CN103131800B - Primer group for identifying peste des petits ruminant virus wild strain and vaccine strain and application thereof - Google Patents
Primer group for identifying peste des petits ruminant virus wild strain and vaccine strain and application thereof Download PDFInfo
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- CN103131800B CN103131800B CN201310097627.9A CN201310097627A CN103131800B CN 103131800 B CN103131800 B CN 103131800B CN 201310097627 A CN201310097627 A CN 201310097627A CN 103131800 B CN103131800 B CN 103131800B
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Abstract
The invention discloses a diagnostic kit for identifying a peste des petits ruminant virus wild strain and a vaccine strain and an application thereof. The diagnostic kit provided by the invention comprises two pairs of primers, i.e. a primer pair which is combined with an H gene in a peste des petits ruminant virus Nigeria 75/1 vaccine strain genome (GenBank Accession Number X74443) and a primer pair which is combined with the H gene in a peste des petits ruminant virus Tibet 07 wild strain genome (GenBank Accession Number JF939201). The diagnostic kit for identifying the peste des petits ruminant virus wild strain and the vaccine strain has high detection sensitivity, can detect 200 replicated target ribonucleic acids (RNA) to the minimum extent, has good specificity, is simple and convenient to operate, can effectively identify peste des petits ruminant virus vaccine immune sheep and wild virus infected sheep, and is in particular suitable for monitoring and detecting peste des petits ruminants.
Description
Technical field
The invention belongs to biotechnology detection technique field, be specifically related to a kind of primer sets and the application thereof of differentiating the wild strain of PPR virus and vaccine strain.
Background technology
PPR be caused by the PPR virus of paramyxovirus section Morbillivirus (peste des petits ruminants virus, PPRV) a kind ofly infect goat, sheep and wild little strong, contagious disease of ruminating beast.Non-north African, the Middle East, West Asia and South Asia region during PPR is mainly distributed in.China Ali, Tibet imported PPR virus in 2007, caused heavy losses to local sheep husbandry.Although this disease is controlled at present, but, because PPR is still popular in surrounding countries such as India, Bhutan, Nepal, in addition Southwestern China border region wildlife cross-borderly to migrate, frequent activity, PPR is still seriously endangered the Livestock Production in the provinces, border such as Xie Zhe China Tibet, Xinjiang, Yunnan.
PPR virus has a serotype, according to the hereditary property of N or F gene, PPR virus strain is divided into 4 gene lines, 1 is be distributed in West Africa with 2,3 is viral prevalence in East Africa and Arabic (Oman and Yemen), and 4 is be popular in the Middle East, Arab, the Indian subcontinent and north African.What China once imported into Tibet region is gene 4 is PPR virus.In the prevention and control of PPR, what China taked is vaccine immunization is main policy, and the PPR vaccine used at present is gene 2 is attenuated vaccine strain Nigeria 75/1 strain.
Can produce after attenuated vaccine immunity sheep and protect completely, but, because PPR virus only has a serotype, so PPR virus cannot be distinguished infect sheep and vaccine immunity sheep in serology, bring very large difficulty to differential diagnosis.Therefore, adopt special, responsive, fast molecular detection technology accurately distinguish vaccine strain and street strain, and then distinguish PPR immune sheep and wild virus infection sheep, PPR made a definite diagnosis there is important practical significance.
Summary of the invention
The object of this invention is to provide a kind of primer sets and the application thereof of differentiating the wild strain of PPR virus and vaccine strain, and comprise PPR virus street strain and the vaccine strain differential diagnosis kit of this primer sets, test kit provided by the invention is based on polymerase chain reaction (polymerase chain reaction, PCR) technology, can differentiate PPR virus street strain and vaccine strain fast.
The invention provides a kind of primer sets for differentiating the wild strain of PPR virus and vaccine strain, including for detecting the primer pair of the wild strain of PPR virus and the primer pair for detecting PPR virus vaccine strain;
Wherein street strain's primer pair is made up of upstream primer H1a and downstream primer H2b:
H1a 5'-CCTGGATAGAGAACGCTTGGTC-3' SEQ ID NO:3；
H2b 5'-CCGAGAGTCAATGATTGCAGCT-3' SEQ ID NO:4；
Described vaccine strain primer pair is made up of upstream primer H3c and downstream primer H5e:
H3c 5'-CGACGAAGGACCTGAGGTACATA-3' SEQ ID NO:5；
H5e 5'-TAGGGGATGGCTCGGAGGC-3' SEQ ID NO:6。
Wherein street strain's primer pair amplifies clip size is 477 ± 3 bp, and vaccine strain primer pair amplifies clip size is 324 ± 3 bp;
Primer sets of the present invention is for the preparation of the goods differentiating the wild strain of PPR virus and vaccine strain;
The above-mentioned detection kit that is prepared as includes PCR reaction buffer, archaeal dna polymerase, reversed transcriptive enzyme;
Also positive control nucleic acid is included in test kit, be made up of vaccine strain positive control and street strain's positive control, described vaccine strain positive control can be the RNA etc. of PPR virus Nigeria 75/1 vaccine strain virus or its RNA or the synthetic containing PPR virus Nigeria 75/1 vaccine strain H gene fragment.Described street strain positive control can be the RNA etc. of PPR virus street strain virus or its RNA or the synthetic containing PPR virus street strain H gene fragment.
Described test kit can comprise negative control, can be DEPC process water, phosphoric acid buffer etc.
Primer sets of the present invention specially can detect PPR virus vaccine strain and street strain, to foot and mouth disease virus and sheep braxy virus reactionless; Susceptibility is high, can detect 10
-3the object RNA of ng/ μ L.Test kit practical value of the present invention is high, can effectively the strain of PPR virus vaccine immunity and the strain of PPR virus wild virus infection be distinguished, and can be applicable to the clinical medicine detection that laboratories carries out.
Accompanying drawing explanation
Fig. 1: the electrophorogram of the embodiment of the present invention 2, swimming lane 1-3 are positive reference substance, and 4-6 is negative controls, and 7-9 is Healthy Sheep sample, and 10-12 is disease sheep sample, and 13-15 is immune sheep sample;
Fig. 2: the electrophorogram of the embodiment of the present invention 3, every 6 pipes are a reaction group, totally 6 reaction groups, and reaction group I-IV street strain positive reference substance concentration 10 times of serial dilutions, are followed successively by 2 × 10
5-2 × 10
0ng/ μ L, each reaction group each reaction tubes vaccine strain positive reference substance concentration 10 times of serial dilutions, are followed successively by 2 × 10
5-2 × 10
0ng/ μ L;
Fig. 3: the electrophorogram of the embodiment of the present invention 4, swimming lane 1-3 are positive reference substance, and 4-6 is negative controls, and 7-9 is foot and mouth disease virus sample, and 10-12 is sheep of virus sample.
Embodiment
Following embodiment is convenient to understand the present invention better, but is not limited to the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is routine biochemistry reagent.
Applicant is the genome sequence of street strain and Nigeria 75/1 vaccine strain by detailed comparison PPR virogene 4, and finding that H gene homology is minimum, is 91.7%, so select H gene as designing the right target gene of diagnostic primers.By the H gene that comparison all genes 4 recent years are PPR virus street strain and Nigeria 75/1 vaccine strain, find out sequence highly divergent isolate region, design vaccine strain primer pair; Meanwhile, comparison all genes 4 recent years are the H gene of PPR virus street strain, find the conserved regions that gene 4 is PPR virus street strain, design street strain primer pair.So above-mentioned primer can the wild strain of specific discriminating PPR virus and vaccine strain.Detected result shows that two pairs of primers match completely with aim sequence respectively, can guarantee thoroughly carrying out of reaction, also ensure that the specificity of detection method.
Primer sets of the present invention is made up of following two pairs of pcr amplification the primers; Pair of primers is the vaccine strain primer pair that can be combined with H gene in PPR virus Nigeria 75/1 vaccine strain geneome RNA (GenBank AccessionNumber X74443), and the nucleotides sequence of this subregion is classified as SEQ ID NO:1; Pair of primers is street strain's primer pair that can be combined with H gene in PPR virus street strain geneome RNA (GenBank Accession Number JF939201), and the nucleotides sequence of this partial sequence is classified as SEQ ID NO:2.
Concrete, street strain's primer pair is made up of upstream primer H1a and downstream primer H2b, and vaccine strain primer pair is made up of upstream primer H3c and downstream primer H5e, and its concrete sequence is as follows:
H1a 5'-CCTGGATAGAGAACGCTTGGTC-3' SEQ ID NO:3，
H2b 5'-CCGAGAGTCAATGATTGCAGCT-3' SEQ ID NO:4，
H3c 5'-CGACGAAGGACCTGAGGTACATA-3' SEQ ID NO:5，
H5e 5'-TAGGGGATGGCTCGGAGGC-3' SEQ ID NO:6。
Table 1: the calmodulin binding domain CaM of the H gene of primer of the present invention and PPR virus
Primer sets of the present invention can complete the amplification to template ribonucleic acid under the effect of ThermoScript II and archaeal dna polymerase.Amplification procedure is divided into two stages, specific as follows:
(1) the reverse transcription stage
Under the effect of ThermoScript II, sample rna is inverted records into cDNA.
(2) the polymerase chain reaction (PCR) amplification stage
Under the effect of polysaccharase, primer is combined with template and starts the synthesis of DNA.
Deposition condition is 80V, 30 min, after electrophoresis terminates, sepharose is placed in gel imaging system and takes pictures.Wherein the primer amplification clip size of PPR virus vaccine strain is 324bp, and the amplified fragments size of PPR virus street strain is 477 bp, so just can determine the kind of detected sample.
Primer sets of the present invention can be used for preparing to be differentiated PPR virus street strain and vaccine strain and/or detects the test kit that sample to be checked contains PPR virus street strain and/or vaccine strain.
Described test kit also can comprise 2 × reaction buffer, and water-soluble for solute (as distilled water) obtains by 2 × reaction buffer; Described solute and concentration as follows: MgSO
4be 2.8 ~ 4 mM, four kinds of each 0.4mM, RNase inhibitor of dNTP are 2 U/ μ L, described upstream primer H1a is 0.4 ~ 1.2 μM, described downstream primer H2b is 0.4 ~ 1.2 μM, and described upstream primer H3c is 0.4 ~ 1.2 μM, and described downstream primer H5e is 0.4 ~ 1.2 μM.
Described test kit can comprise enzyme mixation, by solute and solvent composition; Described solvent is water (as distilled water); Described solute and concentration as follows: archaeal dna polymerase is 1U/ μ L, and ThermoScript II is 10U/ μ L.
Described test kit can comprise centrifuge tube (as 0.2 ml specification), disposable self-suction trace suction nozzle (10 ± 2 μ L specification) etc.
Described test kit can comprise positive control, is made up of vaccine strain positive control and street strain's positive control.Described vaccine strain positive control can be the RNA etc. of PPR virus Nigeria 75/1 vaccine strain virus or its RNA or the synthetic containing PPR virus Nigeria 75/1 vaccine strain H gene fragment.Described street strain positive control can be the RNA etc. of PPR virus street strain virus or its RNA or the synthetic containing PPR virus street strain H gene fragment.Described positive control specifically can be prepared as follows: extract PPR virus Nigeria 75/1 vaccine strain RNA, carry out pcr amplification, carry out purifying to PCR primer with PPRH3cT7 and PPRH5e primer, order-checking qualification.The PCR primer of getting 200ng carries out in-vitro transcription according to the explanation of RiboprobeIn vitro Transcription Systems.Product, through DNase I enzymic digestion, 3M NaAc alcohol settling, is dissolved in appropriate DEPC treated water, be then vaccine strain engineered rna, measures concentration and is converted to copy number.Extract PPR virus Tibet 07 street strain RNA, carry out pcr amplification with PPRH1aT7 and PPRH2b primer, purifying is carried out to PCR primer, order-checking qualification.As mentioned above, preparation street strain engineered rna, measures concentration and is converted to copy number.Respectively get appropriate vaccine strain engineered rna and street strain's engineered rna mixes, final concentration is respectively 10
5copy/μ L, is positive reference substance.
Described test kit can comprise negative control, can be DEPC process water, phosphoric acid buffer etc.Described DEPC process glassware for drinking water body can be prepared as follows: preparation bi-distilled water, through Millipore MILLI-Q PFPLUS pure water instrument purifying, requires purifying resistivity of water >=18.0M Ω .cm, stores in aseptic bottle; It is 0.1% that Millipore-Q purified water adds DEPC to final concentration, mixing, 37oC process 12h, 151bf/in2(1.034 × 105Pa) high pressure steam sterilization 15min.Be packed as 200 μ L/ to manage.
Present invention also offers a kind of differential diagnosis from the method for whether carrying vaccine strain or street strain's PPR virus in the sample of infected animal,, with described primer special or described test kit, reverse transcription-polymerase chain reaction is carried out to the total serum IgE of the sample from infected animal, detect amplified production, determine in sample, whether to carry vaccine strain or street strain's PPR virus; Described reverse transcription-polymerase chain reaction condition is: 40 oC ~ 55oC carry out reverse transcription reaction 30 ~ 60 min; 93oC ~ 95oC carries out the activation 45sec ~ 5min of Taq enzyme; The PCR reaction (93oC ~ 98oC sex change 30sec ~ 1 min, 45oC ~ 70oC annealing 20sec ~ 1 min, 68oC ~ 72oC extend 20sec ~ 2 min) of 20 ~ 50 circulations; 72oC extends 7 min eventually.
The condition of described reverse transcription-polymerase chain reaction specifically can be: 50oC reverse transcription 30 min; 94oC carries out activation 2 min of Taq enzyme; The PCR reaction (94oC sex change 30sec, 63oC annealing 30sec, 72oC extend 30sec) of 30 circulations; 72oC extends 7 min eventually.
Applying primer special provided by the invention or test kit differential diagnosis vaccine strain or street strain's PPR virus, can come by carrying out agarose gel electrophoresis to reverse transcription-polymerase chain reaction amplified production.Electrophoresis method is: negate transcription-Polymerase Chain reaction product 5 μ L carries out electrophoresis in 1.5% sepharose of ethidium bromide staining, and deposition condition is 80V, 30min.After electrophoresis terminates, sepharose is placed in gel imaging system and takes pictures.Positive reference substance has size to be two specific amplification bands of 324 bp and 477 bp, and negative controls is without specific amplification band (illustrative experiment credible result); There is the amplified band consistent with 324 bp stripe size in positive control in measuring samples, then illustrates that measuring samples is the PPR virus vaccine strain positive (containing PPR virus vaccine strain in this sample); If the amplified band consistent with 477 bp stripe size in positive control appears in measuring samples, then illustrate that measuring samples is the PPR virus street strain positive (containing PPR virus street strain in this sample).Without amplified band in sample to be checked, then illustrate that the detected result of measuring samples is for negative.
Test kit provided by the invention can be used for detecting the PPR virus in multiple sample (as blood, discharge of eye, nasal discharge, mouth secretory product, lymphoglandula, spleen, lung, tonsilla, kidney, the heart, liver etc.).Useful commercial test kit extracts the nucleic acid of sample to be tested, and gained nucleic acid solution directly uses as template.
When detecting with described test kit, 2 × reaction buffer and enzyme mixation can be mixed, obtain reaction mixture, then appropriate template, positive control and negative control be mixed with reaction mixture respectively, carry out RT-PCR reaction.
The preparation of embodiment 1, PPR virus vaccine strain and street strain's differential diagnosis kit
One, the synthesis of primer
2 pairs of primers below synthetic:
H1a: 5'-CCTGGATAGAGAACGCTTGGTC-3'
H2b: 5'-CCGAGAGTCAATGATTGCAGCT-3'
H3c: 5'-CGACGAAGGACCTGAGGTACATA-3'
H5e: 5'-TAGGGGATGGCTCGGAGGC-3'
Two, the preparation of positive control
Extract PPR virus Nigeria 75/1 vaccine strain RNA, carry out pcr amplification with PPRH3cT7 and PPRH5e primer, purifying is carried out to PCR primer, order-checking qualification.The PCR primer of getting 200ng carries out in-vitro transcription according to the explanation of Riboprobe In vitro Transcription Systems.Product, through DNase I enzymic digestion, 3M NaAc alcohol settling, is dissolved in appropriate DEPC treated water, be then vaccine strain engineered rna, measures concentration and is converted to copy number.Extract PPR virus Tibet 07 street strain RNA, carry out pcr amplification with PPRH1aT7 and PPRH2b primer, purifying is carried out to PCR primer, order-checking qualification.As mentioned above, preparation street strain engineered rna, measures concentration and is converted to copy number.Respectively get appropriate vaccine strain engineered rna and street strain's engineered rna mixes, final concentration is respectively 10
5copy/μ L, is positive reference substance, and-70oC saves backup.
Three, RT-PCR reaction solution is prepared
Water-soluble for solute (as distilled water) obtains by 2 × reaction buffer; Solute and concentration as follows: MgSO
4be 2.8 ~ 4 mM, four kinds of each 0.4mM, RNase inhibitor of dNTP are 2 U/ μ L, and described upstream primer H1a is 0.4 μM, and described downstream primer H2b is 0.4 μM, and described upstream primer H3c is 0.4 μM, and described downstream primer H5e is 0.4 μM.
Enzyme mixation is by solute and solvent composition; Described solvent is water (as distilled water); Described solute and concentration as follows: archaeal dna polymerase is 1U/ μ L, and ThermoScript II is 10U/ μ L.
Four, the assembling of test kit
Test kit is made up of following material: 2 × reaction buffer, enzyme mixation, positive control and DEPC treated water
The application of embodiment 2, PPR virus vaccine strain and street strain's differential diagnosis kit
Get 1 and be diagnosed as the sick sheep of PPR virus infection, 1 PPR virus vaccine immunity sheep and 1 Healthy Sheep, adopt mouth and nose cotton swab respectively, the test kit adopting embodiment 1 to prepare detects sample.
One, the process of sample
The mouth and nose swab of collection is put into 2ml screw socket centrifuge tube, adds 1ml PBS, roll, after the centrifugal 5min of 3000rpm, get 200ul supernatant and extract nucleic acid with commercial RNA extraction test kit (QIAampViral RNA Kit, Qiagen), gained nucleic acid solution can directly use as template.
Adopt the mouth and nose swab of disease sheep to carry out above-mentioned process, get in nucleic acid-templated each 5 μ L to 3 0.2mL centrifuge tube, as I group reaction pipe.Adopt the mouth and nose swab of immune sheep to carry out above-mentioned process, get in nucleic acid-templated each 5 μ L to 3 0.2mL centrifuge tube, as II group reaction pipe.Adopt the mouth and nose swab of Healthy Sheep to carry out above-mentioned process, get in nucleic acid-templated each 5 μ L to 3 0.2mL centrifuge tube, as III group reaction pipe.Get in DEPC treated water each 5 μ L to 3 0.2mL centrifuge tube, as negative control pipe.Get in positive reference substance each 5 μ L to 3 0.2mL centrifuge tube, as positive control pipe.
Two, RT-PCR reaction
Preparation feedback premixed liquid, in 25 μ L reaction systems, adds 2 × reaction buffer 12.5 μ L, enzyme mixation 1 μ L, DEPC treated water 6.5 μ L.
Get I group reaction pipe, II group reaction pipe, III group reaction pipe, negative control pipe, positive control pipe, often pipe adds 20 μ L premixed liquids.
Carry out RT-PCR reaction, reaction conditions is: 50oC reverse transcription 30 min; 94oC carries out activation 2 min of Taq enzyme; The PCR reaction (94oC sex change 30sec, 63oC annealing 30sec, 72oC extend 30sec) of 30 circulations; 72oC extends 7 min eventually.
Three, the detection of RT-PCR product
Get RT-PCR reaction product 5 μ L and carry out electrophoresis in 1.5% sepharose of ethidium bromide staining.The results are shown in Figure 1.Positive control Guan Jun has size to be two specific amplification bands of 330 bp and 477bp, and negative controls is without specific amplification band.3 side reaction Guan Junwu amplified bands of Healthy Sheep, the 3 side reaction Guan Jun of sick sheep have the amplified band consistent with 477 bp stripe size in positive control, 3 side reaction Guan Jun of immune sheep have the amplified band consistent with 324 bp stripe size in positive control, and the sequencing result of band also demonstrate that the specificity that primer pair of the present invention detects.
The detection of embodiment 3, PPR virus vaccine strain and street strain's differential diagnosis kit sensitivity
One, the preparation of positive criteria product
With the step 2 of embodiment 1, preparing final concentration is 10
6the vaccine strain engineered rna of copy/μ L is as vaccine strain positive criteria product, and preparing final concentration is 10
6street strain's engineered rna of copy/μ L is as street strain's positive criteria product.
Two, the preparation of positive criteria product serial dilutions
With DEPC treated water, street strain's positive criteria product and vaccine strain positive criteria product are carried out serial dilution respectively, preparation concentration is respectively 2 × 10
5copy/μ L, 2 × 10
4copy/μ L, 2 × 10
3copy/μ L, 2 × 10
2copy/μ L, 2 × 10
1copy/μ L and 2 × 10
0the serial dilutions of copy/μ L.
Test set I: in 6 0.2mL centrifuge tubes, respectively adding 2.5 μ L concentration is 2 × 10
5street strain's positive criteria product of copy/μ L, then to add 2.5 μ L concentration be respectively 2 × 10
5(No. 1 pipe), 2 × 10
4(No. 2 pipes), 2 × 10
3(No. 3 pipes), 2 × 10
2(No. 4 pipes), 2 × 10
1(No. 5 pipes), 2 × 10
0the vaccine strain positive criteria product of (No. 6 pipes) copy/μ L.
Test set II: in 6 0.2mL centrifuge tubes, respectively adding 2.5 μ L concentration is 2 × 10
4street strain's positive criteria product of copy/μ L, then to add 2.5 μ L concentration be respectively 2 × 10
5(No. 1 pipe), 2 × 10
4(No. 2 pipes), 2 × 10
3(No. 3 pipes), 2 × 10
2(No. 4 pipes), 2 × 10
1(No. 5 pipes), 2 × 10
0the vaccine strain positive criteria product of (No. 6 pipes) copy/μ L.
Test set III: in 6 0.2mL centrifuge tubes, respectively adding 2.5 μ L concentration is 2 × 10
3street strain's positive criteria product of copy/μ L, then to add 2.5 μ L concentration be respectively 2 × 10
5(No. 1 pipe), 2 × 10
4(No. 2 pipes), 2 × 10
3(No. 3 pipes), 2 × 10
2(No. 4 pipes), 2 × 10
1(No. 5 pipes), 2 × 10
0the vaccine strain positive criteria product of (No. 6 pipes) copy/μ L.
Test set IV: in 6 0.2mL centrifuge tubes, respectively adding 2.5 μ L concentration is 2 × 10
2street strain's positive criteria product of copy/μ L, then to add 2.5 μ L concentration be respectively 2 × 10
5(No. 1 pipe), 2 × 10
4(No. 2 pipes), 2 × 10
3(No. 3 pipes), 2 × 10
2(No. 4 pipes), 2 × 10
1(No. 5 pipes), 2 × 10
0the vaccine strain positive criteria product of (No. 6 pipes) copy/μ L.
Test set V: in 6 0.2mL centrifuge tubes, respectively adding 2.5 μ L concentration is 2 × 10
1street strain's positive criteria product of copy/μ L, then to add 2.5 μ L concentration be respectively 2 × 10
5(No. 1 pipe), 2 × 10
4(No. 2 pipes), 2 × 10
3(No. 3 pipes), 2 × 10
2(No. 4 pipes), 2 × 10
1(No. 5 pipes), 2 × 10
0the vaccine strain positive criteria product of (No. 6 pipes) copy/μ L.
Test set VI: in 6 0.2mL centrifuge tubes, respectively adding 2.5 μ L concentration is 2 × 10
0street strain's positive criteria product of copy/μ L, then to add 2.5 μ L concentration be respectively 2 × 10
5(No. 1 pipe), 2 × 10
4(No. 2 pipes), 2 × 10
3(No. 3 pipes), 2 × 10
2(No. 4 pipes), 2 × 10
1(No. 5 pipes), 2 × 10
0the vaccine strain positive criteria product of (No. 6 pipes) copy/μ L.
Three, RT-PCR reaction
Preparation feedback premixed liquid, in 25 μ L reaction systems, adds 2 × reaction buffer 12.5 μ L, enzyme mixation 1 μ L, DEPC treated water 6.5 μ L.
In each pipe of test set I ~ V, respectively add 20 μ L and react premixed liquid, carry out the detection of RT-PCR reaction and product.
Four, result
Detected result is shown in Fig. 2.1 ~ No. 4 Guan Jun of test set I ~ IV has size to be two specific amplification bands of 330bp and 477bp, and 5 ~ No. 6 pipes only have the amplified band of 477bp size.1 ~ No. 4 of test set V ~ VI pipe only has size to be the specific amplification band of 330bp, and 5 ~ No. 6 pipes do not have amplified band.
Embodiment 4, PPR virus vaccine strain and the specific detection of street strain's differential diagnosis kit
Extract foot and mouth disease virus and sheep of virus nucleic acid RNA respectively, the test kit adopting embodiment 1 to prepare carries out specific detection.
One, the process of sample
Above-mentioned virocyte nutrient solution sample, commodity in use RNA extracts reagent (QIAamp ViralRNA Kit, Qiagen) and extracts nucleic acid, and gained nucleic acid solution directly uses as template.Get in foot and mouth disease virus nucleic acid-templated each 5 μ L to 3 0.2mL centrifuge tube, as I group reaction pipe.Get in sheep of virus nucleic acid-templated each 5 μ L to 3 0.2mL centrifuge tube, as II group reaction pipe.Get in DEPC treated water 5 μ L to 0.2mL centrifuge tube, as negative control pipe.Get in positive reference substance 5 μ L to 0.2mL centrifuge tube, as positive control pipe.
Two, RT-PCR reaction
Preparation feedback premixed liquid, in 25 μ L reaction systems, adds 2 × reaction buffer 12.5 μ L, enzyme mixation 1 μ L, DEPC treated water 6.5 μ L.
In I ~ II test set, positive control pipe, negative control pipe, often manage and add 20 μ L reaction premixed liquids.
Carry out the detection of RT-PCR reaction and product.
Three, result
Detected result is shown in Fig. 3, and positive control Guan Jun has size to be two specific amplification bands of 330bp and 477bp, and negative controls is without specific amplification band.The each Guan Jun of test set I and II does not have specificity to expand band, thus proves that primer sets of the present invention has good specificity.
Above-described embodiment is the present invention's preferably embodiment, but embodiments of the present invention are not restricted to the described embodiments, other are any does not deviate from change, modification that spirit of the present invention and principle do, substitute, combine, simplification etc., all should be the substitute mode of equivalence, be included in of the present invention comprising within scope.
Claims (4)
1., for differentiating a primer sets for the wild strain of PPR virus and vaccine strain, include for detecting the primer pair of the wild strain of PPR virus and the primer pair for detecting PPR virus vaccine strain; The nucleotides sequence wherein detecting street strain's primer pair is classified as SEQ ID NO:3 and SEQ ID NO:4; The nucleotides sequence detecting vaccine strain primer pair is classified as SEQ ID NO:5 and SEQ ID NO:6; Wherein street strain's primer pair amplifies object product clip size is 477 ± 3bp, and vaccine strain primer pair amplifies object product clip size is 324 ± 3bp.
2. primer sets according to claim 1 is preparing the application in the goods differentiating the wild strain of PPR virus and vaccine strain.
3. for differentiating a test kit for the wild strain of PPR virus and vaccine strain, it is characterized in that, described test kit includes primer sets according to claim 1; Described test kit also includes the positive control nucleic acid of PCR reaction buffer, archaeal dna polymerase and reversed transcriptive enzyme, the wild strain of PPR virus and vaccine strain; The positive control nucleic acid of the wild strain of described PPR virus is the RNA of PPR virus street strain virus or its RNA or the synthetic containing PPR virus street strain H gene fragment; Described PPR virus vaccine strain positive control nucleic acid is the RNA of PPR virus Nigeria75/1 vaccine strain virus or its RNA or the synthetic containing PPR virus Nigeria75/1 vaccine strain H gene fragment.
4. test kit as claimed in claim 3, it is characterized in that described test kit also includes amplification negative control, described amplification negative control is DEPC process water or phosphoric acid buffer.
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| CN105063236B (en) * | 2015-07-31 | 2016-06-01 | 中国动物卫生与流行病学中心 | A kind of differentiate that PPR virus vaccine strain and gene 4 are the method for street strain |
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| CN102178947A (en) * | 2011-03-16 | 2011-09-14 | 中国人民解放军军事医学科学院军事兽医研究所 | Live vector vaccine for expressing peste des petits ruminants virus (PPRV) H gene and preparation method thereof |
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