CN103717756B - For detecting the test of the nucleotide subsitution in the genome of Sa guest oral polio virus vaccine virus - Google Patents
For detecting the test of the nucleotide subsitution in the genome of Sa guest oral polio virus vaccine virus Download PDFInfo
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Abstract
The invention provides for specifically and detect multiple SNP test method and the primer of the sudden change in the genome of the poliovirus that attenuation oral polio virus vaccine (Sa Bin) the alive virus of all three kinds of serotypes, vaccine sample and vaccine derive simultaneously.Importantly, primer of the present invention and test eliminate expensive, time-consuming and complicated process, as the demand of complete gene order-checking.This test newly can at very short several sample of time build-in test.Therefore, this test is very simple, quick and cost-efficient.Additionally provide the test kit comprising this new primer.
Description
Invention field:
The present invention broadly belongs to biological technical field, and be specifically related to the new primer of the sudden change of the specific site in the genome for detecting the poliovirus that attenuation oral polio virus vaccine (Sa Bin) alive virus, vaccine sample and vaccine derive, and the test kit containing above-mentioned primer.
Background of invention:
Doctor AlbertBSabin have developed the attenuation oral polio virus vaccine alive for immunity antagonism paralytic spinal poliomyelitis.He is by careful laboratory work, isolates the virus strain of sudden change from the disease of the wild poliovirus of all three kinds of serotypes of generation very carefully.By the strain of these attenuations (non-neurotoxic) poliovirus for generation of attenuation oral polio vaccine of living, this vaccine has employed more than 50 year in the whole world.Sa Bin have also developed the test of the neurotoxicity (NVT) for evaluating poliovirus in monkey.The standard quality that NVT has become for proving oral polio virus vaccine attenuation controls test.Have developed the neurotoxicity test in transgenic mice recently, as the replacement scheme of monkey NVT.
NVT can check the biological character (attenuation and toxicity) of poliovirus.Although NVT can by the classification of severity of toxicity, it can not determine the genome proterties of virus.
In the eighties in 20th century, describe the antigen and molecule marker of distinguishing wild and Sa guest OPV strain.Genome sequencing, clone and recombinant DNA technology are used for the genome difference between that understand Sa guest attenuation and that parent is wild poliovirus.These researchs cause the detection of the specific sudden change relevant to the attenuation proterties of Sabin vaccine poliovirus.
To give most possible attenuation site in following table 1:
Have developed molecular testing, that is, by the mutation analysis of PCR and restriction enzyme cutting (MAPREC), with the sudden change on Single locus in quantitative Sabin vaccine strain (the main attenuation site in the 5 ' untranslated district) and with the associating of NVT result.WHO has recommended this test for testing the quality of poliovirus vaccine.Recently, have developed the abrupt climatic change by PCR in real time.Also have developed the Microarray of the sudden change that can detect in whole virogene of poliomyelitis group.
United States Patent (USP) 5585477 and 5691134 relates to the Auele Specific Primer for detecting the poliovirus in clinical sample, and thus itself and non-poliomyelitis enterovirus (NPEV) are made a distinction, but the specific test method distinguishing poliovirus based on its inherited character (that is, specifically based on attenuation site) is not described.
Complete gene order-checking and full-length genome micromatrix can determine the exact nucleotide (A, T, G or C) in all known poliomyelitis attenuation sites, but still there is not fast and easily test and be used as above-mentionedly in single test, detecting poliovirus 1(six sites simultaneously), 2(tri-sites) and 3(2 site) the costliness of sudden change in all known attenuation site and the alternative of complex technology.
Goal of the invention:
The object of the application is to be provided for detect the multiplex PCR test of the sudden change of the specific site in the genome of the poliovirus that attenuation oral polio vaccine (Sa Bin) virus alive of these three kinds of serotypes, vaccine sample and vaccine derive simultaneously.
Another free-revving engine of the present invention is to provide the new primer of the sudden change in the specific attenuation site in the genome of the poliovirus for detecting these three kinds of serotypes simultaneously.
Another object of the present invention is to provide for the test kit comprising above-mentioned primer in multiplex PCR test, the sudden change of the specific site in the genome of the poliovirus that described test is used for detecting that the attenuation oral polio vaccine (Sa Bin) alives of these three kinds of serotypes is viral simultaneously, vaccine sample and vaccine derive.
Invention statement
According to the present invention, provide the sudden change of specific site in the genome of the poliovirus that attenuation oral polio vaccine (Sa Bin) alive virus, vaccine sample and vaccine for detecting these three kinds of serotypes simultaneously derive or the test of SNP and primer.
Summary of the invention:
The present invention relates to the test of the sudden change of the specific site in the genome of the poliovirus that attenuation oral polio vaccine (Sa Bin) virus alive for detecting these three kinds of serotypes simultaneously, vaccine sample and vaccine derive and new primer.Present invention also offers the test kit of the primer of the sudden change on all specific sites in the genome comprised for detecting above-mentioned virus.
the summary of the present invention of reference table/schema/embodiment etc.:
Fig. 1 & 1A depicts for the result of Sa guest poliovirus 1 with the sudden change-SNaP firing test (shotassay) of the Nucleotide of the specific position of contrast target.
Fig. 2 & 2A depicts for the result of Sa guest poliovirus 2 with the sudden change-SNaP firing test of the Nucleotide of the specific position of contrast target.
Fig. 3 & 3A depicts for the result of Sa guest poliovirus 3 with the sudden change-SNaP firing test of the Nucleotide of the specific position of contrast target.
Fig. 4 depicts the first step multiplex PCR result on 2% sepharose.
Table 1 provides attenuation site most possible in the genome of poliovirus.
Table 2 provides the primer of DNA cloning for the preparation of desired specificities region.
with reference to the detailed description of the present invention of accompanying drawing/embodiment
Reference table, accompanying drawing etc. and specific embodiment describe the present invention; This description is not want to explain in a limiting sense.When with reference to description of the invention, the various embodiment for replacing of the present invention will be that those skilled in the art are apparent.Therefore expect that these can form a part of the present invention for the embodiment of replacing.
The invention provides a kind of test method; A set of specific new primer and the test kit comprising these Auele Specific Primers, for detecting the Nucleotide in all known attenuation site of the poliovirus of all three kinds of hypotypes in single test simultaneously, described three kinds of hypotypes and poliovirus 1(six sites), 2(tri-sites) and 3(2 site).
Other advantages of this test are that same principle can be utilized to extend further to comprises other site.This test based on multiple reverse transcription PCR, with virogene of poliomyelitis group RNA is transformed into cDNA and DNA cloning son.Then PCR primer is used for multiple SNP to test.Use GeneticAnalyzer to resolve SNP and test product, to identify the Nucleotide (Fig. 1 to 3) in the attenuation site in virogene of poliomyelitis group.This test is applicable to a large amount of sample, and for test vaccine and field isolates.This test method can be transformed into quantitative test.
the detailed content of test:
1. poliovirus sample (known serotype, such as, poliovirus 1,2 or 3) is obtained from various source.(be separated 1,2 and 3 type polioviruses from faecal samples, obtain oral polio virus vaccine (Sa Bin) strain from NIBSc.PK).
2. use antisense PCR primer synthesis cDNA, and produce DNA cloning in single multiple reverse transcription and PCR reaction.
3. purified pcr product (DNA cloning).
4. use " new primer " of the present invention to carry out multiple single nucleotide polymorphism detection experiment [test at present for 1 type poliovirus comprises 6 primer pairs, and 2 types comprise 3 primer pairs, and 3 types comprise 2 primer pair polyads].
5. resolve SNP by the electrophoresis on GeneticAnalyzer, retrieve data, and use these data of GeneMapper software analysis.
Novelty of the present invention and creative in new design of primers, for being intrinsic in the test kit that detects the convenience of the Multiple experiments of the sudden change in all attenuation sites and specificity and comprise these Auele Specific Primers simultaneously.In order to understand the present invention better, provide further detailed content as follows:
Table 1: attenuation site most possible in the genome of poliovirus
I.
the design of primer: for the new PCR primer of detection specificity sudden change
A. a primer Nucleotide shorter in attenuation site to be studied
B. devised 5 ' end tail of variable-length, and be combined in primer.
C. genome sequence reaction (complementation) of cauda Duan Buyu primer target.
D. design the primer (base pair) of increasing lengths, when making to resolve in GeneticAnalyzer, first represent first attenuation site with ascending order, and last site in the end.
2,
test method
A. this test can identify the allelotrope of all nucleotide positions of target, that is, can detect the Nucleotide in all attenuation sites in poliovirus 1,2 and 3.
If b. should novel site be comprised, can in conjunction with other primers in a Multiple experiments.
C. for three kinds of poliovirus serotypes, different primer sets is designed.
D. all attenuation sites can be studied in a test.
Table 2: for the preparation of the primer of DNA cloning in desired specificities region
1-type
1.PV128-5′CATGGGACGCTAGTTGTGAA3′
2.PVI-IA-5'ATGAAACCTGAGCACCCAIT3'
3.Y7-5'GGGTTTGTGTCAGCCTGTAATGA3'
4.81-5'TGGGACGACTACACATGGCA3'
5DG53-5'TGGCTGCITATGGTGACAAT3'
6.DC21-5'TCA'GGTAATITCCACCACCA3'
7.PVI-J38-5'AAGTCATCGGGATGCATGIT3'
8，PV112A-5'CTGGCCAGCATAGTGGTCTA3'
2-type
1.PV23218-5'TGAGTCTGGACATTCCTCACC3'
2.PV2-575A.5′GTCACCATAAGCAGCCATGA3'
3.PV2-38308-5'CAACTGAGACGCAAACTGGA3'
4.PV2-3018A-5'AGAGGACGTCAGCCACGTAD'
3-type
I.PV3-285'ACGGGACGCTAGITGTGAAC3'
2.PV3-2A5'GCTCCCATTGTGACACTGAA3'
3.PV3-585'GAGCTCGCCGAGATAGACAC3'
4.PV3-5A5'AGATGAITGGAGGCCAAGAD'
The following provide the detailed content of the primer for SNP test:
For the SNP primer of 1-type (Sa guest virus)
Primer: PVIIF-1, nucleotide position-480
Serial ID 1:
5'GACTGACTAATGCGGCTAATCCCAACCTCGG3′
Tm-67.6, GC-56.5%, length-31nt
Primer: PVI_2F-I, nucleotide position-935
Serial ID 2:
5'GACTGACTGCCCATCAAGGATGTCCTGATAAAAACA3'Tm-66.4, GC-42.9%, length-36nt
Primer: PVI_3F-1, nucleotide position-2438
Serial ID 3:
5'GACTGACTGACTGACTTGTAATGACTTCAGCGTGCGCTTG3'Tm-65.2, GC-50%, length-40nt
Primer: PVI_4F-1, nucleotide position-2795
Serial ID 4:
5'GACTGACTGACTCAGCTTCCACCAAGAATAAGGATAAGCTATTT3'Tm-64.4, GC-37.5%, length-44nt
Primer: PVI_5F-I, nucleotide position-2879
Serial ID 5:
5'GACTGACTGACTGACTGACTTTCACCTATTCTAGATTTGATATGGAA3'Tm-55 .7, GC-29.6%, length-47nt
Primer: PVI_6F1, nucleotide position-6203
Serial ID 6:
5'GACTGACTGACTGACTGACTACGTGGGTAACAAAATTACTGAAGTGGATGAG3T m-66.3, GC-40.6%, length-52nt
For the SNP primer of (2-type poliomyelitis)
Primer: PV2_2A, nucleotide position-481
Serial ID 7:
5'GACTGACTCGGCTAATCCTAACCACGGA3'
Tm-58.7, GC-55%, length-28nt
Primer: PV2_28, nucleotide position-2908
Serial ID 8:
5'GACTGACTGACTTTTTGTGGTCACCTCAAACTAC3'
Tm-53.2, GC-40.9%, length-34nt
Primer: PV2_2C, nucleotide position-2909
Serial ID 9:
5'GACTGACTGACTGACTTTTTGTGGTCACCTCAAACTACA3'Tm-56, GC-39%, length-39nt
For the SNP primer of (3-type poliomyelitis)
Primer: PV3_3A, nucleotide position-472
Serial ID 10:
5'GACTGACTCCCCTGAATGCGGCTAAT3'
Tm-57.2, GC-55.6%, length-26nt
Primer: PV3_38, nucleotide position-2034
Serial ID 11:
5'GACTGACTGACTGCTTGTCACTATCCCCAGCAT3'
Tm-57.8, GC-52.4%, length-33nt
According to one of them embodiment, concrete test method disclosed in the present invention can identify the allelotrope of all nucleotide positions of target and study all attenuation sites in a test, and therefore can be used for the Nucleotide in all attenuation sites of identifying in poliovirus 1,2 and 3 genome.If need to comprise new site, additional primers can be incorporated in a Multiple experiments.This test method make use of the different primers group into these three kinds of poliovirus serotype designs.This test method is applicable to a large amount of sample, and may be used for detecting vaccine and field isolates.In addition, this test method can be transformed into quantitative test, and easily extensible is to comprising other mutational site.
Another embodiment of the invention relates to the PCR primer in the mutation specific site in the genome of the poliovirus that attenuation oral polio virus vaccine (Sa Bin) virus alive, vaccine sample and vaccine for detecting all three kinds of serotypes derive.The feature of this primer is that they have change over its length, and is a Nucleotide shorter in attenuation site to be studied.This primer has been designed to 5 ' the end tail in conjunction with variable-length, and the genome sequence of this cauda section and primer institute target is non-complementary.
Devise the primer of increasing lengths (base pair), when making to resolve in GeneticAnalyzer, first represent first attenuation site with ascending order, and last site in the end.
Another aspect more of the present invention relates to test kit, it contains above-mentioned primer, for detecting the sudden change (nucleotide subsitution) of the specific site in the genome of the poliovirus that attenuation oral polio virus vaccine (Sa Bin) the alive virus of all three kinds of serotypes, vaccine sample and vaccine derive, comprise above-mentioned primer.This test kit comprises deoxy-ribonucleoside triphosphate, polysaccharase, divalent cation and/or damping fluid further.
Embodiment:
Following examples are for illustration of of the present invention, instead of are intended to limit the scope of the invention by any way.
Embodiment 1:
DNA cloning is prepared from the poliovirus of three kinds of serotypes
Obtain the poliovirus of each serotype (1,2 or 3 type) individually.In single multiple reverse transcription and PCR reaction (Roche), antisense PCR primer and DNA cloning is used to produce, from freezing infection culture synthesis poliovirus sample, cDNA.By PCR purification kit (Quagcn) purified pcr product (DNA cloning).
RT-PCR-analyzes for RT-PCR, directly obtains the 67mMTris/HCl(pH8.8 in freezing infection culture (1fll) and 50fll cumulative volume), 17mM ammonium sulfate, 6mMEDTA, 2mMMgCh, 200Mm often plant dNTP, 1mM dithiothreitol (DTT), 1mM and often plant primer, 10U placenta RNA enzyme inhibitors (Roche), 3U avian meloblastosis virus (AMV) reversed transcriptive enzyme (Roche) and 5UTaqDNA polysaccharase (RocheAppliedScience).Reaction is hatched 30 minutes at 50 DEG C, then at 94 DEG C, hatches 3 minutes.35 thermal cyclings have been carried out, 94 DEG C, 30s, 42 DEG C, 30s, 72 DEG C, 30s in 9700-type thermal cycler (AppliedBiosystems).Then 5 minutes are hatched at 72 DEG C after thermal cycling.Carry out analytical reaction product by electrophoresis in 10% polyacrylamide TBE gel, and dye with 0.5flg Ethidum Eremide mrl.Purify DNA is carried out by QIA QIAquick PCR purification kit (Qiagen).
Embodiment 2:
For the identification of Single nuclear polymorphism test (SNP test) of sudden change
Namely reaction mixture (5lll), primer mixture (3lll) (final concentration is being used containing SNaPshot is multiple, 0.15-0.6Ilmolll) with in the 10III final volume of template (2Ill) carried out 10 SNP reactions, described template is made up of the multiple PCR products of above-mentioned QIAquickPCR purification kit (Qiagcn) purifying.By the multiple PCR products set of increasing from viral RNA, and before SNaPshot reaction, at identical purified on columns.Cycling program comprises 25 circulations, 96 DEG C of 10s, 50 DEG C of 5s and 60 DEG C 30s.By hatching IS-minute with 1U shrimp alkaline phosphotase at 37 DEG C, at 75 DEG C, hatching IS-minute subsequently, making enzyme-deactivating carry out purifying extension products.The product (0.5Ill) of purifying is mixed with 9III methane amide and 0.5IIIGeneScan-120LIZ size standards (AppliedBiosystems), and by capillary electrophoresis (ABIPRISM3130GeneticAnalyzer; AppliedBiosystems) be separated.With GeneMapper3.0 software (AppliedBiosystems) analytical results.
Design SNP primer by this way, make each primer Nucleotide shorter in attenuation site to be studied, and synthesize from Sigma.The primer of design increasing lengths (base pair), make first to represent first attenuation site, and last site in the end.Multiple single nucleotide polymorphism detection experiment has been carried out by SnaPshot test kit (AppliedBiosystem).By GeneticAnalyzer3130(AppliedBiosystem) on capillary electrophoresis resolve SNP, and regain data, and by GeneMapper software analysis.
Advantage of the present invention
1. can study all attenuation sites in a test, and detect the Nucleotide in all attenuation sites in poliovirus 1,2 and 3 genome.
2. this test method can detect the allelotrope (base of mixing) in all research sites.
3. this test method can identify the allelotrope of the nucleotide position of all targets.
4. this test comprises multiple reaction.
5. this test can operate a large amount of sample.
6. this test can be used for vaccine detection and field isolates detection.
7. this test can be transformed into quantitative test.
8. this test easily extensible is to comprising other site.
Claims (8)
- The PCR primer of 1.SEQID1 to 11 for the preparation of detect in single reaction simultaneously three kinds of serotypes poliovirus genome in the sudden change of specific site or the purposes of the test kit of SNP, the implementation Process of wherein said detection by comprising the following steps:A. poliovirus sample is obtained from various source,B. by using DNA cloning of the poliovirus of the synthesis of antisense PCR primer cDNA, generating step a,C. DNA cloning of purification step b,D. use the PCR primer of SEQID1 to 11, carry out multiple single nucleotide polymorphism testing process,E. SNP is resolved by electrophoresis,F. retrieve data, and use these data of GeneMapper software analysis.
- 2. the purposes of claim 1, wherein said virus is selected from 1-type poliovirus, 2-type poliovirus and 3-type poliovirus.
- 3. the purposes of claim 1, wherein saidPrimer SEQID1 to 6 detects the SNP in 1-type poliovirus,Primer SEQID7 to 9 detects the SNP in 2-type poliovirus, andPrimer SEQID10 to 11 is for detecting the SNP in 3-type poliovirus.
- 4. the purposes of claim 1, wherein said primer detects the SNP in the poliovirus that the attenuation oral polio virus vaccine alive virus of three kinds of serotypes, vaccine sample and vaccine derive.
- 5. the purposes of claim 1, wherein said primer has been designed to 5 ' the end tail in conjunction with variable-length.
- 6. the purposes before any one of claim, wherein with the Design of length primer increased progressively, making when resolving in GeneticAnalyzer, first represent first attenuation site, and last site in the end with ascending order.
- 7. the purposes of claim 1, if wherein will comprise new site, then can be incorporated to additional primers in a Multiple detection process.
- 8., for detecting the sudden change of specific site in the genome of the poliovirus that the attenuation oral polio virus vaccine alive virus of all three kinds of serotypes, vaccine sample and vaccine derive or the test kit of SNP in single reaction simultaneously, wherein this test kit comprises the primer of SEQID1 to 11.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/IN2012/000160 WO2013132504A1 (en) | 2012-03-07 | 2012-03-07 | Assay for the detection of nucleotide substitutions in genomes of sabin oral poliovirus vaccine viruses |
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| CN103717756A CN103717756A (en) | 2014-04-09 |
| CN103717756B true CN103717756B (en) | 2016-03-30 |
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| CN (1) | CN103717756B (en) |
| BR (1) | BR112013030390A2 (en) |
| HK (1) | HK1194438A1 (en) |
| WO (1) | WO2013132504A1 (en) |
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| CN110923359A (en) * | 2019-11-29 | 2020-03-27 | 中国食品药品检定研究院 | Mass spectrum detection primer group for detecting I-type spinal cord ash attenuated live vaccine nucleic acid mutation and method thereof |
| WO2023002505A1 (en) * | 2021-07-19 | 2023-01-26 | Indian Council Of Medical Research | Assay for detection of epidemiologically important sars- cov-2 variants |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101522216A (en) * | 2006-08-10 | 2009-09-02 | 英国国家生物标准委员会 | Attenuated polio viruses |
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| US5728519A (en) * | 1990-11-06 | 1998-03-17 | The United States Of America As Represented By The Department Of Health And Human Services | Assay for virulent revertants of attenuated live vaccines and kits therefor |
| US5585477A (en) | 1993-07-13 | 1996-12-17 | Us Health | Poliovirus specific primers |
| GB9323819D0 (en) * | 1993-11-19 | 1994-01-05 | Smithkline Beecham Biolog | Anlytical method |
-
2012
- 2012-03-07 BR BR112013030390A patent/BR112013030390A2/en not_active Application Discontinuation
- 2012-03-07 CN CN201280023280.7A patent/CN103717756B/en not_active Expired - Fee Related
- 2012-03-07 WO PCT/IN2012/000160 patent/WO2013132504A1/en active Application Filing
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101522216A (en) * | 2006-08-10 | 2009-09-02 | 英国国家生物标准委员会 | Attenuated polio viruses |
Non-Patent Citations (3)
| Title |
|---|
| ABI Applied Biosystems.ABI PRISM？ SNaPshot™Multiplex Kit.《http://tools.invitrogen.com/contents/sfs/manuals/cms_041203.pdf》.2010,第9页，第29页. * |
| Mechanisms of genetic variation in polioviruses;Carita Savolainen-Kopra等;《Rev. Med. Virol.》;20101015;第20卷;第363页右栏最后一段至第365页表2，第365页右栏第2段， * |
| SNaPshot技术检测乙型肝炎病毒基因组P基因区单核苷酸多态性的研究;陈勇等;《中华实验和临床病毒学杂志》;20050630;第19卷(第2期);162-164 * |
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| HK1194438A1 (en) | 2014-10-17 |
| WO2013132504A1 (en) | 2013-09-12 |
| CN103717756A (en) | 2014-04-09 |
| BR112013030390A2 (en) | 2016-09-06 |
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