CN108796128B - GII.8 type norovirus genome amplification primer and amplification method - Google Patents
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Abstract
The invention discloses an amplification primer and an amplification method of a GII.8 type norovirus genome. Aiming at the genome characteristics of the GII.8 type norovirus, the invention adopts a 4+1+1 segmented amplification strategy, designs six groups of amplification primers and two genome terminal sequencing primers in a conserved region, performs RT-PCR amplification and sequencing by taking target virus RNA as a template, and obtains the genome sequence of the GII.8 type norovirus through splicing comparison. The amplification method is applied to actual detection samples, and the GII.8 type norovirus genome sequence derived from China is successfully obtained. The method has the advantages of simple operation, short period, easy popularization and the like, and can be widely applied to the fields of medical treatment, health care, inspection and quarantine, etc. with norovirus detection requirements and corresponding scientific researches.
Description
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to a GII.8 type norovirus genome amplification primer and an amplification method.
Background art:
norovirus is an important nonbacterial pathogen of global acute gastroenteritis and can infect people of all ages. Norovirus infection is a self-limiting disease, and patients will heal themselves within 72 hours, but there is a risk of dehydration death for infants, the elderly, and people with immunodeficiency. In addition, norovirus is very easy to cause epidemic outbreak, and all susceptible people in semi-closed environments such as schools, hospitals, nursing homes, large ships and the like can be infected in 24. The virus is reported to cause about 2.3 million infections per year, costing billions of dollars per year for related treatments in developed countries, and is particularly severe in developing countries, resulting in at least 20 million deaths of children under 5 years of age. Norovirus poses a great threat to public health safety, but no effective antiviral drugs and treatment means exist so far.
Norovirus, an RNA virus, has a rich genetic diversity and can be divided into six genomes (genoreup) GI-GVI according to the amino acid sequence of its capsid protein VP1, where GI, GII and GVI can infect humans. Norovirus within the same genome can be further divided into different genotypes, e.g., GI encompasses at least 9 genotypes and GII encompasses 22 genotypes. Norovirus type gii.4 is the major prevalent genotype worldwide, with about 80% of norovirus infections caused by this type of strain. However, other genotypes are also found during virus monitoring, such as gii.2, gii.3, gii.6, gii.8, etc., and can also lead to outbreaks of epidemics. However, these non-major epidemic genotypes are still of less concern and therefore, it is of great interest to enhance the information gathering efforts of norovirus of different genotypes.
The GII.8 type norovirus, one of the non-major prevalent genotypes common in epidemiological studies, was first reported in Japan in 1967 and was thereafter found in various countries such as the Netherlands, Turkey, Korea, New Zealand, Brazil, and Kamylon. Meanwhile, GII.8 type norovirus is often detected at a low infection rate in the clinical monitoring of China, mainly in Guangdong, Jiangsu and the like. In addition, gii.8 genotype was also found in oyster, surface water monitoring. However, only one piece of genomic information has been reported for this genotype (AB039780| U25), the strain was found in japan in 1997, and the complete capsid protein VP1 sequence has only 3 pieces. The accumulation of virus genetic information is an important basis for deeply researching virus pathogenicity and evolution mechanisms, so that the development of a virus genome amplification and sequencing method has important significance.
As a RNA virus which is extremely easy to mutate, continuous monitoring of the epidemic level and the mutation condition of the virus is an important basis for understanding the norovirus. The genome of GII norovirus is approximately 7.5kb in length, including 3 open reading frames. The 4+1+1 amplification strategy established for the GII.4 type norovirus, the GII.17 type norovirus and the GII.P12/GII.3 recombinant norovirus in earlier work has the characteristics of simple operation, short period, low cost and high sensitivity, so that a genome amplification primer and an amplification method aiming at the GII.8 type norovirus are developed according to the novel amplification strategy, and a powerful research tool is provided for accumulating genome resources of the GII.8 type norovirus in China.
The invention content is as follows:
the invention aims to provide a GII.8 type norovirus genome amplification primer and an amplification method.
Specifically, the amplification primers of the GII.8 type norovirus genome provided by the invention comprise six pairs of amplification primers and two sequencing primers at the tail ends of the genome:
primer pair 1: II-1F: 5'-GTGAATGAAGATGGCGTCTAAC-3', respectively;
II.8-1R：5'-CTGTAGGCATCCCAGTGATC-3'；
and (3) primer pair 2: II.8-2F: 5'-TCATCTTCGCCTGACATCGT-3', respectively;
II.8-2R：5'-TCCTCTTCACAGAAGTTCTC-3'；
and (3) primer pair: II.8-3F: 5'-CACACTGCATTCTCCAGCAA-3', respectively;
II.8-3R：5'-CAATCCTCCAAATGCCCGA-3'；
and (3) primer pair 4: II.8-4F: 5'-GAGGAAGCCAAGAAGACAGT-3', respectively;
II-4R：5'-CCRCCNGCATRHCCRTTRTACAT-3'；
and (3) primer pair 5: II-5F: 5'-GGAGGGCGATCGCAATC-3', respectively;
II-5R：5'-CCRGCRAAGAAAGCTCCAGCCAT-3'；
and (3) primer pair 6: II.8-6F: 5 '-TCYAACAGTGGGGACCACC-3';
II.8-6R：5'-TCACTAAGCCCGTGACTCC-3'；
sequencing primer: II.8-seq 1R: 5'-AACTCACCATCCCACATCTC-3', respectively;
II.8-seq6F：5'-GACAAGGAGATGTTGAATGC-3'；
r represents A/G, H represents A/C/T, N represents base A/C/T/G, and Y represents C/T.
The invention also provides an amplification method of the GII.8 type norovirus genome, which specifically comprises the following steps: respectively taking the primer pairs II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R and II.8-6F/II.8-6R as upstream and downstream primers of amplification primers, carrying out RT-PCR amplification by taking RNA of the GII.8 type norovirus as a template to respectively obtain amplification products, respectively carrying out nucleic acid sequence sequencing on the corresponding amplification products by adopting each pair of the amplification primers, the sequencing primers II.8-seq1R and II.8-seq6F, and carrying out splicing comparison to obtain the GII.8 type norovirus genome sequence.
Further, the reaction system of the RT-PCR is as follows: contains 10 mu L of 2 Xone-step RT-PCR mixture, 0.6 mu L of each of 10 mu mol/L upstream primer and 10 mu mol/L downstream primer, 0.8 mu L of MLV/RNase/HS-Taq enzyme mixture, 2 mu L of RNA template, and the balance of the mixture is made up to 20 mu L by double distilled water; the reaction conditions are as follows: reverse transcription is carried out at 50 ℃ for 30min, pre-denaturation is carried out at 94 ℃ for 3min, then extension is carried out at 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 75s for 30 cycles, and finally extension is carried out at 72 ℃ for 7 min.
Compared with the prior art, the invention has the following beneficial effects:
aiming at the common GII.8 type norovirus genotypes, the invention adopts a segmented amplification strategy of '4 +1+ 1' covering the whole genome, and obtains the GII.8 type norovirus genome sequence by applying to amplification, sequencing and sequence splicing of an actual detection sample. The method for amplifying and sequencing GII.8 type norovirus by using the primer provided by the invention has the characteristics of simple operation, short period, low cost, high sensitivity and the like, and can be widely applied to mechanisms with norovirus detection requirements, such as medical health, inspection and quarantine, and the like, and the corresponding scientific research field.
Description of the drawings:
FIG. 1 shows the GII.8 type norovirus genome amplification strategy and the design positions of the corresponding primers.
FIG. 2 shows the optimized RT-PCR annealing temperature for the primers for GII.8 norovirus genome amplification, with the electrophoresis sequence of M, 1-24, where M is DNA Ladder, 1-4, 5-8, 9-12, 13-16, 17-20, and 21-24 are the electrophoresis results of the amplification products of the genome amplification primers II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R, and II.8-6F/II.8-6R at different annealing temperatures (45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, respectively).
FIG. 3 shows the concentration optimization of RT-PCR primers suitable for GII.8 type norovirus genome amplification, where in FIGS. A-F show the optimization results of primers II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R, and II.8-6F/II.8-6R, respectively, and the electrophoresis sequence is M, 1-9, where M is DNA Ladder, 1-3 is the electrophoresis result of 0.2. mu.L of primers respectively diluted 10 times with RNA stock solution and 100 times with RNA stock solution, 4-6 is the electrophoresis result of 0.6. mu.L of primers respectively diluted 10 times with RNA stock solution and 100 times with RNA stock solution, and 7-9 is the electrophoresis result of 1.0. mu.L of primers respectively amplified with RNA stock solution and RNA stock solution, And (3) taking the result of electrophoresis of the template after the RNA stock solution is diluted by 10 times and the RNA stock solution is diluted by 100 times.
FIG. 4 shows the RT-PCR sensitivity evaluation of the primers for GII.8 type norovirus genomic amplification, the electrophoresis sequence is M, 1-35, wherein M is DNA Ladder, 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35 are genome amplification primers II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R, II.8-6F/II.8-6R and detection primers G2SKF/G2SKR at different degrees of viral RNA dilution (sequentially 10 degrees of dilution)1-105Double dilution) ofElectrophoresis results of the amplification products.
FIG. 5 shows the amplification effect of GII.8 type norovirus in the actual sample, the electrophoresis sequence is M, 1-6, wherein M is DNA Ladder, and 1-6 are the electrophoresis results of amplifying 6 genomic fragments of the actual sample L601.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof. The following examples are given without specifying the particular experimental conditions and methods, and the technical means employed are generally conventional means well known to those skilled in the art.
Example 1 amplification strategy for GII.8 type norovirus genomes and design of corresponding primers
The genome of the GII.8 norovirus is about 7.5kb in size and includes three ORFs, wherein ORF1 is about 5.1kb in length, ORF2 is about 1.6kb in length, and ORF3 is about 0.8kb in length. Based on the one-generation Sanger deoxynucleotide sequencing method, the size of each amplified fragment is set to be in the range of 1.3kb to 1.6kb, wherein ORF1 is divided into 4 fragments, and ORF2 and ORF3 are both 1 fragment. In addition, in order to obtain complete sequences at the 5 'end and the 3' end of the genome, fragments with the amplification length of 100-800bp are designed at both ends of the genome respectively, and corresponding primers are named as II.8-Seq1R and II.8-Seq6F respectively. Specific genome segmentation strategies and corresponding primer positions can be seen in FIG. 1.
Under the above restriction conditions, Oligo software was used to design corresponding primers, and the specific primer information is shown in table 1. Wherein R in the nucleotide sequence of the primer represents A/G, H represents A/C/T, N represents base A/C/T/G, and Y represents C/T.
Table 1: GII.8 type norovirus genome amplification primers and sequencing primer information
aPrimer position referenced GII.8 type norovirusRepresentative sequence GenBank accession No. AB 039778.
Example 2 RT-PCR annealing temperature optimization of primers for GII.8 type norovirus genome amplification
(1) Virus sample treatment and nucleic acid extraction: the collected sample to be treated (GII.8 type norovirus positive sample L601) was diluted to a concentration of 10% (w/v) by PBS solution (DEPC treatment), shaken and mixed well, centrifuged at 12000 Xg for 10min, and 140. mu.L of supernatant was collected and viral RNA in the sample was extracted by RNA extraction kit for 60. mu.L.
(2) Genome segmentation amplification method: namely, the amplification is divided into 6 sections, and the primer pairs used are as follows: II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R, II.8-6F/II.8-6R, and a pair of primers is selected for each RT-PCR reaction. Adopting a 20 mu L one-step RT-PCR reaction system, containing 2 Xone-step RT-PCR mix 10 mu L, 10 mu mol/L upstream primer and 10 mu mol/L downstream primer 0.6 mu L each, MLV/RNase/HS-Taq enzyme mixed solution 0.8 mu L, sample RNA template 2 mu L, and ddH for the rest2And (4) complementing O.
The reaction conditions are as follows: reverse transcription is carried out at 50 ℃ for 30min, pre-denaturation is carried out at 94 ℃ for 3min, then 30s at 94 ℃, 30s at 45-60 ℃ and 75s at 72 ℃ are carried out for 30 cycles, and finally extension is carried out at 72 ℃ for 7 min.
The annealing temperatures were selected to be 45 deg.C, 50 deg.C, 55 deg.C, and 60 deg.C, respectively.
(3) Electrophoresis: mu.L of the amplification product was taken, subjected to agarose gel nucleic acid electrophoresis, and the result was observed by a gel imaging system. According to the sequence of primer pairs II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R and II.8-6F/II.8-6R, the GII.8 type norovirus genome amplification bands are 1629bp, 1469bp, 1509bp, 1569bp, 1671bp and 902bp in sequence. The electrophoresis result is shown in FIG. 2, and the results show that six pairs of genomic amplification primers have a wider annealing temperature range, and in order to reduce non-specific amplification in an actual sample, 55 ℃ is finally selected as the application annealing temperature.
Example 3 optimization of RT-PCR primer concentrations for amplification of GII.8 norovirus genomes
(1) Virus sample treatment and nucleic acid extraction: collection by dilution with PBS solution (DEPC treatment)The sample to be treated (GII.8 type norovirus positive sample L601) is shaken and mixed evenly to the concentration of 10 percent (w/v), centrifuged for 10min at 12000 Xg, 140 mu L of supernatant is collected, 60 mu L of virus RNA in the sample is extracted by an RNA extraction kit, and nuclease-free ddH is adopted2O was diluted with a 10 Xgradient, and RNA stocks (stock concentration: 10) were each selected4RTPCRU), 10-fold dilution of RNA stock, and 100-fold dilution of RNA stock were used as amplification templates. It should be noted that, the norovirus content of the invention is defined by using RTPCRU units, that is, the virus solution diluted by 10 times gradient is detected by using detection primers G2SKF/G2SKR through a standard RT-PCR method, and the virus concentration is one RTPCRU when diluted to be detected.
(2) Genome segmentation amplification method: namely, the amplification is divided into 6 sections, and the primer pairs used are as follows: II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R, II.8-6F/II.8-6R, and a pair of primers is selected for each RT-PCR reaction. Adopting a 20 mu L one-step RT-PCR reaction system containing 2 Xone-step RT-PCR mix 10 mu L, 10 mu mol/L upstream primer and 10 mu mol/L downstream primer, respectively adding 0.2 mu L, 0.6 mu L, 1.0 mu L, 0.8 mu L of MLV/RNase/HS-Taq enzyme mixed solution, 2 mu L of sample RNA template with different dilution, and the rest is ddH2And (4) complementing O.
The reaction conditions are as follows: reverse transcription is carried out at 50 ℃ for 30min, pre-denaturation is carried out at 94 ℃ for 3min, then extension is carried out at 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 75s for 30 cycles, and finally extension is carried out at 72 ℃ for 7 min.
(3) Electrophoresis: mu.L of the amplification product was taken, subjected to agarose gel nucleic acid electrophoresis, and the result was observed by a gel imaging system. According to the sequence of primer pairs II-1F/II.8-1R, II.8-2F/II.8-2R, II.8-3F/II.8-3R, II.8-4F/II-4R, II-5F/II-5R and II.8-6F/II.8-6R, the GII.8 type norovirus genome amplification bands are 1629bp, 1469bp, 1509bp, 1569bp, 1671bp and 902bp in sequence. The electrophoresis results are shown in FIG. 3, and the results show that the requirements of different primer pairs on primer concentration are inconsistent, but the optimal conditions are or include 0.6. mu.L, therefore, 0.6. mu.L is finally selected as the addition amount of the primers in the amplification system.
Example 4 evaluation of RT-PCR sensitivity of primers for amplification of GII.8 type norovirus genome
(1) Virus sample treatment and nucleic acid extraction: diluting the collected sample to be treated (GII.8 type norovirus positive sample L601) to 10% (w/v) by PBS solution (DEPC treatment), shaking and mixing uniformly, centrifuging at 12000 Xg for 10min, collecting supernatant 140. mu.L, extracting viral RNA in the sample by RNA extraction kit for 60. mu.L, and adopting nuclease-free ddH2Appropriate dilution of O with a gradient of 10 (10 in sequence)1-105Double dilution) treatment.
(2) Genome segmentation amplification method: namely, the amplification is divided into 6 sections, and the primer pairs used are as follows: II-1F/II.2-1R, II.2-2F/II.2-2R, II.2-3F/II.2-3R, II.2-4F/II-4R, II-5F/II-5R, II.2-6F/II.2-6R, and a pair of primers is selected for each RT-PCR reaction. Adopting a 20 mu L one-step RT-PCR reaction system, containing 2 Xone-step RT-PCR mix 10 mu L, 10 mu mol/L upstream primer and 10 mu mol/L downstream primer 0.6 mu L each, MLV/RNase/HS-Taq enzyme mixed solution 0.8 mu L, sample RNA template 2 mu L, and ddH for the rest2And (4) complementing O.
The reaction conditions are as follows: reverse transcription is carried out at 50 ℃ for 30min, pre-denaturation is carried out at 94 ℃ for 3min, then extension is carried out at 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 75s for 30 cycles, and finally extension is carried out at 72 ℃ for 7 min.
The detection primers G2SKF/G2SKR (G2 SKF: 5 '-CNTGGGAGGGCGATCGCAA-3'; G2 SKR: 5'-CCRCCNGCATRHCCRTTRTACAT-3') were used as a control, and the reaction was carried out in accordance with the above system and reaction conditions.
(3) Electrophoresis: taking 5 mu L of amplification product, carrying out agarose gel nucleic acid electrophoresis, observing the result through a gel imaging system, wherein the content of the norovirus is defined by RTPCRU unit of a detection primer, namely, the virus RNA which is diluted by 10 multiplied by gradient is detected by using a common detection primer G2SKF/G2SKR, and the virus concentration is one RTPCRU when the virus RNA is diluted to be detected. According to the sequence of the primer pairs II-1F/II.2-1R, II.2-2F/II.2-2R, II.2-3F/II.2-3R, II.2-4F/II-4R, II-5F/II-5R and II.2-6F/II.2-6R, the GII.8 type norovirus genome amplification bands are 1629bp, 1469bp, 1509bp, 1569bp, 1671bp and 902bp in sequence, the electrophoresis result is shown in figure 4, and the result is shown as follows: the sensitivity of the six fragment amplification primers reaches 1RTPCRU, and compared with the detection primer G2SKF/G2SKR, all the primers reach the sensitivity of the detection primer.
Example 5 actually examined the effect of amplifying the genome of GII.8-type norovirus in a sample
(1) Virus sample treatment and nucleic acid extraction: a GII.8 type norovirus positive sample L601 is taken, a sample to be treated is diluted to a concentration of 10% (w/v) by a PBS solution (DEPC treatment), the mixture is uniformly shaken and centrifuged at 12000 Xg for 10min, 140 mu L of supernatant is collected, and 60 mu L of viral RNA in the sample is extracted by an RNA extraction kit.
(2) Genome segmentation amplification method: namely, the amplification is divided into 6 sections, and the primer pairs used are as follows: II-1F/II.2-1R, II.2-2F/II.2-2R, II.2-3F/II.2-3R, II.2-4F/II-4R, II-5F/II-5R, II.2-6F/II.2-6R, and a pair of primers is selected for each RT-PCR reaction. Adopting a 20 mu L one-step RT-PCR reaction system, containing 2 Xone-step RT-PCR mix 10 mu L, 10 mu mol/L upstream primer and 10 mu mol/L downstream primer 0.6 mu L each, MLV/RNase/HS-Taq enzyme mixed solution 0.8 mu L, sample RNA template 2 mu L, and ddH for the rest2And (4) complementing O.
The reaction conditions are as follows: reverse transcription is carried out at 50 ℃ for 30min, pre-denaturation is carried out at 94 ℃ for 3min, then extension is carried out at 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 75s for 30 cycles, and finally extension is carried out at 72 ℃ for 7 min.
(3) The amplification product was taken in an amount of 5. mu.L by electrophoresis, subjected to agarose gel nucleic acid electrophoresis, and the result was observed by a gel imaging system. According to the sequence of the primer pair II-1F/II.2-1R, II.2-2F/II.2-2R, II.2-3F/II.2-3R, II.2-4F/II-4R, II-5F/II-5R and II.2-6F/II.2-6R, the GII.8 type norovirus genome amplification bands are in the range of 1629bp, 1466bp, 1529bp, 1569bp, 1701bp and 927bp in sequence, the electrophoresis result is shown in figure 5, and the result shows that the amplification of the sample L601 by using the amplification primers is successful.
(4) Nucleic acid sequencing and genome splicing alignment: the amplification products of the L601 sample correspondingly amplified are sequenced and spliced by respectively adopting the six pairs of amplification primers, the two sequencing primers II.8-SEQ1R and II.8-SEQ6F, the length of the genome sequence is 7476bp, the genome nucleotide sequence is shown as SEQ ID NO.1, and the genome sequence is submitted to BLAST analysis, and the result shows that only one homologous sequence with the genome coverage and similarity of more than 90 percent of the L601 sample is AB039780/U25/JP (the coverage is 99 percent and the similarity is 94 percent).
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Guangdong province institute for microbiology (Guangdong province center for microbiological analysis and detection)
GUANGDONG HUANKAI MICROBIAL SCI & TECH. Co.,Ltd.
<120> GII.8 type norovirus genome amplification primers and amplification method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7476
<212> DNA
<213> norovirus GII.8L 601(norovirus GII.8L 601)
<400> 1
tgtgaatgaa gatggcgtct aacgacgcta ccgctgccgc tggcaacttt aacattgcca 60
acaacagtga aaacactccc cctcccgtta aggaagaggc gagtgtccta tccaacgtaa 120
aagttggatt caagaaaata ttgggggctg tgcctaagca gcccaagcca ccagaagtga 180
aaaccaaaga tcaacccagg gtgctggtca atggtaaggc cctggaggtg ccgcccccac 240
caccaaatgg tgaagacgtg gtttactata acagcaagga tgacactgtg cacggcctac 300
caaatctaac aacagtcagc tgtgaaggcg aaaaccttcc gtacacagtt cctcctctca 360
gcgagagaga gcacagggca gcaccagaac cattacctgg caccatacta gagatgtggg 420
atggtgaatt ctaccattat gcgatatatg tctctggtgg taaggccata ggggtgcaca 480
aacccccagc tgcaatcagc cttgccacca tcgagcttac acctatatcg ctttactgga 540
ggcctgttta tgtccccaat tacttggtcc accctgacac actcaagagc ttagctggtg 600
aaaaatttcc atacaccgca tttagcaata attgttacaa tttctgttgc tgggttcttg 660
acttgaaaga ctcgtggctt aaccgcagat caataactag aacaaccggt ttcttcaagc 720
cttatcaacc ctggaacaag aaaccacttc cgacggtgga tgatggaaaa atcaagaaag 780
tcgccaatgc agtcctgtgt gcccttggtt cactattctc aaaacctatt aaggacttga 840
tcgggaagct gaaaccactg aatttcttga atctcctcgc ttcgtgcgac tggaccttct 900
ccggagtggt tgagaccgtc atactagctg ctgaactatt cggtgtgttt tggacccccc 960
ccgacgtgtc gaacttcatc gcgtcactca tcggggactt cgagttgcag ggtccagagg 1020
atttagctgt ggagttggtg ccgattatca tgggcggcat cgggatggtg ctgggattca 1080
ccgccgagaa gatcggccga atgctatcat cagctgcatc gacccttagg gcctgtaaag 1140
acctaggtaa ctacgccctt gacatactta aattggtcat gaagtggttc ttcccaaaga 1200
aggaggagaa ggcagagatg gagaccttga gggccataga ggatgctgtc ttagacatgg 1260
aagcgattgg aaataatcac ctcactgctc tgctgaaaga cagggacagc ctcctaacct 1320
acatgaagac tctagacctc gaagaggaaa aggcaagaaa gttatccaca aaatcatctt 1380
cccctgacat cgtgggtacc atcaatgcca tactagccag aattgccgcg gctaggtctc 1440
tcctacacag agccaaagaa gaaatgtaca gcagacctag gcctgtcgtg gtgatgatat 1500
caggaagacc aggggttggt aaaacccata tggctagaca cctcgccaag agtgtggcgg 1560
gcaccatgag cggtgaccaa agggtgggcc tcatcccccg taatggagtt gatcactggg 1620
atgcctacag gggggaacga gttgtcttgt gggacgacta tggcatgggt aacccagtga 1680
aggacgccct tgctctgcaa gaactagcag acacttgtcc agtcaccctc aattgtgaca 1740
ggatagagaa taagggaaag atgttcgata gcgaggtaat aattataaca accaatctgg 1800
ccaaccccgc cccgcttgat tatgtcaact ttgaggcatg ttcccgcaga attgacttcc 1860
tcgtctacgc agaggccccg accattgaga aggcgaagaa ggatttcccc gggcaaccag 1920
acatgtggaa ggagcacttc aaagcagact tctctcatct caaactcaca ctggctccgc 1980
aaggcggttt tgacaagaac ggcaacacgc cacatgggaa ggggactatg aagaccttaa 2040
cacagggttc cctcacagca agagtggctg ggcttgtgca cgaaaggaaa gacgagttcc 2100
agttacaggg tggggagttg caggtctaca actttgacac aaacaaggta tcggctttca 2160
ggaagcttgc tgcagacaac aactatggat ttctggagac aatgagagtc ggctcctcac 2220
tcaaggacgt caagacagtg gaagatctta aatgtgccct caggagtgtc agtttcaaag 2280
aatgtgagat aatctacagg aatgtcaagt accgcatatc atcagacggt tgcggtaagg 2340
tcagcataga gaaactggca gataacaact cccaaacaac cagagagatt atcacggccg 2400
tcactagact acaacaggct cgtgctaggt actacatcag ctgtttccaa gacctagtct 2460
acacactctt gcaggttgcc ggagcctcat ttgtcattaa tagaatagtt aagaagttca 2520
actgggaacg atgggtcaag gctccggaaa cagatgaacc cacaccaagt aatgacaagc 2580
ccgacactga agaggaatgg gagatcgccc ccaaagatgt ggactctgag ggtaaaaagg 2640
ggaagaacaa gaagggcagg ggacgtaagc acactgcatt ctccagcaaa gggcttagtg 2700
atgaggagta tgatgaattc aaaagactga gggaagagaa acagggccgc tacacaatag 2760
aggagtacct ccaggacaga gaccggtact atgaagagct ggcagtggcc cgggccactg 2820
aagagaactt ctgtgaagag gaagagataa agatcaggca aagaatattc cgccccacca 2880
aaaagcagag gaaggaggag cggggggttc tgggactcgt cacaggagca gacatcagga 2940
aacggagacc cgatgatttc caaccaaaag gaaaactgtg ggcagatgat tctaggagcg 3000
tcgactacaa tgagaagatc gagtttgaag ctcctcctag cgtctggtca agaatcgtcc 3060
cgcttgggac tggttgggga ttctgggtct catccaactt actaatcacc accacacatg 3120
tgctgcccaa aggcatcaaa gagctttttg gggtggacat caaacaagtc caagttcaca 3180
aatcaggtga attttgcaga ttcaggttcc caaaaccagt gaggccagat gtctctgggt 3240
tggtactaga agaaggggcc cccgaaggca cagtgtgctc tgttctcatc aagaggtcaa 3300
caggagagat gatccccctt gcagttagga tgggtaccca cgcctctatg aagatccagg 3360
ggcgcactgt tggcggccag atgggcatgc tgctcactgg agccaacgct aagaacatgg 3420
atttgggtac cggccccggg gattgtggtt gcccgtacat atacaagagg ggcaatgaca 3480
tcgtggtggc cggcgtccac acagcagcag cgcgtggggg caacacggtc atctgtgcaa 3540
cccaaggacc tgatggtgag gcagtgttgg aagggggaga ggaccacggt acttactgtg 3600
gagccccaat attagcccct ggtaaagcgc cgaagcttag cacaaagact aagttttggc 3660
gctcatcccc tgattccctt ccccctggga cgtatgaacc agcttacctt gggggtaagg 3720
atccccgtgt ggagaaaggg ccatcactcc aacaagtgat gcgagaccaa ctcaaaccct 3780
tcacagaacc caggggcaag ccaccgaggc cgtcagtgct ggaggaagcc aagaagacag 3840
tcatgaatgt actggagcag accatagaac cagctaagcc atggacctac tctcaagcct 3900
gtgcttcatt ggataaaaca acatccagtg gcagtcctca ccacgtcagg aagaatgaac 3960
actggaacgg ggagtctttc actgggcctc ttgcagatca agcctcaaaa gccaacctga 4020
tgtatgagga gggtaaacac atggacccta tgtacacagc agccctcaag gacgagctgg 4080
tgaaaactga caaaatctat aagaaaatca agaagaggtt gttgtggggg tctgatctgg 4140
ggacaatggt gaggtgtgct cgagcgtttg gaggattgct cgacagcatg aaagagagct 4200
gcatctcctt accctgtagg gttggcatga acataaatga agatggacca ctcatatttg 4260
aaaaacattc caaatacacc tatcactatg atgctgatta ctctaggtgg gactcaacac 4320
agcagagggc tgtcctgagt gcagccatgg aggtaatggt gaaattctct gctgagccgg 4380
aactggctca ggtggttgcc gaagacctcc ttgcacccag ccgtcttgac gtgggtgact 4440
tcgtcatctc cgttcaagag ggtcttccat cgggtgtccc ctgcacgtca cagtggaact 4500
caatcgctca ctggatcctg actcttagtg ctatgtctga ggtctctggt ctttcccctg 4560
aggttgtgca agccaactcc tgtttctcat tctatgggga tgatgagata gtcagcacag 4620
atataaacct agacccagaa aaactcacca ggaaactgag ggagtatggc ctcgtcccaa 4680
caaggccaga caaaactgag ggcccacttg tgatcactca ggatttgaat ggtctcacat 4740
tcttgaggcg aaccatagtg cgggaccccg caggttggtt tggaaaattg gaccgtgatt 4800
ccattctaag gcagttatac tggaccagag gacccaatca tgagaacccc tttgaaagca 4860
tgatccccca ctcccagaga gcaacccagt taatggccct tcttggggaa gcctcgttgc 4920
atggtcccca gttttacaag aaggtgagta aaatggtcat caatgagatc aagagtggtg 4980
gtctggagtt ttacgtgccc agacaggagg ccatgtttag atggatgaga ttttcagacc 5040
tcagcacgtg ggagggcgat cgcaatctgg ctcccgagaa tgtgaatgaa gatggcgtcg 5100
aatgacgcag ctccatcgaa tgatggcgcg gctggcctcg taccagagat caaccatgag 5160
gtcatggcca tagagcctgt tgcaggggcc tctttagcag cccctgtcgt aggacaactt 5220
aatataattg atccctggat tagaaataat tttgtacaag cccctgctgg agaattcact 5280
gtttcgccta gaaatgctcc aggtgaattt ttgttagatc tagagttagg tccagaactg 5340
aatccttatc ttgctcacct tgcacgcatg tataatgggc atgcaggtgg tatggaggtg 5400
cagatagtgc ttgctgggaa tgcgttcaca gcgggcaaaa tactgtttgc agtcatacca 5460
cctggattcc cctatgaaaa tttgtcacct gcacaattga ccatgtgccc gcatgtagtg 5520
gtggatgtga gacagcttga accaatcctg ctgcccatgc cagacatccg taatacattc 5580
ttccattaca atcagagcaa tggaccaaaa ctcagattag tagccatgct ttacacaccc 5640
ctaagggcta acaatgctgg tgatgatgtc ttcacagtct cctgcagggt tttgactcgt 5700
ccgtcaccag attttgaatt caattttctt gtccccccat ctgttgaatc taagactaaa 5760
gctttcactc ttccaattct taagatatct gagatgacaa attcaagatt tcccatacca 5820
gttgaccaga tgtatactag taggaatgaa aatatagttg ttcaaccaca gaatggcagg 5880
gtgacacttg atggagagtt acagggcaca accacacttc aaccggtgtc catttgtggc 5940
ttccgaggga cgctgcagac caggctggca gatcaaccaa attacacata ccaagtgcac 6000
cttgagaatt tggacggctc acccgtcgat ccgacagatg aagtcccagc ccccctgggg 6060
acgccagatt ttcaggccca gctatttgga gttgtcagtc aaaggagtag tgataatgcc 6120
accagggcac atgaggctag ggtcaacacc aacgatccta ccttcgcgcc ccagatcgcc 6180
caggtgcgtt tcaaatcacc atcccacgac ttctttgaca atgaacccat caagttcaca 6240
ccagttggaa ttagtgttga tagcgaaaac agctacaacc aatggctcct tcccaggtat 6300
ggcgggcact tgacaaacaa cacccacctg gccccctctg tgtccccaat gttccccggc 6360
gagcagattc tcttcttccg ctcgttcatg ccaggtgcca gtggtcatac tgatggtgct 6420
attgactgcc tcttaccaca agagtgggtt gcccacttct atcaggaggc cgcaactgcg 6480
caaacagatg tggctctgat tagatttgtg aaccccgaca caggcagagt gctatttgaa 6540
gggaagctac acaagcgggg ctttataacc atctccaaca gtggggacca ccccattgtt 6600
atgcctgcta atggatactt caggtttgag gcatgggtca accaatttta ctctctcgcc 6660
cccgtgggaa ctggaagtgg gcgcagaagg gttcaataat ggctggagct ttctttgcgg 6720
gtctggctgg tgatgtggtg acctctggca tcgggtcgct catcaatgca ggtgctaatg 6780
cagtgaacca aaaggttgaa tatgacttca ataggcaact tcaagaagct tcttttaaac 6840
atgacaagga gatgttgaat gctcaaatta atgccacaac caaattgcag agggacatga 6900
ttgccatcaa gcagggggtt ttgaccgctg gcggcttttc ccccactgat gctgcaagag 6960
gttccatcaa tgctccaacc accaaaattc ttgactggaa tggatctagg tactgggcgc 7020
caggatcaat gagaacaaca gcatactctg gcaatttcat atcacaggga gttaagcaac 7080
ctacacccat tgttagatct gtgaaacctc aagtacagca atctcctgcc ccttcttctg 7140
tttattctgt aggttctgtt aagtcacaat caacacaatc aactcaatta acagggtcag 7200
ggactgtatc aagggcaccc accccctcaa caaccacaac actgtccaga acaagtgagt 7260
gggtgaggag ccagaatgat aggctgagcc cctttatggg aggcgcactc cagacagctt 7320
tcatcacgcc cccatcgagt aaggcttcat cctcatcagg aacagtctca accgttccta 7380
gagaaatttt ggactcctgg actcctgcat ttaacacacg caggcagccg ctcttcgccc 7440
acatgcgtag gcgaggggag tcacgggctt agtgaa 7476
Claims (3)
1. A GII.8 type norovirus genome amplification primer is characterized by comprising six pairs of amplification primers and two sequencing primers at the ends of a genome:
amplification primer pair 1:
II-1F：5'-GTGAATGAAGATGGCGTCTAAC-3'；
II.8-1R：5'-CTGTAGGCATCCCAGTGATC-3'；
amplification primer pair 2:
II.8-2F：5'-TCATCTTCGCCTGACATCGT-3'；
II.8-2R：5'-TCCTCTTCACAGAAGTTCTC-3'；
amplification primer pair 3:
II.8-3F：5'-CACACTGCATTCTCCAGCAA-3'；
II.8-3R：5'-CAATCCTCCAAATGCCCGA-3'；
amplification primer pair 4:
II.8-4F：5'-GAGGAAGCCAAGAAGACAGT-3'；
II-4R：5'-CCRCCNGCATRHCCRTTRTACAT-3'；
amplification primer pair 5:
II-5F：5'-GGAGGGCGATCGCAATC-3'；
II-5R：5'-CCRGCRAAGAAAGCTCCAGCCAT-3'；
amplification primer pair 6:
II.8-6F：5'-TCYAACAGTGGGGACCACC-3'；
II.8-6R：5'-TCACTAAGCCCGTGACTCC-3'；
sequencing primer:
II.8-seq1R：5'-AACTCACCATCCCACATCTC-3'；
II.8-seq6F：5'- GACAAGGAGATGTTGAATGC-3'；
r represents A/G, H represents A/C/T, N represents base A/C/T/G, and Y represents C/T.
2. A GII.8 type norovirus genome amplification method is characterized in that six pairs of amplification primers of claim 1 are respectively used as upstream and downstream primers, RNA of GII.8 type norovirus is used as a template to carry out RT-PCR amplification to respectively obtain amplification products, then each pair of amplification primers and sequencing primers II.8-seq1R and II.8-seq6F of claim 1 are respectively used for carrying out nucleic acid sequence sequencing on the corresponding amplification products, and then the full-length sequence of the GII.8 type norovirus genome is obtained through splicing comparison.
3. The amplification method of claim 2, wherein the reaction system of the RT-PCR is as follows: contains 10 mu L of 2 Xone-step RT-PCR mixture, 0.6 mu L of each of 10 mu mol/L upstream primer and 10 mu mol/L downstream primer, 0.8 mu L of MLV/RNase/HS-Taq enzyme mixture, 2 mu L of RNA template, and the balance of the mixture is made up to 20 mu L by double distilled water; the reaction conditions are as follows: reverse transcription is carried out at 50 ℃ for 30min, pre-denaturation is carried out at 94 ℃ for 3min, then extension is carried out at 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 75s for 30 cycles, and finally extension is carried out at 72 ℃ for 7 min.
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