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A kind of gii.4 type norovirus genome amplification primer and amplification method

A virus genome and amplification primer technology, which is applied in the field of GII.4 norovirus genome amplification primers and amplification, can solve the problems of difficult sensitivity, large fragment length, and increased difficulty of amplification of direct amplification methods, etc.

Active Publication Date: 2018-04-03
GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the direct amplification method is often difficult to have high sensitivity, and has high requirements on the quality of the sample to be processed, so it does not have a high success rate.
In addition, the commonly used segmented amplification methods include the three-segment method and the multi-segment method, and the primers of the multi-segment method are generally designed according to the genome sequence with a high similarity to the virus in the sample to be processed, so the broad-spectrum of the primers also caused problems. Certain restrictions; the length of the fragments amplified in the three-segment method is relatively large (>3K), which not only increases the difficulty of amplification, but also makes it difficult to sequence directly, thus reducing the success rate of obtaining viral genome sequences

Method used

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  • A kind of gii.4 type norovirus genome amplification primer and amplification method
  • A kind of gii.4 type norovirus genome amplification primer and amplification method
  • A kind of gii.4 type norovirus genome amplification primer and amplification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Optimization of RT-PCR annealing temperature for primers suitable for GII.4 norovirus genome amplification

[0041] (1) Virus sample processing and nucleic acid extraction: Dilute the collected sample to be processed (containing GII.4 Norovirus L10) to a concentration of 10% (w / v) with PBS solution (pH7.4, treated with DEPC), shake fully Mix well, and centrifuge at 12000×g for 10 min to collect 140 μL of the supernatant, and extract 60 μL of viral RNA in the sample with an RNA extraction kit.

[0042](2) "4+1+1" genome segment amplification method (that is, amplification in 6 segments, the primers used are as follows: P1F / P1596R, P1258F / P2805R, P2689F / P4248R, P4099F / G2SKR, NV2of2 / GV132 and P6484F / P7513R, select a pair of primers for each RT-PCR reaction): use 20 μL of one-step RT-PCR reaction system, containing 10 μL of 2×one-stepRT-PCR mixture, upstream primer and downstream primer (10 μmol / L) 0.6 μL each, 0.8 μL of MLV / RNasin / HS-Taq enzyme mixture, 2 μL of sample vi...

Embodiment 2

[0046] Embodiment 2: RT-PCR sensitivity analysis applicable to primers for GII.4 type norovirus genome amplification

[0047] (1) Virus sample processing and nucleic acid extraction: Dilute the collected sample to be processed (containing GII.4 Norovirus L10) to a concentration of 10% (w / v) with PBS solution (pH7.4, treated with DEPC), shake fully Mix evenly, collect 140 μL of supernatant by centrifugation at 12000×g for 10 min, and extract 60 μL of viral RNA in the sample with an RNA extraction kit, and perform appropriate dilution of 10× gradient.

[0048] (2) "4+1+1" genome segment amplification method (that is, amplification in 6 segments, the primers used are as follows: P1F / P1596R, P1258F / P2805R, P2689F / P4248R, P4099F / G2SKR, NV2of2 / GV132 and P6484F / P7513R, select a pair of primers for each RT-PCR reaction): Use 20 μL of one-step RT-PCR reaction system, containing 10 μL of 2×one-stepRT-PCR mixture, upstream primer and downstream primer (10 μmol / L) 0.6 μL each, 0.8 μL of...

Embodiment 3

[0052] Embodiment 3: Amplification effect of virus genome in actual sample

[0053] (1) Virus sample processing and nucleic acid extraction: take GII.4 norovirus positive samples L10, L62, L65, L106, L232, and dilute the sample to be treated to 10% (w) by PBS solution (pH7.4, DEPC treatment) / 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.

[0054] (2) "4+1+1" genome segment amplification method (that is, amplification in 6 segments, the primers used are as follows: P1F / P1596R, P1258F / P2805R, P2689F / P4248R, P4099F / G2SKR, NV2of2 / GV132 and P6484F / P7513R, select a pair of primers for each RT-PCR reaction): Use 20 μL of one-step RT-PCR reaction system, containing 10 μL of 2×one-stepRT-PCR mixture, upstream primer and downstream primer (10 μmol / L) 0.6 μL each, 0.8 μL of MLV / RNasin / HS-Taq enzyme mixture, 2 μL of sample RNA template, and the rest by ddH 2 O ...

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Abstract

The invention discloses a GII.4 type norovirus genome amplification primer and an amplification method. In the present invention, six pairs of primers are used as the upstream and downstream primers of the amplification primers, and the RNA of GII.4 type norovirus is used as the template to carry out RT-PCR amplification to obtain amplification products respectively, and then perform nucleic acid sequence sequencing on the amplification products , and then spliced ​​and compared to obtain the full-length sequence of the GII.4 norovirus genome. According to the main popular genotypes of GII.4 Norovirus, the present invention designs a "4+1+1" segmentation amplification strategy according to the three open reading frames contained in the genome, and designs corresponding amplification according to the conserved region Primer; it can effectively amplify the whole genome sequence of GII.4 type norovirus. The invention can be widely used in medical and health care, inspection and quarantine, etc., which have norovirus detection requirements and corresponding scientific research fields.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a GII.4 type norovirus genome amplification primer and an amplification method. Background technique: [0002] Norovirus is an important pathogen of global epidemic and sporadic acute gastroenteritis, causing about 230 million people to be infected every year, especially in developing countries, causing at least 200,000 deaths of children under the age of 5, which poses a serious threat to public health security. a great threat. Norovirus infection is a self-limiting disease, but there is a risk of dehydration death for infants, the elderly, and immunocompromised people. So far, there is no effective antiviral drug and treatment. [0003] As an RNA virus, norovirus is highly mutated and has rich genetic diversity. According to the sequence homology of their capsid proteins, the viruses can be divided into 6 genomes (Genogroup), and the nucleic acid sequence homology b...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/10
Inventor 薛亮吴清平蔡伟程寇晓霞张菊梅
Owner GUANGDONG INST OF MICROBIOLOGY GUANGDONG DETECTION CENT OF MICROBIOLOGY
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