Primers for determining full-gene sequence of influenza A H1N1 viruses and determination method

An influenza virus and whole gene technology, applied in the field of primers and kits for determining the whole gene sequence of influenza A (H1N1) virus, can solve the problems of poor primer specificity and sensitivity that affect the monitoring of the whole gene sequence of influenza A (H1N1) virus Low-level problems, to achieve the effect of stable results, simple sampling and high sensitivity

Active Publication Date: 2013-03-06
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, we also found that according to the 46 pairs of whole gene sequencing primers recommended by WHO (http: / / www.who.int / csr / disease / swineflu / en / index.html), more than one-third of the primers have poor specificity and sensitivity The low phenomenon seriously affects the progress of monitoring the whole gene sequence determination of influenza A (H1N1) virus

Method used

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  • Primers for determining full-gene sequence of influenza A H1N1 viruses and determination method
  • Primers for determining full-gene sequence of influenza A H1N1 viruses and determination method
  • Primers for determining full-gene sequence of influenza A H1N1 viruses and determination method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Design of primers and probes

[0031] By comparing and analyzing the gene sequences of 100 known A / H1N1 virus strains in all GenBank databases, a highly conserved segment was selected, and 48 pairs of primers and probes were designed, as described in SEQ ID NO: 1-96. The principles of primer design are as follows:

[0032] (1) The length of the primer is about 20-23nt, and the GC content is between 45%-55%.

[0033] (2) The length of PCR amplification is generally between 400-600 bp.

[0034] (3) For the same template fragment, design primers so that each site is covered by 2-3 PCR amplified fragments, so as to avoid point mutations in the PCR reaction from affecting the interpretation of the results.

[0035] Analyze the information of the above-mentioned virus-specific genes, and use the sequence analysis software DNASTAR to exclude inter-primer / inner dimers, and verify the specificity of the primers and the homology of similar pathogens by BLAST, and d...

Embodiment 2

[0036] Embodiment 2: Construction and optimization of PCR reaction system

[0037] 1. Sample Collection

[0038] Our laboratory collected samples from three suspected cases on May 17 and May 28, 2009, including the first suspected imported H1N1 case in Guangdong Province and the first and second second-generation suspected cases of H1N1 in my country Nasal / throat swab specimens of Dai and Xue.

[0039] 2. Extraction of RNA from nasal / throat swab specimens

[0040] Use the QIAmp MiniElute Virus Spin Kit (Qiagen, Chatsworth, CA) to extract RNA from nasal / pharyngeal swab specimens, and follow the instructions:

[0041] (1) When using this kit for the first time, add 100% ethanol to the AW1 and AW2 buffers according to the volume indicated on the reagent bottle, add 25 ml absolute ethanol to 19 ml AW1, add 30 ml absolute ethanol to 13 ml AW1 Ethanol; add 28 μg / ml carrier RNA to AL.

[0042] (2) Put 25 μl Qiagen Protease into a 1.5 ml centrifuge tube.

[0043] (3) Add 200 μl ...

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Abstract

The invention discloses full-gene sequence primers for determining influenza A H1N1 viruses and a determination method. The invention relates to 48 pairs of primers with strong specificity, high sensitivity and good stability, which are used for determining 8 fragments of gene sequences of the influenza A H1N1 viruses, wherein the 8 fragments comprise HA, NA, NP, M1, M2, PA, PB1 and PB2. The method for detection by utilizing the 48 pairs of primers has the advantages of simple operation, short time consumption, strong specificity and high sensitivity and is suitable for identification of the influenza A H1N1 viruses, epidemiological survey and research and the like.

Description

technical field [0001] The invention relates to a detection technology of type A (H1N1) influenza virus, in particular to a primer and a kit for measuring the whole gene sequence of type A (H1N1) influenza virus. Background technique [0002] Influenza (flu) is a respiratory infection caused by the influenza virus (flu virus). Influenza viruses belong to the Orthomyxoviridae family and are negative single-stranded RNA viruses. Influenza viruses are divided into three types: A, B, and C according to the antigenicity of nucleoprotein (NP) and matrix protein (MP), all of which can infect humans. Among them, influenza A virus has the strongest ability to mutate and can cause influenza seasonal epidemics or influenza pandemics. Influenza B virus has a weak ability to mutate, and generally causes seasonal influenza epidemics. The antigenicity of influenza C virus is relatively stable, and it is less likely to cause epidemics. [0003] Influenza A virus spread from birds to hum...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 黎孟枫曹开源吴珏珩朱勋何振健袁洁李隽张定梅徐霖杨纨李蔓英
Owner SUN YAT SEN UNIV
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