CN105039330A - GII.4 type norovirus genome amplimer and amplification method - Google Patents
GII.4 type norovirus genome amplimer and amplification method Download PDFInfo
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Abstract
The invention discloses a GII.4 type norovirus genome amplimer and an amplification method. Six pairs of primers respectively serve as upstream and downstream primers of the amplimer, RNA of GII.4 type norovirus serves as a template for performing RT-PCR amplification to obtain amplification products respectively, then the amplification products are subjected to nucleotide sequence sequencing, then splicing comparison is performed to obtain a full length sequence of the GII.4 type norovirus genome. Aimed at a main epidemic genotype of the GII.4 type norovirus, a 4+1+1 subsection amplification strategy is designed according to three open reading frames contained in the genome, and corresponding amplimers are designed according to a conservation region; the whole genome sequence of the GII.4 type norovirus can be effectively amplified. The amplimer disclosed by the invention can be widely applied to corresponding scientific research fields of medical hygiene, inspection quarantine, and the like having norovirus detection demand.
Description
Technical field:
The invention belongs to biological technical field, be specifically related to a kind of GII.4 type norovirus genome amplification primer and amplification method.
Background technology:
Norovirus is the important pathogen of global pandemic and acute sporadic hepatitis E virus infected patients gastro-enteritis, causes about 2.3 hundred million person-times to catch an illness every year, particularly serious in developing country, at least causes the death of 200,000 example, less than 5 years old children, causes great threat to public health security.Norovirus infects for self-limited disease, but can have the danger of dehydration property death for infant, old man and immune deficiency crowd.To so far, also there is no effective antiviral and treatment means.
As RNA viruses, norovirus very easily makes a variation, and has abundant genetic diversity.Virus can be divided into 6 genomes (Genogroup) according to its capsid protein sequence homology, the nucleic acid sequence homology between genome about 46%, wherein G I, G II and G IV is people source virus.Nucleic acid homology in same gene group is 69% ~ 97%, and be divided into different genotype (Genotype) further, such as GI contains 8 genotype, and GII contains 19 (and constantly increasing).Wherein, GII.4 type is current global Major Epidemic genotype, and the norovirus of nearly 80% infects and caused by this type strain, and this virus will produce new variant every 1 year, caused the viral prevalence on a large scale of a new round simultaneously.
Although had breakthrough recently in the norovirus cultivation of people source, the suitable system that copies still has lacked.Therefore, the acquisition of genomic information is significant for norovirus research.Norovirus genome is about 7.5-7.8kb, comprises 3 open reading frame.The genome amplification method of current report has direct TRAP and segmentation TRAP.Wherein, direct amplification method is often difficult to have higher sensitivity, and the quality treating process sample has higher requirement, does not thus have higher success ratio.In addition, that commonly uses in segmentation TRAP comprises three-stage process and multiple process, and the general basis of the primer of the multiple process genome sequence high with viral similarity in pending sample designs, and thus also result in certain restriction to the broad spectrum of primer; Increase in three-stage process the fragment length comparatively large (>3K) obtained, and this not only adds the difficulty of amplification, and is difficult to direct Sequencing, thus reduces the success ratio obtaining virus genome sequence.
Summary of the invention:
The object of this invention is to provide a kind of highly sensitive GII.4 type norovirus genome amplification primer and amplification method.
GII.4 type norovirus genome amplification primer of the present invention, is characterized in that, comprise six pairs of amplimers:
Primer pair 1:P1F:5 '-GTGAATGAAGATGGCGTCTAAC-3 ';
P1596R：5′-TCGACGCCATTGCGTGGGA-3′；
Primer pair 2:P1258F:5 '-CAACCATATGACCACCTTGCT-3 ';
P2805R：5′-ATGGCCACCTCCTCATAGTA-3′；
Primer pair 3:P2689F:5 '-AGGTCTCAGTGATGAAGAG-3 ';
P4248R：5′-GGGCCATCCTCATTCATATTCA-3′；
Primer pair 4:P4099F:5 '-TGGTAAGATCAAGAAGAGGCT-3 ';
G2SKR：5′-CCRCCNGCATRHCCRTTRTACAT-3′；
Primer pair 5:NV2of2:5 '-GGAGGGCGATCGCAATC-3 ';
GV132：5′-CCRGCRAAGAAAGCTCCAGCCAT-3′；
Primer pair 6:P6484F:5 '-CAGCACAATCTGATGTGGC-3 ';
P7513R：5′-TTACACCCGTGACTCCCCT-3′。
Second object of the present invention is to provide the genomic amplification method of a kind of GII.4 type norovirus, it is characterized in that, respectively using above-mentioned primer P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132 and P6484F/P7513R as the upstream and downstream primer of amplimer, with the RNA of GII.4 type norovirus for template carries out RT-PCR amplification, obtain amplified production respectively, then determining nucleic acid sequence is carried out to amplified production, and then splice comparison, obtain GII.4 type norovirus full-length genome sequence.
Preferably, described RT-PCR, its reaction system is: containing 2 × one-stepRT-PCRmixture10 μ L, 10 μm of ol/L upstream primers and 10 μm of each 0.6 μ L of ol/L downstream primer, MLV/RNasin/HS-Taq enzyme mixation 0.8 μ L, RNA template 2 μ L, all the other complement to 20 μ L by distilled water, reaction conditions is: 50 DEG C of reverse transcription 30min, 94 DEG C of denaturation 3min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 80s, after totally 30 circulations, 72 DEG C finally extend 7min.
The present invention is directed to GII.4 type norovirus Major Epidemic genotype, three open reading frame comprised according to genome devise the segmentation amplification strategy of " 4+1+1 ", and design corresponding amplimer according to conservative region; On the basis of Optimal reaction conditions, each pair of primer all reaches the amplification sensitivity identical with conventional sense primer, effectively can amplify the whole genome sequence of GII.4 type norovirus.The present invention can be widely used in health care, inspection and quarantine etc. and have norovirus detection demand and corresponding scientific research field.
Accompanying drawing illustrates:
Fig. 1 is the RT-PCR annealing temperature optimization being applicable to GII.4 type norovirus genome amplification primer, electrophoresis order is M, 1-36, wherein M is DNALadder, 1-6,7-12,13-18,19-24,25-30,31-36 be respectively the electrophoresis result of primer P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132, P6484F/P7513R amplified production under different annealing temperature (being followed successively by 50 DEG C, 52 DEG C, 54 DEG C, 56 DEG C, 58 DEG C, 60 DEG C).
Fig. 2 is the RT-PCR sensitivity analysis being applicable to GII.4 type norovirus genome amplification primer, and electrophoresis order is M, 1-42.Wherein M is DNALadder, 1-6,7-12,13-18,19-24,25-30,31-36,37-42 be respectively genome amplification primer P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132, P6484F/P7513R and (be followed successively by 10 with detection primer G2SKF/G2SKR at the different extent of dilution of viral RNA
1-10
6doubly dilution) under the electrophoresis result of amplified production.
Fig. 3 is virus genomic expanding effect in actual sample, and electrophoresis order is M, 1-30.The electrophoresis result that wherein M is DNALadder, 1-5,6-10,11-15,16-20,21-25,26-30 are followed successively by actual sample L10, L62, L65, L106, L232 carry out 4+1+1 strategy amplification gene group 6 products.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
The GII.4 type norovirus genome amplification primer that following examples use, specific as follows:
Primer pair 1:P1F:5 '-GTGAATGAAGATGGCGTCTAAC-3 ';
P1596R：5′-TCGACGCCATTGCGTGGGA-3′；
Primer pair 2:P1258F:5 '-CAACCATATGACCACCTTGCT-3 ';
P2805R：5′-ATGGCCACCTCCTCATAGTA-3′；
Primer pair 3:P2689F:5 '-AGGTCTCAGTGATGAAGAG-3 ';
P4248R：5′-GGGCCATCCTCATTCATATTCA-3′；
Primer pair 4:P4099F:5 '-TGGTAAGATCAAGAAGAGGCT-3 ';
G2SKR：5′-CCRCCNGCATRHCCRTTRTACAT-3′；
Primer pair 5:NV2of2:5 '-GGAGGGCGATCGCAATC-3 ';
GV132：5′-CCRGCRAAGAAAGCTCCAGCCAT-3′；
Primer pair 6:P6484F:5 '-CAGCACAATCTGATGTGGC-3 ';
P7513R：5′-TTACACCCGTGACTCCCCT-3′。
Embodiment 1:
Be applicable to the RT-PCR annealing temperature optimization of GII.4 type norovirus genome amplification primer
(1) Virus Sample process and nucleic acid extraction: by PBS solution (pH7.4, DEPC process) dilute the pending sample (containing GII.4 type norovirus L10) of collection to 10% (w/v) concentration, abundant vibration mixing, under 12000 × g, centrifugal 10min collects supernatant liquor 140 μ L, and extracts viral RNA totally 60 μ L in test kit extracting sample by RNA.
(2) " 4+1+1 " genome segmentation TRAP (namely divides 6 sections of amplifications, the primer combo used is as follows: P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132 and P6484F/P7513R, each RT-PCR selecting response pair of primers): the One step RT-PCR reaction system adopting 20 μ L, containing 2 × one-stepRT-PCRmixture10 μ L, upstream primer and downstream primer (10 μm of ol/L) each 0.6 μ L, MLV/RNasin/HS-Taq enzyme mixation 0.8 μ L, sample virus RNA template 2 μ L, all the other are by ddH
2o supplies.
Reaction conditions is: 50 DEG C of reverse transcription 30min, 94 DEG C of denaturation 3min, then 94 DEG C of 30s, 50-60 DEG C of 30s, 72 DEG C of 80s, and after totally 30 circulations, 72 DEG C finally extend 7min.
Annealing temperature is chosen as 50 DEG C, 52 DEG C, 54 DEG C, 56 DEG C, 58 DEG C, 60 DEG C respectively.
(3) electrophoresis, PCR primer sequencing: get amplified production 5 μ L, the sepharose (containing 0.05 ‰ GoldView nucleic acid dye) of 1.0% carries out electrophoresis, and by gel imaging system observations.By primer pair: the order of P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132 and P6484F/P7513R, norovirus genome amplification band is followed successively by 1595bp, 1548bp, 1560bp, 1283bp, 1680bp, 1030bp.Electrophoresis result is as Fig. 1, and result shows that the annealing temperature of P2689F/P4248R primer is chosen as 54 DEG C, and the annealing temperature of all the other primers may be selected to be 56 DEG C.
Embodiment 2: the RT-PCR sensitivity analysis being applicable to GII.4 type norovirus genome amplification primer
(1) Virus Sample process and nucleic acid extraction: by PBS solution (pH7.4, DEPC process) dilute the pending sample (containing GII.4 type norovirus L10) of collection to 10% (w/v) concentration, abundant vibration mixing, under 12000 × g, centrifugal 10min collects supernatant liquor 140 μ L, and extract viral RNA totally 60 μ L in test kit extracting sample by RNA, and carry out the suitable dilution process of 10 × gradient.
(2) " 4+1+1 " genome segmentation TRAP (namely divides 6 sections of amplifications, the primer combo used is as follows: P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132 and P6484F/P7513R, each RT-PCR selecting response pair of primers): the One step RT-PCR reaction system adopting 20 μ L, containing 2 × one-stepRT-PCRmixture10 μ L, upstream primer and downstream primer (10 μm of ol/L) each 0.6 μ L, MLV/RNasin/HS-Taq enzyme mixation 0.8 μ L, sample virus RNA template 2 μ L, all the other are by ddH
2o supplies.
Reaction conditions is: 50 DEG C of reverse transcription 30min, 94 DEG C of denaturation 3min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 80s, and after totally 30 circulations, 72 DEG C finally extend 7min.
With detect primer G2SKF/R (G2SKR:5 '-CCRCCNGCATRHCCRTTRTACAT-3 '; G2SKF:5 '-CNTGGGAGGGCGATCGCAA-3 ') in contrast, carry out according to above-mentioned system and reaction conditions.
(3) electrophoresis, PCR primer sequencing: get amplified production 5 μ L, the sepharose (containing 0.05 ‰ GoldView nucleic acid dye) of 1.0% carries out electrophoresis, and by gel imaging system observations.By primer pair: the order of P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132 and P6484F/P7513R, norovirus genome amplification band is followed successively by 1595bp, 1548bp, 1560bp, 1283bp, 1680bp, 1030bp.Electrophoresis result as Fig. 2, result show with detection primer G2SKF/R (G2SKR:5 '-CCRCCNGCATRHCCRTTRTACAT-3 '; G2SKF:5 '-CNTGGGAGGGCGATCGCAA-3 ') contrast, only the amplification sensitivity of P2689F/P4248R pair of primers is lower than an order of magnitude, and other primers all can reach the sensitivity exceeding and detect primer.
Embodiment 3: virus genomic expanding effect in actual sample
(1) Virus Sample process and nucleic acid extraction: get GII.4 type norovirus positive sample L10, L62, L65, L106, L232, by PBS solution (pH7.4, DEPC process) dilute pending sample to 10% (w/v) concentration, abundant vibration mixing, under 12000 × g, centrifugal 10min collects supernatant liquor 140 μ L, and extracts viral RNA totally 60 μ L in test kit extracting sample by RNA.
(2) " 4+1+1 " genome segmentation TRAP (namely divides 6 sections of amplifications, the primer combo used is as follows: P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132 and P6484F/P7513R, each RT-PCR selecting response pair of primers): the One step RT-PCR reaction system adopting 20 μ L, containing 2 × one-stepRT-PCRmixture10 μ L, upstream primer and downstream primer (10 μm of ol/L) each 0.6 μ L, MLV/RNasin/HS-Taq enzyme mixation 0.8 μ L, sample rna masterplate 2 μ L, all the other are by ddH
2o supplies.
Reaction conditions is: 50 DEG C of reverse transcription 30min, 94 DEG C of denaturation 3min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 80s, and after totally 30 circulations, 72 DEG C finally extend 7min.
(3) electrophoresis, PCR primer sequencing: get amplified production 5 μ L, the sepharose (containing 0.05 ‰ GoldView nucleic acid dye) of 1.0% carries out electrophoresis, and by gel imaging system observations.By primer pair: the order of P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132 and P6484F/P7513R, norovirus genome amplification band is followed successively by 1595bp, 1548bp, 1560bp, 1283bp, 1680bp, 1030bp.Electrophoresis result is as Fig. 3, and result shows, except L62 sample (virus quantity is extremely low), all the other sample standard deviations successfully obtain amplification.
(4) genome sequence splicing and comparison: check order to the amplified production of wherein L10 and L232 sample and splice, obtain genome sequence respectively, length is 7513bp.Two genome sequences are submitted to respectively and carries out BLAST analysis, result shows, L10 (genomic nucleotide sequence is as shown in SEQIDNO.1) and L232 (genomic nucleotide sequence is as shown in SEQIDNO.2) with Genbank accession number are respectively: the strain genome sequences such as KC175323.1 (Hong Kong), KJ196279.1 (Japan), KC577175.1 (Jiangsu) and KJ196283.1 (Taiwan) reach the similarity of 99%.
Claims (3)
1. a GII.4 type norovirus genome amplification primer, is characterized in that, comprises six pairs of amplimers:
Primer pair 1:P1F:5'-GTGAATGAAGATGGCGTCTAAC-3';
P1596R：5'-TCGACGCCATTGCGTGGGA-3'；
Primer pair 2:P1258F:5'-CAACCATATGACCACCTTGCT-3';
P2805R：5'-ATGGCCACCTCCTCATAGTA-3'；
Primer pair 3:P2689F:5'-AGGTCTCAGTGATGAAGAG-3';
P4248R：5'-GGGCCATCCTCATTCATATTCA-3'；
Primer pair 4:P4099F:5'-TGGTAAGATCAAGAAGAGGCT-3';
G2SKR：5'-CCRCCNGCATRHCCRTTRTACAT-3'；
Primer pair 5:NV2of2:5'-GGAGGGCGATCGCAATC-3';
GV132：5'-CCRGCRAAGAAAGCTCCAGCCAT-3'；
Primer pair 6:P6484F:5'-CAGCACAATCTGATGTGGC-3';
P7513R：5'-TTACACCCGTGACTCCCCT-3'。
2. the genomic amplification method of GII.4 type norovirus, it is characterized in that, respectively using primer pair P1F/P1596R, P1258F/P2805R, P2689F/P4248R, P4099F/G2SKR, NV2of2/GV132 and P6484F/P7513R according to claim 1 as the upstream and downstream primer of amplimer, with the RNA of GII.4 type norovirus for template carries out RT-PCR amplification, obtain amplified production respectively, then nucleotide sequence order-checking is carried out to amplified production, and then splice comparison, obtain GII.4 type norovirus full-length genome sequence.
3. amplification method according to claim 2, it is characterized in that, described RT-PCR, its reaction system is: containing 2 × one-stepRT-PCRmixture10 μ L, 10 μm of ol/L upstream primers and 10 μm of each 0.6 μ L of ol/L downstream primer, MLV/RNasin/HS-Taq enzyme mixation 0.8 μ L, RNA template 2 μ L, all the other complement to 20 μ L by distilled water, and reaction conditions is: 50 DEG C of reverse transcription 30min, 94 DEG C of denaturation 3min, then 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 80s, after totally 30 circulations, 72 DEG C finally extend 7min.
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Cited By (9)
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| CN105525039A (en) * | 2016-01-28 | 2016-04-27 | 中国医学科学院医学生物学研究所 | Amplification method for whole genome aiming at different enterovirus serotypes |
| CN105925573A (en) * | 2016-06-30 | 2016-09-07 | 广东省微生物研究所 | GII.P12/GII.3 recombinant norovirus genome amplification primer and amplification method |
| CN106834282A (en) * | 2016-12-02 | 2017-06-13 | 华南农业大学 | A kind of primer sets and method for being segmented amplification Porcine epidemic diarrhea virus full-length genome |
| CN108796129A (en) * | 2018-06-21 | 2018-11-13 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of GI.Pb/GI.6 recombinant types norovirus genome amplification primer and amplification method |
| CN108796130A (en) * | 2018-06-21 | 2018-11-13 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of GII.2 types norovirus genome amplification primer and amplification method |
| CN108796128A (en) * | 2018-06-21 | 2018-11-13 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of GII.8 types norovirus genome amplification primer and amplification method |
| CN108823331A (en) * | 2018-06-21 | 2018-11-16 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of GII.6 type norovirus genome amplification primer and amplification method |
| CN109825643A (en) * | 2019-03-12 | 2019-05-31 | 中国人民解放军东部战区疾病预防控制中心 | Bat source parvovirus genome, amplimer and amplification method |
| CN112725543A (en) * | 2021-03-04 | 2021-04-30 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Characteristic nucleic acid identification primer group, kit and detection method for human norovirus GII.4 |
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| CN105525039A (en) * | 2016-01-28 | 2016-04-27 | 中国医学科学院医学生物学研究所 | Amplification method for whole genome aiming at different enterovirus serotypes |
| CN105925573A (en) * | 2016-06-30 | 2016-09-07 | 广东省微生物研究所 | GII.P12/GII.3 recombinant norovirus genome amplification primer and amplification method |
| CN106834282A (en) * | 2016-12-02 | 2017-06-13 | 华南农业大学 | A kind of primer sets and method for being segmented amplification Porcine epidemic diarrhea virus full-length genome |
| CN108823331A (en) * | 2018-06-21 | 2018-11-16 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of GII.6 type norovirus genome amplification primer and amplification method |
| CN108796130A (en) * | 2018-06-21 | 2018-11-13 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of GII.2 types norovirus genome amplification primer and amplification method |
| CN108796128A (en) * | 2018-06-21 | 2018-11-13 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of GII.8 types norovirus genome amplification primer and amplification method |
| CN108796129A (en) * | 2018-06-21 | 2018-11-13 | 广东省微生物研究所（广东省微生物分析检测中心） | A kind of GI.Pb/GI.6 recombinant types norovirus genome amplification primer and amplification method |
| CN108796128B (en) * | 2018-06-21 | 2021-07-27 | 广东省微生物研究所（广东省微生物分析检测中心） | GII.8 type norovirus genome amplification primer and amplification method |
| CN108823331B (en) * | 2018-06-21 | 2021-08-13 | 广东省微生物研究所（广东省微生物分析检测中心） | GII.6 type norovirus genome amplification primer and amplification method |
| CN108796129B (en) * | 2018-06-21 | 2021-09-03 | 广东省微生物研究所（广东省微生物分析检测中心） | GI.Pb/GI.6 recombinant norovirus genome amplification primer and amplification method |
| CN109825643A (en) * | 2019-03-12 | 2019-05-31 | 中国人民解放军东部战区疾病预防控制中心 | Bat source parvovirus genome, amplimer and amplification method |
| CN112725543A (en) * | 2021-03-04 | 2021-04-30 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Characteristic nucleic acid identification primer group, kit and detection method for human norovirus GII.4 |
| CN112725543B (en) * | 2021-03-04 | 2022-05-10 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Characteristic nucleic acid identification primer group, kit and detection method for human norovirus GII.4 |
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