, and Ct value > 32.0 or nothing, and the Ct value of positive control should≤29.0, and occur specific amplification curve, judge that detected result is set up.Be discontented with as negative control and positive control result and be enough to upper condition, it is invalid that this time experiment is considered as.
Under the prerequisite that detected result is set up, if sample detection result Ct value≤29.0, and there is specific amplification curve, be judged to be the positive; Ct value > 32.0 or nothing, and without specific amplification curve, be then judged to be feminine gender; 32.0 >=Ct value > 29.0 and to have the sample of specific amplification curve to be judged to be suspicious, again test suspicious specimen, result is that positive is judged to be the positive, and result is that negative patient is judged to be feminine gender; Again test suspicious specimen, result answers this animal sample of Resurvey again to test for suspicious person again.
Embodiment 3 differentiates the evaluating characteristics of porcine pseudorabies virus vaccine strain gD gene and street strain gE gene duplex fluorescent PCR detection method
1, the foundation of sensitivity test and typical curve
PGEM-T/gD and the pGEM-T/gE recombinant plasmid of embodiment 2 gained is 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 FQ-PCR reaction conditions optimized by embodiment 2 carries out the double FQ-PCR sensitivity test of gD/gE gene.Respectively with the logarithm of pGEM-T/gD and pGEM-T/gE recombinant plasmid starting template number for X-axis, with FQ-PCR cycle index C
tvalue makes regression curve for Y-axis, sets up the typical curve of double FQ-PCR method.
Result shows double FQ-PCR detection gD and gE minimum detectability and is 1.0 × 10
1copy/μ L (see Fig. 2,4).GD gene typical curve is set up by sensitivity Detection result, relation conefficient is 0.993, slope is-3.35, and intercept is 34.9, thus can draw the linear relationship expression formula between copy number (X) and Ct value: Ct=-3.35 × log copy number+34.9; GE gene typical curve is set up by sensitivity Detection result, relation conefficient 0.998, slope is-3.44, and intercept is 34.70, thus can draw the linear relationship expression formula between copy number (X) and Ct value: Ct=-3.44 × log copy number+34.70.The initial copy number (see Fig. 3,5) that expression formula just can calculate gD gene and gE gene is substituted into according to the Ct value that instrument reads.
2, specific test
By method described in embodiment 2, porcine reproductive and respiratory syndrome virus (PRRSV), Pestivirus suis (CSFV) are extracted to RNA and carried out reverse transcription, porcine circovirus 2 type (PCV2), pig parvoviral (PPV) then extracting directly DNA, set PRV type strain equal cell culture mixture through deactivation as positive control simultaneously, normal PK-15 cell and mdck cell are negative control, carry out FQ-PCR amplification with the specificity verifying the method by the FQ-PCR reaction conditions of embodiment 2.Result display positive control gD and the Ct value of gE gene are respectively 20.2 and 20.8 (set PRV type strain equal cell culture mixture as positive control) through deactivation, and occur specific amplification curve; But all there is not specific amplification curve in normal PK-15 cell, mdck cell, porcine reproductive and respiratory syndrome virus (PRRSV), Pestivirus suis (CSFV), porcine circovirus 2 type (PCV2), pig parvoviral (PPV), sees Fig. 6,7,8.
3, stability and replica test
Equivalent plasmid mixture of being recombinated by pGEM-T/gD and pGEM-T/gE of 10 of 3 concentration times of serial dilutions respectively increases according to the FQ-PCR reaction conditions of embodiment 2, and each series setting 3 repetitions, to verify stability and the repeatability of the method.Replica test result shows, three concentration gD/gE gene recombination plasmid equal amount of mixture, 3 test-results are the positive, negative control experiments result is feminine gender, and error less (see Fig. 9) between 3 repetitions, illustrate that the gD/gE gene double FQ-PCR method set up has good stability and repeatability.
Embodiment 4 clinical application
The double FQ-PCR detection method of PRV gD/gE gene set up according to the embodiment of the present invention 2 and duplex PCR method, preparation detection reagent, with the PRV type strain equal cell culture mixture of deactivation for positive control, normal Pk-15 cell and mdck cell are negative control, (sample number into spectrum is: 1 ~ 47) carried out applying detection to infect sample to 47 parts of clinical doubtful PRV, analysis is compared to the detected result of these two kinds of methods, and to differentiate with traditional method PRV virus purification and sequencing result compares.Result shows, adopts the double FQ-PCR of gD/gE gene of the present invention to detect, and 16 parts is that gD/gE Gene Double is positive, and numbering is respectively 8 ~ 10,13,17,21,33 ~ 39,41,42,47; Common duplex PCR is adopted to detect (the 8th national member representative assembly of livestock contagious disease branch of Chinese animal and veterinary association and the 15th scientific seminar's collection of thesis; Differentiate foundation and the application of the duplex PCR diagnostic method of PRV (Pseudorabies virus) gE gene-deleted vaccine and wild virus infection; Zhao Xueli, Yan Ruoqian, Chen Huijuan, Zhao Mingjun etc.; On June 9th, 2014) 12 parts be that gD/gE Gene Double is positive, numbering is respectively 8,21,24,26,33 ~ 37,39,41,47 (see Figure 10,11).Show that the gD/gE double FQ-PCR method that the present invention sets up is higher than the susceptibility of PCR method, this FQ-PCR method detected result and gD/gE virus purification differentiate and PRV gD gene, PRV 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 (10)
1. one kind for differentiating the Auele Specific Primer pair of porcine pseudorabies virus vaccine strain gD gene and street strain gE gene, and its nucleotide sequence is containing, for example the sequence shown in SEQ ID NO.1 ~ 4.
2. with claim 1 described in primer with the use of fluorescent probe, its nucleotide sequence is containing, for example the sequence shown in SEQ ID NO.5 ~ 6.
3. differentiate a detection reagent for porcine pseudorabies virus vaccine strain and street strain, it is characterized in that, containing 2 pairs of Auele Specific Primers to 2 fluorescent probes; The right nucleotide sequence of the 2 pairs of Auele Specific Primers is as shown in SEQ ID NO.1 ~ 4, and the nucleotide sequence of 2 fluorescent probes is as shown in SEQ ID NO.5 ~ 6.
4. detection reagent as claimed in claim 3, it is characterized in that, also comprise: DNA cleavage liquid, fluorescent quantitation reaction solution, negative template, positive template, described negative template is distilled water, and described positive template is porcine pseudorabies virus genomic dna.
5. detection reagent as claimed in claim 3, it is characterized in that, its 25 μ L total working system is:
6. detection reagent as claimed in claim 3, it is characterized in that, its working routine is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 15sec, 60 DEG C of annealing 30sec, 40 circulations.
7. the application in porcine pseudorabies toxic vaccine strain and street strain's detection reagent differentiated by probe described in primer described in claim 1 and claim 2 in preparation.
8. differentiate a method for porcine pseudorabies virus street strain and vaccine strain, it is characterized in that, adopt 2 pairs of Auele Specific Primers to carry out the detection of bifluorescence quantifying PCR method to 2 fluorescent probes to sample template; The right nucleotide sequence of described 2 pairs of Auele Specific Primers is as shown in SEQ ID NO.1 ~ 4, and the nucleotide sequence of 2 fluorescent probes is as shown in SEQ ID NO.5 ~ 6; If detect in sample, gE and the gD Gene Double of PRV is positive, then there is street strain in sample; If the gD gene masculine of PRV only detected in the sample to which, the gE gene of PRV is negative, then contain vaccine strain in sample.
9. method as claimed in claim 8, is characterized in that, according to Ct value judged result.
10. method as claimed in claim 8, it is characterized in that, the program of described bifluorescence quantifying PCR method is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 15sec, 60 DEG C of annealing 30sec, 40 circulations.
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| CN105483287A (en) * | 2015-12-22 | 2016-04-13 | 广东省实验动物监测所 | HRM detection method and primer for quickly differentiating porcine pseudorabies virus vaccine strain Bartha-K61 from other strains |
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| CN107460194A (en) * | 2017-09-19 | 2017-12-12 | 中国动物疫病预防控制中心 | The dual droplet digital pcr absolute quantitation detection kit of Pseudorabies virus gB, gE |
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| CN111154917A (en) * | 2020-02-22 | 2020-05-15 | 南京农业大学 | PCR primer and method for identifying porcine pseudorabies virus variant strain |
| CN112011646A (en) * | 2020-09-11 | 2020-12-01 | 北京市动物疫病预防控制中心 | Primer, probe and kit for combined detection of African swine fever virus and wild strain of porcine pseudorabies virus and application of primer, probe and kit |
| CN112522446A (en) * | 2020-12-25 | 2021-03-19 | 河南宏信检测技术有限公司 | Detection primer pair and kit for wild strain of porcine pseudorabies virus |
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