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GII.P12/GII.3 recombinant norovirus genome amplification primer and amplification method

A virus genome, amplification primer technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial assay/inspection, etc., can solve the problem of lack of antiviral drugs and treatment methods, etc.

Inactive Publication Date: 2016-09-07
GUANGDONG INST OF MICROORGANISM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there are no effective antiviral drugs and treatments

Method used

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  • GII.P12/GII.3 recombinant norovirus genome amplification primer and amplification method
  • GII.P12/GII.3 recombinant norovirus genome amplification primer and amplification method
  • GII.P12/GII.3 recombinant norovirus genome amplification primer and amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: GII.P12 / GII.3 Recombinant Norovirus Genome Amplification Strategy and Design of Corresponding Primers

[0031] (1) The genome of type GII norovirus is about 7.5 kb, including three ORFs, wherein ORF1 is about 5.1 kb in length, ORF2 is about 1.6 kb in length, and ORF3 is about 0.8 kb in length. Based on the first-generation Sanger deoxynucleotide sequencing method, each amplified fragment is designed to be 1.3kb-1.6kb, of which ORF1 is divided into 4 fragments, and ORF2 and ORF3 are both 1 fragment. In addition, in order to obtain the complete sequence of the 5' and 3' ends of the genome, fragments with a length of 100-800 bp were designed and amplified at both ends of the genome, and the corresponding sequencing primers were named II.12-Seq1R and II.3-Seq6F, respectively. The specific genome segmentation strategy and corresponding primer positions can be seen figure 1 , the overlapping regions between fragments 1 to 6 are 220bp, 347bp, 185bp, 340bp, 541bp, r...

Embodiment 2

[0037] Example 2: RT-PCR Sensitivity Analysis of Primers Suitable for GII.P12 / GII.3 Recombinant Norovirus Genome Amplification

[0038] (1) Virus sample processing and nucleic acid extraction: the sample to be processed (containing GII.P12 / GII.3 recombinant norovirus positive sample GZ2013-L20) was diluted to 10% by PBS solution (pH7.4, DEPC treatment) (w / v) concentration, fully shake and mix, centrifuge at 12000×g for 10 minutes to collect 140 μL of supernatant, and extract 60 μL of viral RNA in the sample with RNA extraction kit, and perform appropriate dilution of 10× gradient.

[0039] (2) "4+1+1" genome segment amplification method: amplification in 6 segments, the primer pairs used are as follows: II-1F / II.12-1R, II.12-2F / II.12 -2R, II.12-3F / II.12-3R, II.12-4F / II-4R, II-5F / II-5R and II.3-6F / II.3-6R, each RT-PCR The reaction selects a pair of primers. Use 20 μL of one-step RT-PCR reaction system, containing 10 μL of 2×one-step RT-PCR mixture, 0.6 μL of upstream primer a...

Embodiment 3

[0043] Embodiment 3: Amplification effect of virus genome in actual sample

[0044] (1) Virus sample processing and nucleic acid extraction: Take the GII.P12 / GII.3 recombinant norovirus positive sample GZ2013-L20 obtained in Guangzhou area, and dilute the sample to be treated with PBS solution (pH7.4, DEPC treatment) to 10 % (w / v) concentration, shake and mix well, centrifuge at 12000×g for 10 min to collect 140 μL of supernatant, and extract 60 μL of viral RNA in the sample by RNA extraction kit.

[0045] (2) "4+1+1" genome segment amplification method: amplification in 6 segments, the primer pairs used are as follows: II-1F / II.12-1R, II.12-2F / II.12 -2R, II.12-3F / II.12-3R, II.12-4F / II-4R, II-5F / II-5R and II.3-6F / II.3-6R, each RT-PCR The reaction selects a pair of primers. Use 20 μL of one-step RT-PCR reaction system, containing 10 μL of 2×one-step RT-PCR mixture, 0.6 μL of upstream primer and downstream primer (10 μmol / L), 0.8 μL of MLV / RNasin / HS-Taq enzyme mixture, sample ...

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Abstract

The invention discloses a GII.P12 / GII.3 recombinant norovirus genome amplification primer and an amplification method. The amplification method comprises the following steps: by taking six pairs of amplification primers as forward and reverse primers of amplification primers respectively, and by taking RNA of GII.P12 / GII.3 recombinant norovirus as a template, performing RT-PCR amplification so as to obtain amplification products respectively, performing nucleotide sequencing on the amplification products by using the six amplification primers and two sequencing primers II.12-Seq1R and II.3-Seq6F, and then carrying out jointing and comparison, thereby obtaining full-length sequences of a GII.P12 / GII.3 recombinant norovirus genome. For the GII.P12 / GII.3 recombinant norovirus which occurs frequently in China, a sectional amplification strategy of '4+1+1'is designed according to three opening reading frames included in the genome, corresponding amplification primers are designed according to conserved regions, the amplification primers are applied to practical detection samples, and GII.P12 / GII.3 recombinant norovirus genome sequences can be acquired. The GII.P12 / GII.3 recombinant norovirus genome amplification primer and the amplification method can be widely applied to fields such as medical treatment and public health and inspection and quarantine with norovirus detection requirements, and related scientific fields.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a GII.P12 / GII.3 recombinant norovirus genome amplification primer and an amplification method. Background technique [0002] Norovirus (Norovirus) is an important non-bacterial pathogen of acute gastroenteritis and has been reported all over the world. Norovirus infects people of all ages, and the main clinical manifestations are vomiting, diarrhea, fever, etc. It is also easy to cause dehydration death in children, the elderly and immunocompromised people. According to reports, norovirus causes at least 230 million infections every year, especially in developing countries, causing more than 200,000 deaths of children under the age of 5 every year, posing a great threat to public health security. So far, there is no effective antiviral drug and treatment. The harm of NoV mainly lies in its extremely strong transmission ability, and the US Centers for Disease Control and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/11C12N15/10
CPCC12Q1/701C12Q1/686
Inventor 薛亮吴清平蔡伟程蔡淑珍张菊梅
Owner GUANGDONG INST OF MICROORGANISM
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