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Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing

A pyrosequencing and influenza virus technology is applied in the detection of the pathogenicity of influenza A H1N1 virus, and the detection of the pathogenicity of influenza A H1N1 virus based on pyrosequencing can solve the problem of time-consuming and laborious detection of virus gene mutations. , to achieve the effect of preventing the further expansion of the epidemic, easy operation and convenient monitoring

Inactive Publication Date: 2014-06-04
山东国际旅行卫生保健中心
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Problems solved by technology

[0004] In order to strengthen the control of the rapid spread of H1N1 and avoid its outbreak, the development of rapid, reliable and high-throughput virus mutation detection technology is a research hotspot. The traditional automatic DNA sequencing technology based on the Sanger method is time-consuming in the detection of virus gene mutations. , laborious disadvantages
Pyrosequencing technology is a new generation of short-segment DNA sequence analysis technology, which can perform short DNA sequence analysis quickly, accurately and in real time, with high detection throughput, simple and convenient operation, and programmable. There is no report on the detection of the cleavage site of HA gene

Method used

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  • Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing
  • Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing
  • Method for detecting pathogenicity of influenza A (H1N1) virus based on pyrosequencing

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Embodiment Construction

[0040] The content of the present invention will be further described in detail below by way of examples.

[0041] 1. RNA extraction

[0042] MDCK subcultured cells infected with freshly propagated virus were used as templates for RNA extraction. Viral RNA was extracted using the HighPure Viral RNA Kit produced by Roche. The specific operation was performed according to the kit instructions. The extracted RNA was immediately used for RT-PCR amplification or placed in- Store at 80°C.

[0043] 2. RT-PCR reaction to amplify the HA1 gene fragment

[0044] The first round of amplification was carried out with the PCR primers having the sequence indicated by H1F and the PCR primers having the sequence indicated by H1R, and the extracted viral RNA was used as a template. In the 50μL reaction system, the optimal concentration of the first primer is 50nmol / L. The RT-PCR reaction conditions were: reverse transcription at 50°C for 30min; pre-denaturation at 94°C for 2min; 45 cycles of...

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Abstract

The invention relates to a method for detecting the pathogenicity of an influenza A (H1N1) virus based on pyrosequencing. The method comprises the steps of carrying out RT-PCR (Reverse Transcription-Polymerase Chain Reaction) amplification on a hemagglutinin (HA) gene of an H1N1 virus; carrying out pyrosequencing on a PCR amplification product to judge whether the cleavage site of the HA gene of the influenza A (H1N1) virus has a mutation, wherein the pyrosequencing is carried out in an SQA (Sequence Analysis) mode, and a nucleotide sampling sequence is shown as AGCT. The mutation of the cleavage site of the HA gene of the influenza A (H1N1) virus can be detected at rather high accuracy through comparing a detected result with the sequence of the cleavage site of the HA gene of a standard strain of the influenza A / H1N1 virus. The invention provides a simple and rapid experimental scheme for determining the virulence, pathogenicity and host range of the virus, the complex experiment steps related to complete genome sequencing are omitted, the variation direction of the virus can also be accurately mastered, the virus is conveniently monitored, an infection source is isolated, a transmission route is cut off, and the further development of an epidemic situation is stopped.

Description

technical field [0001] The invention relates to a method for detecting the pathogenicity of influenza A (H1N1) virus, in particular to a method for detecting the pathogenicity of influenza A (H1N1) virus based on pyrosequencing, and belongs to the technical field of virus detection. Background technique [0002] Influenza A (H1N1), which broke out in March 2009, was another major public health event after SARS and highly pathogenic avian influenza, which caused great panic to people all over the world. Influenza A virus is a single-strand negative-segmented RNA virus, which is prone to antigenic drift, antigenic conversion and genetic recombination, especially the mutation and recombination of the viral outer membrane protein hemagglutinin (HA), which can form various mutations Strains or recombinant strains lead to changes in the virulence and host range of the virus, thereby causing influenza epidemics of varying degrees. Whether hemagglutinin (HA) can be cut into HA1 and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68
CPCC12Q1/6869C12Q1/70C12Q1/701C12Q2600/142C12Q2600/156C12Q2531/113C12Q2565/301
Inventor 陈晓光程琳郭宁张瑾姜华金燕梁洁张娟
Owner 山东国际旅行卫生保健中心
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