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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 establish a rapid detection method for the mutation of hemagglutinin (HA) gene of influenza A (H1N1) virus based on pyrosequencing technology and to explore its clinical value. Methods The HA fragment of the hemagglutinin gene of A (H1N1) virus was amplified by RT-PCR. Based on the bioinformatics analysis, the HA gene of A (H1N1) PCR and primer sequencing. The pyrosequencing technology was used to sequence the gene fragment containing the H1N1 virus cleavage site, and the genetic characteristics of the cleavage site of influenza A (H1N1) influenza in Qingdao were analyzed. Results A pyrosequencing method based on pyrosequencing was established to detect the mutation site of influenza A H1N1 influenza virus. The high-throughput detection of HA gene cleavage site of influenza A H1N1 influenza virus, the 344 amino acid sequence of influenza A H1N1 influenza virus in Qingdao region No variation occurred. Conclusion The method established in this study has the characteristics of high degree of automation and accurate results. It is suitable for rapid and high-throughput detection of influenza A H1N1 influenza virus cleavage sites.