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焦磷酸测序技术在检测甲型H1N1血凝素基因裂解位点突变中的应用
2014-07-09 15:03:59 中国质量新闻网
    陈晓光,程琳,郭宁,姜华,张瑾,金燕,梁洁,张娟

  山东国际旅行卫生保健中心,山东 青岛 266007

  摘要:目的 基于焦磷酸测序技术建立甲型H1N1流感病毒血凝素(HA)基因裂解位点突变的快速检测方法,探讨其临床应用价值。方法 采用RT-PCR扩增甲型H1N1病毒血凝素基因中的HA片段,在生物信息学分析的基础上,针对甲型H1N1病毒血凝素基因含有裂解位点序列片段,分别设计一套生物素标记的PCR引物和测序引物,运用焦磷酸测序技术对含甲型H1N1病毒裂解位点基因片段进行序列测定,同时分析青岛地区甲型H1N1流感病毒流行株的裂解位点基因特征。结果 建立了基于焦磷酸测序技术检测甲型H1N1流感病毒裂解位点突变方法,实现甲型H1N1流感病毒HA基因裂解位点的高通量检测,青岛地区甲型H1N1流感病毒裂解位点344氨基酸序列没有发生变异。结论 本研究所建立的方法具有自动化程度高和结果准确等特点,适用于甲型H1N1流感病毒裂解位点进行快速高通量检测。

  关键词:焦磷酸测序技术;甲型H1N1流感病毒;血凝素(HA)基因;突变;裂解位点

  中图分类号:R446.5  文献标识码:B

  Application of pyrosequencing technology in hemagglutinincleavage site variant of H1N1 virus

  CHEN Xiao-guang, CHENG Lin, GUO Ning, JIANG Hua, ZHANG Jin, JINYan, LIANG Jie, ZHANG Juan

  Shandong International Travel Healthcare Center, Qingdao,Shandong 266071, China

  Abstract:  Objective  To explore a rapid  assay for detection of cleavage site variant  of the pandemicinfluenza A (H1N1) virus based on pyrosequencing techniques. Methods  The HA gene of these strains was amplified byRT-PCR. According to the published sequences in GenBank,pyrosequencing primers and one pair of PCR primers were designedfor pyrosequencing analysis. The genetic characteristic of cleavagesite of pandemic influenza A(H1N1) virus in Qingdao City werepyrosequenced and analyzed.  Results  A novelpyrosequencing  assay was developed for the rapid andhigh-throughput detection of cleavage site of the pandemicinfluenza A (H1N1)virus. The key amino acid position 344 ofcleavage site of virulent strains in Qingdao City isn’t mutatedcompared with that of strains in 2009.  Conclusion  Thispyrosequencing assay is a rapid and high-throughput method forcleavage site variant of the virus and could be used for thepandemic influenza A(H1N1) virus surveillance.

  Key words:  Pyrosequencing; Pandemic influenza A(H1N1)virus;Hemagglutinins;Variants;Cleavage site

  《中国国境卫生检疫杂志》2014年3期

  
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