CN103131800A - 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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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 application thereof of differentiating the wild strain of PPR virus and vaccine strain.
Background technology
PPR is a kind of goat, sheep and wild little strong, contagious disease of ruminating beast of infecting that the PPR virus (peste des petits ruminants virus, PPRV) by paramyxovirus section Morbillivirus causes.Non-north African, the Middle East, West Asia and South Asia region during PPR mainly is distributed in.China's Ali, Tibet imported PPR virus in 2007, cause heavy losses for local sheep husbandry.Although should disease controlled at present, but, because PPR is still national popular at peripheries such as India, Bhutan, Nepal, in addition Southwestern China border region wildlife cross-borderly migrate, frequent activity, PPR still seriously danger coercing the Livestock Production in the provinces, border such as China Tibet, Xinjiang, Yunnan.
PPR virus has a serotype, hereditary property according to N or F gene, the PPR virus strain is divided into 4 gene lines, 1 is to be to be distributed in West Africa with 2,3 be viral prevalence in East Africa and Arab (Oman and Yemen), 4 is to be popular in the Middle East, Arab, the Indian subcontinent and north African.What China once imported into the Tibet region is that gene 4 is PPR virus.In the prevention and control of PPR, what China taked is that vaccine immunization is main policy, and the PPR vaccine that uses at present is that gene 2 is attenuated vaccine strain Nigeria 75/1 strain.
Can produce protection completely after the attenuated vaccine immunity sheep, still, because PPR virus only has a serotype, infect sheep and vaccine immunity sheep so can't distinguish PPR virus on serology, bring very large difficulty to differential diagnosis.Therefore, adopt special, responsive, molecular detection technology is accurately distinguished vaccine strain and street strain fast, and then distinguishes PPR immune sheep and wild virus infection sheep, PPR is made a definite diagnosis have great practical significance.
Summary of the invention
The purpose of this invention is to provide a kind of primer sets and application thereof of differentiating the wild strain of PPR virus and vaccine strain, and the PPR virus street strain and the vaccine strain differential diagnosis kit that comprise this primer sets, test kit provided by the invention is based on polymerase chain reaction (polymerase chain reaction, PCR) technology can be differentiated 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, include for detection of the primer pair of the wild strain of PPR virus with for detection of the primer pair of PPR virus vaccine strain;
Wherein street strain's primer pair is comprised 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 comprised 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 amplified fragments size is 477 ± 3 bp, and vaccine strain primer pair amplified fragments size is 324 ± 3 bp;
Primer sets of the present invention is for the preparation of the goods of 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 include positive control nucleic acid in test kit, be comprised of vaccine strain positive control and street strain's positive control, described vaccine strain positive control can be PPR virus Nigeria 75/1 vaccine strain virus or its RNA or contains the RNA etc. of the synthetic of PPR virus Nigeria 75/1 vaccine strain H gene fragment.Described street strain positive control can be PPR virus street strain virus or its RNA or contains the RNA etc. of the synthetic of the PPR virus H of street strain gene fragment.
Described test kit can comprise negative control, can be DEPC and processes water, phosphoric acid buffer etc.
Special PPR virus vaccine strain and the street strain of detecting of primer sets energy of the present invention, reactionless to foot and mouth disease virus and sheep braxy virus; Susceptibility is high, can detect 10
-3The purpose 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.
Description of drawings
Fig. 1: the electrophorogram of the embodiment of the present invention 2, the positive reference substance of swimming lane 1-3, the negative reference substance of 4-6,7-9 are the Healthy Sheep sample, and 10-12 is disease sheep sample, and 13-15 is the 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 10 times of serial dilutions of reaction group I-IV street strain's positive reference substance concentration are followed successively by 2 * 10
5-2 * 10
0Ng/ μ L, 10 times of serial dilutions of each reaction tubes vaccine strain positive reference substance concentration are organized in each reaction, are followed successively by 2 * 10
5-2 * 10
0Ng/ μ L;
Fig. 3: the electrophorogram of the embodiment of the present invention 4, the positive reference substance of swimming lane 1-3, the negative reference substance of 4-6,7-9 are the foot and mouth disease virus sample, 10-12 is the sheep of virus sample.
Embodiment
Following embodiment is convenient to understand better the present invention, 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.
The applicant is the genome sequence of street strain and Nigeria 75/1 vaccine strain by detailed comparison PPR virogene 4, finds that the H DNA homolog is minimum, is 91.7%, so select the H gene as the right target gene of design diagnostic primers.Be the H gene of PPR virus street strain and Nigeria 75/1 vaccine strain by comparison all genes 4 recent years, seek out the variation of sequence height regional, design vaccine strain primer pair; Simultaneously, comparison all genes 4 recent years are the H gene of PPR virus street strain, and seeking gene 4 is the conserved regions of PPR virus street strain, designs street strain's primer pair.So, wild strain and vaccine strain that above-mentioned primer can specific discriminating PPR virus.Detected result shows that two pairs of primers match fully with aim sequence respectively, and that can guarantee to react thoroughly carries out, and has also guaranteed the specificity of detection method.
Primer sets of the present invention is comprised 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 Accession Number X74443), and the nucleotides sequence of this subregion is classified SEQ ID NO:1 as; Pair of primers is the street strain's primer pair that can be combined with H gene in PPR virus street strain's geneome RNA (GenBank Accession Number JF939201), and the nucleotides sequence of this partial sequence is classified SEQ ID NO:2 as.
Concrete, street strain's primer pair is comprised of upstream primer H1a and downstream primer H2b, and the vaccine strain primer pair is comprised 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 be completed the amplification to template ribonucleic acid under the effect of ThermoScript II and archaeal dna polymerase.Amplification procedure is divided into two stages, and is 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 is started the synthetic of DNA.
Deposition condition is 80V, 30 min, and electrophoresis is placed in gel imaging system with sepharose and takes pictures after finishing.Wherein the primer amplification clip size of PPR virus vaccine strain is 324 bp, 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 2 * reaction buffer obtains solute water-soluble (as distilled water); Described solute and concentration thereof are as follows: MgSO
4Be 2.8 ~ 4 mM, four kinds of each 0.4mM of dNTP, RNase inhibitor are 2 U/ μ L, described upstream primer H1a is 0.4 ~ 1.2 μ M, described downstream primer H2b is 0.4 ~ 1.2 μ M, and described upstream primer H3c is 0.4 ~ 1.2 μ M, and described downstream primer H5e is 0.4 ~ 1.2 μ M.
Described test kit can comprise enzyme mixation, by solute and solvent composition; Described solvent is water (as distilled water); Described solute and concentration thereof are 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 comprised of vaccine strain positive control and street strain's positive control.Described vaccine strain positive control can be PPR virus Nigeria 75/1 vaccine strain virus or its RNA or contains the RNA etc. of the synthetic of PPR virus Nigeria 75/1 vaccine strain H gene fragment.Described street strain positive control can be PPR virus street strain virus or its RNA or contains the RNA etc. of the synthetic of the PPR virus H of street strain gene fragment.Described positive control specifically can prepare as follows: extract PPR virus Nigeria 75/1 vaccine strain RNA, carry out pcr amplification with PPRH3cT7 and PPRH5e primer, the PCR product is carried out purifying, order-checking is identified.The PCR product of getting 200ng carries out in-vitro transcription according to the explanation of Riboprobe In vitro Transcription Systems.Product is dissolved in appropriate DEPC treated water through the enzymic digestion of DNase I, 3M NaAc ethanol precipitation, is the vaccine strain engineered rna, measures concentration and is converted to copy number.Extract PPR virus Tibet 07 RNA of street strain, carry out pcr amplification with PPRH1aT7 and PPRH2b primer, the PCR product is carried out purifying, order-checking is identified.As mentioned above, prepare street strain's engineered rna, measure concentration and also be converted to copy number.Respectively get appropriate vaccine strain engineered rna and street strain's engineered rna and mix, final concentration is respectively 10
5Copy/μ L is positive reference substance.
Described test kit can comprise negative control, can be DEPC and processes water, phosphoric acid buffer etc.Described DEPC processes the glassware for drinking water body and can prepare as follows: the preparation bi-distilled water, through Millipore MILLI-Q PFPLUS pure water instrument purifying, require purifying resistivity of water 〉=18.0M Ω .cm, and store in aseptic bottle; It is 0.1% to final concentration that the Millipore-Q purified water adds DEPC, mixing, and 37oC processes 12h, the high pressure steam sterilization 15min of 151bf/in2(1.034 * 105Pa).Be packed as 200 μ L/ pipes.
The present invention also provides 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, the total RNA from the sample of infected animal to be carried out reverse transcription-polymerase chain reaction, detect amplified production, determine whether carry vaccine strain or street strain's PPR virus in sample; Described reverse transcription-polymerase chain reaction condition is: 40 oC ~ 55oC carries out reverse transcription reaction 30 ~ 60 min; 93oC ~ 95oC carries out the activation 45sec ~ 5min of Taq enzyme; The PCR of 20 ~ 50 circulations reaction (93oC ~ 98oC sex change 30sec ~ 1 min, 45oC ~ 70oC anneal 20sec ~ 1 min, 68oC ~ 72oC extends 20sec ~ 2 min); 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 of 30 circulations reaction (94oC sex change 30sec, 63oC anneal 30sec, 72oC extends 30sec); 72oC extends 7 min eventually.
Use primer special provided by the invention or test kit differential diagnosis vaccine strain or street strain's PPR virus, can complete by the reverse transcription-polymerase chain reaction amplified production is carried out agarose gel electrophoresis.Electrophoresis method is: get reverse transcription-polymerase chain reaction product 5 μ L and carry out electrophoresis in 1.5% sepharose of ethidium bromide staining, deposition condition is 80V, 30 min.Electrophoresis is placed in gel imaging system with sepharose and takes pictures after finishing.It is two specific amplification bands of 324 bp and 477 bp that positive reference substance has size, and the negative control product are without specific amplification band (illustrative experiment credible result); The amplified band consistent with 324 bp stripe size in positive control appears in sample to be checked, illustrates that sample to be checked is PPR virus vaccine strain positive (containing the PPR virus vaccine strain in this sample); If the amplified band consistent with 477 bp stripe size in positive control appears in sample to be checked, illustrate that sample to be checked is the PPR virus street strain positive (contains in this sample PPR virus street strain).Without amplified band, illustrate that the detected result of sample to be checked is negative in sample to be checked.
Test kit provided by the invention can be used for detecting the PPR virus in multiple sample (as blood, discharge of eye, nasal discharge, mouthful secretory product, lymphoglandula, spleen, lung, tonsilla, kidney, the heart, liver etc.).The useful commercial test kit extracts the nucleic acid of sample to be tested, and the 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 the RT-PCR reaction.
The preparation of embodiment 1, PPR virus vaccine strain and street strain's differential diagnosis kit
One, primer is synthetic
The following 2 pairs of primers of 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, the PCR product is carried out purifying, order-checking is identified.The PCR product of getting 200ng carries out in-vitro transcription according to the explanation of Riboprobe In vitro Transcription Systems.Product is dissolved in appropriate DEPC treated water through the enzymic digestion of DNase I, 3M NaAc ethanol precipitation, is the vaccine strain engineered rna, measures concentration and is converted to copy number.Extract PPR virus Tibet 07 RNA of street strain, carry out pcr amplification with PPRH1aT7 and PPRH2b primer, the PCR product is carried out purifying, order-checking is identified.As mentioned above, prepare street strain's engineered rna, measure concentration and also be converted to copy number.Respectively get appropriate vaccine strain engineered rna and street strain's engineered rna and mix, final concentration is respectively 10
5Copy/μ L is positive reference substance, and-70oC saves backup.
Three, preparation RT-PCR reaction solution
2 * reaction buffer obtains solute water-soluble (as distilled water); Solute and concentration thereof are as follows: MgSO
4Be 2.8 ~ 4 mM, four kinds of each 0.4mM of dNTP, RNase inhibitor are 2 U/ μ L, and described upstream primer H1a is 0.4 μ M, and described downstream primer H2b is 0.4 μ M, and described upstream primer H3c is 0.4 μ M, and described downstream primer H5e is 0.4 μ M.
Enzyme mixation is by solute and solvent composition; Described solvent is water (as distilled water); Described solute and concentration thereof are 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 comprised 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
One, the processing of sample
The mouth and nose swab that gathers is put into 2ml screw socket centrifuge tube, add 1ml PBS, roll, after the centrifugal 5min of 3000rpm, get the 200ul supernatant and extract test kit (QIAamp Viral RNA Kit, Qiagen) extraction nucleic acid with commercial RNA, the gained nucleic acid solution can directly use as template.
Adopt the mouth and nose swab of disease sheep to carry out above-mentioned processing, get in nucleic acid-templated each L to 3 0.2mL centrifuge tube of 5 μ, as I group reaction pipe.Adopt the mouth and nose swab of immune sheep to carry out above-mentioned processing, get in nucleic acid-templated each L to 3 0.2mL centrifuge tube of 5 μ, as II group reaction pipe.Adopt the mouth and nose swab of Healthy Sheep to carry out above-mentioned processing, get in nucleic acid-templated each L to 3 0.2mL centrifuge tube of 5 μ, as III group reaction pipe.Get in each L to 3 0.2mL centrifuge tube of 5 μ of DEPC treated water, as the negative control pipe.Get in each L to 3 0.2mL centrifuge tube of 5 μ of positive reference substance, as the positive control pipe.
Two, RT-PCR reaction
The 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, every pipe adds 20 μ L premixed liquids.
Carry out the RT-PCR reaction, reaction conditions is: 50oC reverse transcription 30 min; 94oC carries out activation 2 min of Taq enzyme; The PCR of 30 circulations reaction (94oC sex change 30sec, 63oC anneal 30sec, 72oC extends 30sec); 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.It is two specific amplification bands of 330 bp and 477bp that positive control Guan Jun has size, and the negative control product are without the 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 has also confirmed 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, the preparation final concentration is 10
6The vaccine strain engineered rna of copy/μ L is as vaccine strain positive criteria product, and the preparation 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 series diluent
With the DEPC treated water, street strain's positive criteria product and vaccine strain positive criteria product are carried out respectively serial dilution, 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 respectively 2.5 μ L concentration be 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 respectively 2.5 μ L concentration be 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 respectively 2.5 μ L concentration be 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 respectively 2.5 μ L concentration be 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 respectively 2.5 μ L concentration be 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 respectively 2.5 μ L concentration be 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
The 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 reaction premixed liquids, carry out the detection of RT-PCR reaction and product.
Four, result
Detected result is seen 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 pipe of test set V ~ VI only has size to be the specific amplification band of 330bp, and 5 ~ No. 6 pipe does not have amplified band.
Extract respectively foot and mouth disease virus and sheep of virus nucleic acid RNA, adopt the test kit of embodiment 1 preparation to carry out specific detection.
One, the processing of sample
Above-mentioned virocyte nutrient solution sample, commodity in use RNA extracts reagent (QIAamp Viral RNA Kit, Qiagen) and extracts nucleic acid, and the gained nucleic acid solution directly uses as template.Get in nucleic acid-templated each L to 3 the 0.2mL centrifuge tube of 5 μ of foot and mouth disease virus, as I group reaction pipe.Get in nucleic acid-templated each L to 3 the 0.2mL centrifuge tube of 5 μ of sheep of virus, as II group reaction pipe.Get in DEPC treated water 5 μ L to 0.2mL centrifuge tubes, as the negative control pipe.Get in positive reference substance 5 μ L to 0.2mL centrifuge tubes, as the positive control pipe.
Two, RT-PCR reaction
The 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, every pipe adds 20 μ L reaction premixed liquids.
Carry out the detection of RT-PCR reaction and product.
Three, result
Detected result is seen Fig. 3, and positive control Guan Jun has size to be two specific amplification bands of 330bp and 477bp, and the negative control product are without the specific amplification band.Each Guan Jun of test set I and II does not have specificity expansion band, thereby proves that primer sets of the present invention has good specificity.
Above-described embodiment is the better embodiment of the present invention, 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, combination, simplification etc., all should be the equivalence substitute mode, be included in of the present invention comprise scope within.
Claims (10)
1. primer sets of be used for differentiating the wild strain of PPR virus and vaccine strain includes for detection of the primer pair of the wild strain of PPR virus with for detection of the primer pair of PPR virus vaccine strain; Wherein detect the nucleotides sequence of street strain's primer pair and classify SEQ ID NO:3 and SEQ ID NO:4 as; Detect the nucleotides sequence of vaccine strain primer pair and classify SEQ ID NO:5 and SEQ ID NO:6 as.
2. primer sets as claimed in claim 1, is characterized in that described street strain primer pair amplification purpose product clip size is 477 ± 3 bp, and vaccine strain primer pair amplification purpose product clip size is 324 ± 3 bp.
3. the application of primer sets claimed in claim 1 in the goods of the preparation discriminating wild strain of PPR virus and vaccine strain.
4. a test kit that is used for differentiating the wild strain of PPR virus and vaccine strain, is characterized in that, described test kit includes primer sets claimed in claim 1.
5. test kit as claimed in claim 4, is characterized in that, also includes PCR reaction buffer, archaeal dna polymerase and reversed transcriptive enzyme.
6. test kit as claimed in claim 5, is characterized in that, also includes the positive control nucleic acid of the wild strain of PPR virus and vaccine strain.
7. test kit as claimed in claim 5, the positive control nucleic acid that it is characterized in that the wild strain of described PPR virus are PPR virus street strain's virus or its RNA or the RNA that contains the synthetic of the PPR virus H of street strain gene fragment.
8. test kit as claimed in claim 5, is characterized in that described PPR virus vaccine strain positive control nucleic acid is PPR virus Nigeria 75/1 vaccine strain virus or its RNA or the RNA that contains the synthetic of PPR virus Nigeria 75/1 vaccine strain H gene fragment.
9. as test kit as described in claim 5-8 any one, characterized by further comprising the amplification negative control.
10. test kit as claimed in claim 9, is characterized in that described amplification negative control is that DEPC processes water or phosphoric acid buffer.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695565A (en) * | 2013-12-27 | 2014-04-02 | 中国检验检疫科学研究院 | Reagent kit for detecting peste des petits ruminant vaccine poison by pyrophosphoric acid sequencing technology |
| CN105063236A (en) * | 2015-07-31 | 2015-11-18 | 中国动物卫生与流行病学中心 | Method for identifying peste des petits ruminants virus vaccine strain and gene 4-line wild 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 |
| CN102108359B (en) * | 2009-12-23 | 2012-12-19 | 上海市农业科学院 | Gene-modified peste des petits ruminants N gene and modification method and chemical synthesis thereof |
-
2013
- 2013-03-25 CN CN201310097627.9A patent/CN103131800B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108359B (en) * | 2009-12-23 | 2012-12-19 | 上海市农业科学院 | Gene-modified peste des petits ruminants N gene and modification method and chemical synthesis thereof |
| 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 |
Non-Patent Citations (2)
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| NCBI: "Peste-des-petits-ruminants virus complete genome", 《GENBANK》, 16 June 2005 (2005-06-16), pages 67906091 * |
| 张玲等: "RT-PCR鉴别小反刍兽疫病毒疫苗株与强毒株方法的建立", 《动物医学进展》, vol. 31, no. 3, 31 December 2010 (2010-12-31), pages 17 - 20 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695565A (en) * | 2013-12-27 | 2014-04-02 | 中国检验检疫科学研究院 | Reagent kit for detecting peste des petits ruminant vaccine poison by pyrophosphoric acid sequencing technology |
| CN103695565B (en) * | 2013-12-27 | 2015-04-08 | 中国检验检疫科学研究院 | Reagent kit for detecting peste des petits ruminant vaccine poison by pyrophosphoric acid sequencing technology |
| CN105063236A (en) * | 2015-07-31 | 2015-11-18 | 中国动物卫生与流行病学中心 | Method for identifying peste des petits ruminants virus vaccine strain and gene 4-line wild strain |
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| Publication number | Publication date |
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| CN103131800B (en) | 2015-02-25 |
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