新型甲型H1N1流感病毒的实验室检测

The laboratory detection of a new human influenza A(H1N1)

目的利用RT-PCR技术,建立并优化快速、敏感、特异的检测新型甲型H1N1流感病毒的方法,并进行初步应用。方法设计高度特异性引物,优化常规RT-PCR和Real-time Fluores-cent Quantitative PCR反应体系,并对2009年惠州市流感样病例咽拭子标本进行甲型H1N1流感病毒RNA的检测。结果荧光定量RT-PCR与常规RT-PCR方法均有较高的特异性,与季节性H1N1、H3N2和B型流感病毒等无交叉反应,两种PCR检测结果的一致率达到99.5%以上,均能快速准确检测出患者咽拭子标本的甲型H1N1流感病毒,且荧光定量RT-PCR最低模板检测限在0.5pg以下。结论常规RT-PCR和荧光定量RT-PCR不仅特异性高,而且比病毒分离法更敏感、简便和快速,适合甲型H1N1流感病毒的快速检测,为早期诊断、定量分析甲型H1N1流感病毒感染程度奠定基础。

Objective To establish and optimize a rapid,sensitive and specific assay for new human influenza A（H1N1） detection with the method of RT-PCR.Methods Primers for H1N1 were designed in highly conserved various subtypes and genotypes of influenza virus A and influenza virus B.The sensitivity of the conventional and real-time PCR assays was optimized by evaluating different concentrations of primers and RNA.These assays were simultaneously used to detect H1N1 for influenza-like illness in Huizhou in 2009.Results Specificities of the conventional RT-PCR and real-time PCR assays were high,and seasonal H1N1,H3N2,influenza B virus did not cross-react with influenza A.The detective result of RT-PCR method was 99.5%,which was in accordance with that of real-time PCR method.The conventional RT-PCR assay and real-time PCR assay successfully detected H1N1 clinical specimens,and the RNA from the sample remained detectable in the real-time PCR assay was below 0.5pg.Conclusions This study shows that conventional and real-time quantitative RT-PCR assays are reliable,sensitive and fast for detecting a novel human influenza A（H1N1） subtype and indicate that the constructed method paves the way for the early and rapid detection of H1N1 and quantitative analysis for the infect degree of H1N1.