基于环介导等温扩增的甲型流感病毒N1/N2亚型检测方法的建立

Establishment of loop-mediated isothermal amplification method for the detection of influenza A virus N1/N2

目的建立用于甲型流感病毒N1/N2亚型的环介导等温扩增方法(loop-mediated isothermal amplification,LAMP),探索该方法在现场快速检测领域的使用前景。方法根据甲型流感病毒N1和N2的基因序列分别设计6条引物,建立LAMP方法,用甲型流感病毒N1/N2感染者的咽拭子为阳性样本,以乙型流感病毒、人鼻病毒感染者的咽拭子样本为对照验证方法的特异性,用市售实时荧光定量PCR试剂盒和LAMP法同时检测106份疑似甲型流感病毒阳性样本,比较LMAP方法的灵敏度和特异性。结果建立的甲型流感病毒N1/N2 LAMP方法检测用时少(30 min)、重复性和特异性好,与乙型流感病毒、人鼻病毒未出现交叉反应。LAMP方法与市售实时荧光定量PCR试剂盒的检测结果有一致性(Kappa=0.706,P<0.0001),特异性和灵敏度分别为90.0%和80.1%。结论建立的甲型流感病毒N1/N2的LAMP检测方法快速、简单,特异性和灵敏度好,适用于现场检测。

Objective To establish a loop-mediated isothermal amplification(LAMP)method for the detection of influenza A virus N1/N2,and explore the application for field rapid test.Methods According to the gene sequence of influenza A virus N1 and N2,six primers were designed,and the LAMP method for detection of influenza A virus N1/N2 was established.The swabs of influenza A virus N1/N2 infections were used as positive samples,and swabs of influenza B virus,human rhinovirus were used as control to test the repeatability and specificity.A total of 106samples of suspected patients infected with influenza A virus N1/N2 were simultaneously detected using commercial Real-time PCR kit and LAMP method,to analyze the specificity and sensitivity of LAMP method.Results The LAMP method for detection of influenza A virus N1/N2 was rapid(30 min),repeatable and specific and had no crossreaction with influenza B virus,human rhinovirus.The detection of throat swabs from patients suspected of influenza A virus infection showed the consistency between LAMP and commercial kit(Kappa=0.706,P<0.0001),as well as the specificity of 90.0%and sensitivity of 80.1%.Conclusion This LAMP method was rapid,simple,specific and sensitive.It can be applied for the field rapid detection of influenza A virus N1/N2.