CN104152586B - Differentiate pseudo-rabies gE gene-deleted vaccine strain and street strain's compound nested polymerase chain reaction diagnostic kit and detection method - Google Patents
Differentiate pseudo-rabies gE gene-deleted vaccine strain and street strain's compound nested polymerase chain reaction diagnostic kit and detection method Download PDFInfo
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Abstract
The discriminating pseudo-rabies gE gene-deleted vaccine strain that the present invention relates to and street strain's compound nested polymerase chain reaction diagnostic kit and detection method, belong to animal medicine animal epidemic diagnostic technique in molecular biology field.Differentiate pseudo-rabies gE gene-deleted vaccine strain and street strain's compound nested polymerase chain reaction diagnostic kit, this test kit expands reaction mixture, negative control and positive controls by an expansion reaction mixture, two and forms.The present invention will adopt sleeve type PCR to increase, and object fragment is less, ensure that the highly sensitive of reaction.Detect and control total time, at 4 hours, to reach the object of rapid detection.The present invention thoroughly can solve pseudo-rabies epidemic isolates and gE gene-deleted vaccine strain differential diagnosis problem, fast, sensitive, specificity is good, be applicable to very much the big area rapid detection generaI investigation of pseudo-rabies epidemic situation, be applicable to the big-and-middle-sized plant that each laboratory, animal epidemic prevention and control center and condition possess.
Description
Technical field
The discriminating pseudo-rabies gE gene-deleted vaccine strain that the present invention relates to and street strain's compound nested polymerase chain reaction diagnostic kit and detection method, belong to animal medicine animal epidemic diagnostic technique in molecular biology field.
Background technology
Pseudoabies (Pseudorabies, PR) aujesky's disease, herpesvirus suis I type etc. is had another name called, by subcutaneous ulcer exanthema virus section (Herpesvirdae), one or more animal infected acute infectious diseases that Pseudorabies virus (PRV) in α-exanthema virus subfamily (Alpherpesvirinae) causes, are still one of main swinery transmissible disease of China so far.Pseudorabies virus can cause farrowing sow miscarriage, stillborn foetus, mummy tire; Newborn piglet exhaustion is dead, and mortality ratio can reach 100%; Adult Pig is many in inapparent infection, and the malicious toxin expelling of long-term band, becomes the major source of infection of this disease, owing to lacking clinical symptom, is with malicious pig to be easy to out in the cold, causes pig farm Pseudorabies virus long-term existence, endanger huge.Since this disease of Hungary's Late Cambrian in 1902, this disease is widely current all over the world.In China, from the pure reported first of Liu Yong in 1984 this after being ill, in succession there is this disease in existing more than 20 province up to now, to the pig industry of China particularly intensive pig production cause tremendous economic loss.In view of the importance of pseudo-rabies, the Ministry of Agriculture of China is classified as two class animal epidemics.
Vaccine immunization is then one of anti-system and even the Main Means eradicating this disease.Since 20 century 70s, the natural attenuated vaccine of artificial screening and the widespread use of engineered deletion vaccine, make significant contribution to anti-pseudoabies processed.At China's widely used Barth-k61 strain attenuated vaccine, have in its gene and comprise whole gE and most of gI gene order disappearance.Gene-deleted vaccine reducing chance that pseudoabies infects, alleviate metainfective symptom, reduce loose poison amount etc. in had very large improvement compared with Attenuate vaccine and deactivation vaccine, thus gene-deleted vaccine has become the Main Means of current prevention and control pseudoabies, and the plan of the elimination pseudoabies of the many countries in west all relies on gene-deleted vaccine and carries out immunization and formulate.
The serological method of the diagnosis pseudorabies that China uses has Virus Isolation, serum neutralization test (SN), agar diffusion test (AGP), latex agglutination test (LA), enzyme linked immunosorbent assay (ELISA) and immunofluorescence technique (IF), and molecular biology method has round pcr and nucleic acid hybridization technique.Virus Isolation is one of the most reliable way of diagnosis pseudo-rabies, but maximum shortcoming is length consuming time, and susceptibility is low.The advantages such as serum neutralization test, agar diffusion test and latex agglutination test are that specificity is good, and shortcoming is that susceptibility is lower.ELISA can distinguish vaccine immunity antibody and wild virus infection antibody, and highly sensitive, is widely used at present.But it be that wild malicious antibody positive does not represent swinery just to infect pseudo-rabies at present wild malicious that ELISA detects swinery, once infected wild poison caused because wild malicious antibody positive also may be swinery.Therefore, the etiologic diagnosis of the wild poison of pseudo-rabies is still needed to rely on the detection of cause of disease.
Along with the develop rapidly of Protocols in Molecular Biology, the advantages such as round pcr is quick with it, sensitivity, easy handling are widely applied in animal epidemic diagnosis.Belak etc., for gB gene design primer, use the method for PCR to detect Pseudorabies virus the earliest.Jestin etc., from nose swab extracting nucleic acid, detect Pseudorabies virus by the gD gene order of the PRV that increases.TalmageTB etc. apply gB gene primer and increase to the tonsil cell of biopsy and parenchymal tissue, can be applied to the inspection of latent infection.Domestic Lou is brilliant, Fan Weixing etc. also establishes the PCR method detecting PRV respectively, can be used for diagnosis and the epidemiology survey of pseudoabies.Because China adopts vaccine prevention to be main prevention and control strategy for many years always, swinery vaccine immunity fraction of coverage is high, causes vaccine strain extensively to exist in swinery.Standard PCR technology can not distinguish vaccine virus and the wild poison of pseudo-rabies, and the judgement of diagnostic result has difficulties.
The domestic scholar of having attempts setting up relevant differential diagnostic method, differentiates the wild poison of porcine pseudorabies and vaccine virus diagnostic method, the nucleic acid-templated amount of 106pg tri-gene-deleted vaccine poison or the wild poison of 756pgPRV can be detected as Liu Lina etc. establishes multiplex PCR.But, author also recognizes, multiple PCR method to the purity of pathological material of disease template and content requirement higher than single PCR method, so to be applied to veterinary clinic, also need further research, and method is not applied to and detects clinical pathology organization material by author, can not determine its performance in clinical application.Lin Fang etc. also establish multiple PCR method, devise 3 pairs of primer pairs, four kinds of pseudo-rabies vaccine sample DNA and carry out multiplexed PCR amplification, and wherein a kind of vaccine obtains and designs 1 specific band conformed to, and all the other are 2.But its maximum defect is still the cell toxicant being only limitted to pure culture, do not carry out the direct-detection test of clinical sample, so can its method be applied to and produce actual, do not need cell cultures to carry out virus isolation and the material such as direct-detection tissue or serum, there is no final conclusion.
Summary of the invention
For solving the problem, the present invention is intended to propose a kind of easy and simple to handle, visual result, the discriminating pseudo-rabies gE gene-deleted vaccine strain that highly sensitive, specificity is good and street strain's compound nested polymerase chain reaction diagnostic kit.
The technical scheme realizing this goal of the invention is as follows: differentiate pseudo-rabies gE gene-deleted vaccine strain and street strain's compound nested polymerase chain reaction diagnostic kit, and this test kit expands reaction mixture, negative control and positive controls by an expansion reaction mixture, two and forms;
Described one expands reaction mixture by sterilizing tri-distilled water, 2 × GCBuffer, 10mmolL
-1dNTP, 25 μm of olL
-1pRgD-1F upstream primer, 25 μm of olL
-1pRgD-1R downstream primer, 25 μm of olL
-1pRgE-1F upstream primer, 25 μm of olL
-1pRgE-1R downstream primer, 5U/ μ LrTaqDNA polysaccharase form, and each reaction volume, than being 7:12.5:1:0.5:0.5:0.5:0.5:0.5, amounts to 23 μ L, and 50 reactions amount to 1150 μ L, dress up 1 pipe;
Described two expand reaction mixture by sterilizing tri-distilled water, 2 × GCBuffer, 10mmolL
-1dNTP, 25 μm of olL
-1pRgD-2F upstream primer, 25 μm of olL
-1pRgD-2R downstream primer, 25 μm of olL
-1pRgE-2F upstream primer, 25 μm of olL
-1pRgE-2R downstream primer, 5U/ μ LrTaqDNA polysaccharase form, and each reaction volume, than being 7:12.5:1:0.5:0.5:0.5:0.5:0.5, amounts to 23 μ L, and 50 reactions amount to 1150 μ L, dress up 1 pipe;
Described negative control is PK-15 normal cell culture supernatant;
Described positive control is gE Gene deletion mutation Batha-k61 cell culture fluid;
Wherein, described primer PRgD-1F sequence is: 5 '-TAAAATTGGGTCGGCGTCC-3 '
Described primer PRgD-1R sequence is: 5 '-CGCCCTCAGGAATCGGAC-3 '
Described primer PRgE-1F sequence is: 5 '-GTTCAGAGCATGCGCCGG-3 '
Described primer PRgE-1R sequence is: 5 '-CGACAGCAGCCAGACGC-3 '
Described primer PRgD-2F sequence is: 5 '-GACCGAGCACGAGGTGCG-3 '
Described primer PRgD-2R sequence is: 5 '-GTCCACGCGCGCCTTGA-3 '
Described primer PRgE-2F sequence is: 5 '-ATCGCGATCGGCGAGTCA-3 '
Described primer PRgE-2R sequence is: 5 '-CGAGTCGAGCGTGCCACTA-3 '.
Another goal of the invention of the present invention: a kind of method of inspection differentiating pseudo-rabies gE gene-deleted vaccine strain and street strain's compound nested polymerase chain reaction diagnostic kit is provided, comprises the following steps:
1) DNAzolReagent reagent method extracts measuring samples DNA, and seasoning in air, with 40 μ L8mmolL
-1naOH dissolves;
2) get an expansion mixed solution 23 μ L, add DNA solution 2 μ L, the laggard performing PCR amplification of Homogeneous phase mixing, reaction conditions is 95 DEG C, denaturation 5min, 94 DEG C of 60s, 62 DEG C of 60s, and 72 DEG C of 60s carry out 35 circulations altogether, and last 72 DEG C extend 10min;
3) get two expansion mixed solution 23 μ L, add the thing 2 μ L that expands production, mix and carry out pcr amplification, reaction conditions is 95 DEG C of denaturation 5min, 94 DEG C of 60s, 65 DEG C of 60s, and 72 DEG C of 60s carry out 35 circulations altogether, and last 72 DEG C extend 10min;
4) 10gL
-1electrophoresis in sepharose;
5) interpretation of result: negative control does not go out band, positive control goes out 217bp band, illustrates that contrast is set up; There is 217bp band in sample, be gE gene-deleted vaccine strain; There are 217bp, 354bp two bands in sample, is pseudo-rabies epidemic isolates and infects.
The proving test of test kit:
1. specific test
Use diagnostic kit of the present invention, illustrate that the DNA of extraction Batha-k61 pseudo-rabies gene-deleted vaccine, the wild DNA virus such as malicious SXHX13 strain, PCV-2, PPV of pseudo-rabies is as template according to DNAzolReagent reagent, expand mixed solutions with an expansion mixed solution and two of test kit and carry out compound sleeve type PCR, electrophoresis observation after amplification.Illustrate according to TRIzolReagent reagent and extract hog cholera lapinised virus living vaccine, porcine reproductive and respiratory syndrome attenuated vaccine and Porcine epidemic diarrhea virus SXJY12 strain total serum IgE, reverse transcription obtains the first chain cDNA, with diagnostic kit one of the present invention expand mixed solution and two expand mixed solutions carry out compound sleeve type PCR, electrophoresis observation after amplification.
Result shows, and 217bp band appears in Batha-k61 pseudo-rabies Gene deletion mutation; There are 217bp, 354bp two bands in the wild malicious SXHX13 strain of pseudo-rabies; All there is not band (see accompanying drawing 1) in PCV-2, PPV, hog cholera lapinised virus living vaccine, porcine reproductive and respiratory syndrome attenuated vaccine and Porcine epidemic diarrhea virus SXJY12 strain.
Reclaim the band of each positive sample, connect pMD18-T carrier after purifying, transform DH5 ɑ competent cell, get PCR and be accredited as positive bacterium liquid, extract plasmid and check order, the sequence recorded and strain gene order used are compared, discovery sequence is completely the same.Result proved test kit and detection method have very high specificity, and can accurately increase PRV gene fragment, and can intuitively distinguish gE Gene deletion mutation and epidemic isolates.
2. sensitivity test
Use diagnostic kit provided by the invention, extract the wild malicious SXHX13 strain DNA of pseudo-rabies, it is 278pgL that ultraviolet spectrophotometer measures concentration
-1, be diluted to 10 according to 10 times of concentration gradients
-9, the expansion mixed solution provided with test kit and two expands mixed solutions and carries out shell type composite PCR, electrophoresis observation after amplification.Result shows, and the limit that test kit of the present invention can detect is 2.78 × 10
-4pgL
-1, prompting present method highly sensitive (see accompanying drawing 2).
3. stability test
Diagnostic kit preservation condition provided by the invention is-20 DEG C, monthly carries out detection experiment, continuous detecting 6 months, the stability that detection kit is deposited with Batha-k61 pseudo-rabies gene-deleted vaccine, the wild malicious SXHX13 strain of pseudo-rabies and contrast 1 time.Result shows, and in 6 months, test kit detected result is completely the same, illustrates that test kit has satisfactory stability.
4. field test
Acquire the clinical doubtful pseudo-rabies in Shaanxi Province and organize pathological material of disease and serum totally 65 parts, diagnose with test kit of the present invention and detection method, carry out Parallel testing test with the PCR method detecting merely gD gene simultaneously.The result positive rate that detects of the present invention is 64.6%(42/65), it is 66.2%(43/65 that the simple PCR method detecting gD gene detects positive rate), two kinds of method coincidence rate 97.6%(42/43), point out two kinds of method coincidence rates high (see accompanying drawing 3).
The present invention will adopt sleeve type PCR to increase, and object fragment is less, ensure that the highly sensitive of reaction.Detect and control total time, at 4 hours, to reach the object of rapid detection.The present invention thoroughly can solve pseudo-rabies epidemic isolates and gE gene-deleted vaccine strain differential diagnosis problem, fast, sensitive, specificity is good, be applicable to very much the big area rapid detection generaI investigation of pseudo-rabies epidemic situation, be applicable to the big-and-middle-sized plant that each laboratory, animal epidemic prevention and control center and condition possess.
Accompanying drawing explanation
Fig. 1 is test kit of the present invention and method specific test electrophorogram;
In figure, M is DL2000DNA molecular mass standard, 1 is Batha-k61 pseudo-rabies Gene deletion mutation, 2 is the wild malicious SXHX13 strain of pseudo-rabies, 3 is PCV-2 strain, 4 is PPV strain, 5 is hog cholera lapinised virus living vaccine strain, and 6 is porcine reproductive and respiratory syndrome attenuated vaccine strain, and 7 is Porcine epidemic diarrhea virus SXJY12 strain.
Fig. 2 is test kit of the present invention and method sensitivity test electrophorogram;
In figure, M is DL2000DNA molecular mass standard, and 1-9 is the wild malicious SXHX13 strain DNA ladder degree dilution of pseudo-rabies, and concentration range is 278 × 10
-1pgL
-1~ 278 × 10
-9pgL
-1.
Fig. 3 is that test kit of the present invention is to partial clinical sample detection result;
In figure, M is DL2000DNA molecular mass standard, and 1-7 is portion of tissue pathological material of disease detected result, and wherein 3,6 is vaccine virus positive findings; 1,2,4,5,7 is the wild malicious positive findings of pseudo-rabies.
Embodiment
Embodiment 1
1, the Design & preparation of primer
With reference to the PRV whole genome sequence that GenBank announces, in conjunction with the PRV gene order that the research of this seminar several years measures, comprehensively analyse and compare with DNAStar biosoftware, 8 primers PRgD-1F, PRgD-1R, PRgE-1F, PRgE-1F, PRgD-2F, PRgD-2R, PRgE-2F and PRgE-2F for gD, gE gene design, the sequence of primer is as follows:
PRgD-1F：5’-TAAAATTGGGTCGGCGTCC-3’
PRgD-1R：5’-CGCCCTCAGGAATCGGAC-3’
PRgE-1F：5’-GTTCAGAGCATGCGCCGG-3’
PRgE-1R：5’-CGACAGCAGCCAGACGC-3’
PRgD-2F：5’-GACCGAGCACGAGGTGCG-3’
PRgD-2R：5’-GTCCACGCGCGCCTTGA-3’
PRgE-2F：5’-ATCGCGATCGGCGAGTCA-3’
PRgE-2R：5’-CGAGTCGAGCGTGCCACTA-3’
Above-mentioned primer is synthesized by Sangon Biotech (Shanghai) Co., Ltd..
2, the preparation of positive and negative contrast
Negative control is the normal PK-15 cell conditioned medium liquid of this laboratory culture, and positive control is gE Gene deletion mutation Batha-k61 cell culture fluid.
3, the preparation of test kit
This test kit is composed of the following components:
1. one expands reaction mixture: by sterilizing tri-distilled water, 2 × GCBuffer, 10mmolL
-1dNTP, 25 μm of olL
-1pRgD-1F upstream primer, 25 μm of olL
-1pRgD-1R downstream primer, 25 μm of olL
-1pRgE-1F upstream primer, 25 μm of olL
-1pRgE-1R downstream primer, 5U/ μ LrTaqDNA polysaccharase form, and each reaction volume, than being 7:12.5:1:0.5:0.5:0.5:0.5:0.5, amounts to 23 μ L, and 50 reactions amount to 1150 μ L, dress up 1 pipe.
2. two expand reaction mixture: by sterilizing tri-distilled water, 2 × GCBuffer, 10mmolL
-1dNTP, 25 μm of olL
-1pRgD-2F upstream primer, 25 μm of olL
-1pRgD-2R downstream primer, 25 μm of olL
-1pRgE-2F upstream primer, 25 μm of olL
-1pRgE-2R downstream primer, 5U/ μ LrTaqDNA polysaccharase form, and each reaction volume, than being 7:12.5:1:0.5:0.5:0.5:0.5:0.5, amounts to 23 μ L, and 50 reactions amount to 1150 μ L, dress up 1 pipe.
3. negative control: be normal PK-15 cell culture supernatant, each reaction need contrast 250 μ L, 50 reactions amount to 12500 μ L, and packing 10 is managed, often pipe 1250 μ L.
4. positive control: be gE gene-deleted vaccine Batha-k61 strain PK-15 cell culture fluid, each reaction need contrast 250 μ L, 50 reactions amount to 12500 μ L, and packing 10 is managed, often pipe 1250 μ L.
Test kit preservation condition of the present invention is-20 DEG C.
Embodiment 2
1, the Design & preparation of primer
With embodiment 1.
2, process and the DNA extraction of samples is organized
That 1. gets doubtful porcine pseudorabies organizes pathological material of disease amygdala, brain or lungs 3 ~ 5g, shred into pasty state, add 5 times of aseptic PBS, fully grind with tissue grinder, collect suspension, 4 DEG C, the centrifugal 15min of 12000r/min, get supernatant liquor 100 μ L to join aseptic EP and manage, the positive and negative contrast provided in test kit is provided simultaneously, adds 1000 μ LDNAzolReagent in above each pipe, put upside down after mixing in 4 DEG C of standing 5min.
2. 4 DEG C, the centrifugal 10min of 12000r/min, supernatant liquor 600 μ L is transferred in another clean EP pipe, adds 500 μ L dehydrated alcohols, puts upside down mixing, and room temperature places 5min.
3. 4 DEG C, the centrifugal 5min of 12000r/min, take out EP pipe, abandoning supernatant, adds 1mL75% alcohol settling, 4 DEG C, the centrifugal 2min of 12000r/min, repetitive scrubbing 2 times.
4. last abandoning supernatant, be inverted EP pipe dry DNA, dissolve with the 8mmol/LNaOH of 40 μ L after dry ,-20 DEG C save backup.
3, differentiate to detect PRV with test kit of the present invention.
1. draw one and expand mixed solution 23 μ L, add DNA solution 2 μ L, mix and carry out pcr amplification, reaction conditions is 95 DEG C of denaturation 5min, 94 DEG C, 60; 62 DEG C, 60s; 72 DEG C of 60s carry out 35 circulations altogether, and last 72 DEG C extend 10min.
2. draw two and expand mixed solution 23 μ L, add the thing 2 μ L that expands production, mix and carry out pcr amplification, reaction conditions is 95 DEG C of denaturation 5min, 94 DEG C of 60s, 65 DEG C of 60s, and 72 DEG C of 60s carry out 35 circulations altogether, and last 72 DEG C extend 10min; Namely amplified production is obtained.
embodiment 3
1, the Design & preparation of primer
With embodiment 1.
2, the process of serum sample and DNA extraction
1. get to be separated and obtain porcine blood serum 100 μ L to be checked and join aseptic EP pipe, the positive and negative contrast provided is provided simultaneously, adds 1000 μ LDNAzolReagent in above each pipe in test kit, put upside down after mixing in 4 DEG C of standing 5min.
2. 4 DEG C, the centrifugal 10min of 12000r/min, supernatant liquor 600 μ L is transferred in another clean EP pipe, adds 500 μ L dehydrated alcohols, puts upside down mixing, and room temperature places 5min.
3. 4 DEG C, the centrifugal 5min of 12000r/min, take out EP pipe, abandoning supernatant, adds 1mL75% alcohol settling, 4 DEG C, the centrifugal 2min of 12000r/min, repetitive scrubbing 2 times.
4. last abandoning supernatant, be inverted EP pipe dry DNA, dissolve with the 8mmol/LNaOH of 40 μ L after dry ,-20 DEG C save backup.
3, differentiate to detect PRV with test kit of the present invention
1. draw one and expand mixed solution 23 μ L, add DNA solution 2 μ L, mix and carry out pcr amplification, reaction conditions is 95 DEG C of denaturation 5min, 94 DEG C, 60s; 62 DEG C, 60s; 72 DEG C; 60s carries out 35 circulations altogether, and last 72 DEG C extend 10min.
2. draw two and expand mixed solution 23 μ L, add the thing 2 μ L that expands production, mix and carry out pcr amplification, reaction conditions is 95 DEG C of denaturation 5min, 94 DEG C of 60s, 65 DEG C of 60s, and 72 DEG C of 60s carry out 35 circulations altogether, and last 72 DEG C extend 10min; Namely amplified production is obtained.
embodiment 4
1, the Design & preparation of primer
With embodiment 1.
2, the process of whole blood sample and DNA extraction
1. get pig whole blood 100 μ L to be checked to join the aseptic EP without RNA enzyme and manage, the positive and negative contrast provided is provided simultaneously, adds 1000 μ LDNAzolReagent in above each pipe in test kit, put upside down after mixing in 4 DEG C of standing 5min.
2. 4 DEG C, the centrifugal 10min of 12000r/min, supernatant liquor 600 μ L is transferred in another clean EP pipe, adds 500 μ L dehydrated alcohols, puts upside down mixing, and room temperature places 5min.
3. 4 DEG C, the centrifugal 5min of 12000r/min, take out EP pipe, abandoning supernatant, adds 1mL75% alcohol settling, 4 DEG C, the centrifugal 2min of 12000r/min, repetitive scrubbing 2 times.
4. last abandoning supernatant, be inverted EP pipe dry DNA, dissolve with the 8mmol/LNaOH of 40 μ L after dry ,-20 DEG C save backup.
3, differentiate to detect PRV with test kit of the present invention
1. draw one and expand mixed solution 23 μ L, add DNA solution 2 μ L, mix and carry out pcr amplification, reaction conditions is 95 DEG C of denaturation 5min, 94 DEG C, 60s; 62 DEG C, 60s, 72 DEG C of 60s carry out 35 circulations altogether, and last 72 DEG C extend 10min;
2. draw two and expand mixed solution 23 μ L, add the thing 2 μ L that expands production, mix and carry out pcr amplification, reaction conditions is 95 DEG C of denaturation 5min, 94 DEG C, 60s; 65 DEG C, 60s; 72 DEG C, 60s carries out 35 circulations altogether, and last 72 DEG C extend 10min; Namely amplified production is obtained.
Result judges
1,1.0g agarose is taken, add in 100mL1 × TAE damping fluid, heating and melting in microwave oven, add 5 μ L(100mg/mL) ethidium bromide Homogeneous phase mixing, pour in the gel dish of horizontal positioned, offset plate thickness is 5mm, to be cooled solidify after extract comb, gel is put into electrophoresis chamber, adds 1 × TAE damping fluid and flood glue face;
2, get 5 μ L above-described embodiment 2 ~ 4 gained pcr amplification product and sample-loading buffer Homogeneous phase mixing respectively, add in well, add 5 μ LDL2000DNA molecular weight standards simultaneously;
3, voltage 80V ~ 100V, or electric current 40mA ~ 50mA, electrophoresis 30min;
4, take out gel, insert in uv analyzer and observe, or insert observation photograph in gel imaging system;
5, using DL2000DNA molecular mass standard as reference, negative control does not go out band, and positive control goes out 217bp band, illustrates that contrast is set up; There are 217bp, 354bp two bands in sample, be pseudo-rabies street strain and infect; Sample goes out 217bp band, is gE gene-deleted vaccine strain.
Claims (1)
1. differentiate pseudo-rabies gE gene-deleted vaccine strain and street strain's compound nested polymerase chain reaction diagnostic kit, it is characterized in that, this test kit expands reaction mixture, negative control and positive controls by an expansion reaction mixture, two and forms;
Described one expands reaction mixture by sterilizing tri-distilled water, 2 × GCBuffer, 10mmolL
-1dNTP, 25 μm of olL
-1pRgD-1F upstream primer, 25 μm of olL
-1pRgD-1R downstream primer, 25 μm of olL
-1pRgE-1F upstream primer, 25 μm of olL
-1pRgE-1R downstream primer, 5U/ μ LrTaqDNA polysaccharase form, and each reaction volume, than being 7:12.5:1:0.5:0.5:0.5:0.5:0.5, amounts to 23 μ L, and 50 reactions amount to 1150 μ L, dress up 1 pipe;
Described two expand reaction mixture by sterilizing tri-distilled water, 2 × GCBuffer, 10mmolL
-1dNTP, 25 μm of olL
-1pRgD-2F upstream primer, 25 μm of olL
-1pRgD-2R downstream primer, 25 μm of olL
-1pRgE-2F upstream primer, 25 μm of olL
-1pRgE-2R downstream primer, 5U/ μ LrTaqDNA polysaccharase form, and each reaction volume, than being 7:12.5:1:0.5:0.5:0.5:0.5:0.5, amounts to 23 μ L, and 50 reactions amount to 1150 μ L, dress up 1 pipe;
Described negative control is PK-15 normal cell culture supernatant;
Described positive control is gE Gene deletion mutation Batha-k61 cell culture fluid;
Wherein, described primer PRgD-1F sequence is: 5 '-TAAAATTGGGTCGGCGTCC-3 '
Described primer PRgD-1R sequence is: 5 '-CGCCCTCAGGAATCGGAC-3 '
Described primer PRgE-1F sequence is: 5 '-GTTCAGAGCATGCGCCGG-3 '
Described primer PRgE-1R sequence is: 5 '-CGACAGCAGCCAGACGC-3 '
Described primer PRgD-2F sequence is: 5 '-GACCGAGCACGAGGTGCG-3 '
Described primer PRgD-2R sequence is: 5 '-GTCCACGCGCGCCTTGA-3 '
Described primer PRgE-2F sequence is: 5 '-ATCGCGATCGGCGAGTCA-3 '
Described primer PRgE-2R sequence is: 5 '-CGAGTCGAGCGTGCCACTA-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410404463.4A CN104152586B (en) | 2014-08-18 | 2014-08-18 | Differentiate pseudo-rabies gE gene-deleted vaccine strain and street strain's compound nested polymerase chain reaction diagnostic kit and detection method |
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| 猪瘟病毒RT-nested PCR检测方法的优化和应用;朱小甫;《西北农林科技大学学报(自然科学版)》;20070630;第35卷(第6期);第12-14页 * |
| 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;赵雪丽;《中国畜牧兽医学会家畜传染病学分会第八届全国会员代表大会暨第十五次学术研讨会论文集》;20131231;429-432 * |
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