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

Application of pyrosequencing technology in hemagglutinin cleavage site variant of H1N1 virus

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。

Objective To explore a rapid assay for detection of cleavage site variant of the pandemic influenza A （H1N1） virus based on pyrosequencing techniques. Methods The HA gene of these strains was amplified by RT-PCR. According to the published sequences in GenBank, pyresequencing primers and one pair of PCR primers were designed for pyrosequencing analysis. The genetic characteristic of cleavage site of pandemic influenza A （H1N1） virus in Qingdao City were pyrosequenced and analyzed. Results Anovel pyrosequencing assay was developed for the rapid and high-throughput detection of cleavage site of the pandemic influenza A （H1N1） virus. The key amino acid position 344 of cleavage site of virulent strains in Qingdao City isn＇t mutated compared with that of strains in 2009. Conclusion This pyrosequencing assay is a rapid and high-throughput method for cleavage site variant of the virus and could be used for the pandemic influenza A（H1N1） virus surveillance.