环介导等温扩增方法检测H9亚型流感病毒的研究

Detection of H9 subtype influenza virus by loop-mediated isothermal amplification

目的:建立环介导等温扩增(LAMP)方法用于H9亚型流感病毒基因的检测,为基层实验室提供简便快速的检测方法。方法:根据GenBank中公布的H9亚型流感病毒血凝素基因序列,选择保守区域应用在线软件PrimerExplorer V4设计LAMP引物。以克隆有H9亚型流感病毒血凝素基因的阳性质粒为模板,建立和优化LAMP检测方法,并进行灵敏度、特异性评价和病毒检测。结果:LAMP检测方法对H9亚型流感病毒基因的检出灵敏度达到5个拷贝,比普通PCR方法高出2个数量级,对H1、H3、H5亚型流感病毒不产生非特异性扩增,显示出良好的特异性。结论:建立的LAMP检测方法能快速、敏感、特异地检测H9亚型病毒,适合在基层实验室推广使用。

Objective：To establish loop-mediated isothermal amplification（LAMP） method for detection of H9 subtype influenza A virus.Methods：The H9 hemagglutinin gene sequence information was obtained from Genbank database.According to alignment analysis of HA gene sequences,a set of specific primers were designed by the LAMP primer design software（PrimerExplorer V4）.The gene of H9 hemagglutinin was cloned and served as the positive standard and the reaction system was established and optimized.Then the sensitivity and specificity of the established method were evaluated.Results：The sensitivity of LAMP method was 100 fold higher than that of conventional PCR,with the detection limit of five copies per reaction.There was no crossing-reaction with other subtype influenza virus including H1,H3 and H5.These results suggest that the present LAMP system provided a new avenue for the recognition of H9 subtype virus.Conclusion：The developed LAMP method is a sensitive and reliable assay for quick detection of H9 subtype influenza virus.It is very suitable for the labs at grass root level.