禽流感病毒RT-PCR及多重RT-PCR检测技术的建立

研究建立了禽流感病毒(AIV)A型、H5、N1、H9、N2亚型特异性RT-PCR及H5/N1、H9/N2、A/H5/N1多重RT-PCR检测技术,用于检测或同时检测和鉴别A型及H5N1、H9N2亚型AIV。所建立的RT-PCR和多重RT-PCR从核酸提取、基因扩增到产物分析在3~4h内即可完成,经对36株AIV及相关病毒分离物检测,与病毒分离鉴定的结果完全一致,且与相关病毒或其他亚型无交叉反应。采用多重RT-PCR检测80份棉拭子样品,并与病毒分离鉴定方法比较,二者H5N1、H9N2亚型的鉴定结果完全吻合。结果表明,该方法具有快速、敏感、特异等优点,为临诊样品中AIV型及H5N1、H9N2亚型鉴定和诊断的有效方法。

Development of RT-PCR and multiplex RT-PCR for detecting avian influenza virus

The type A-specific and H5, N1, H9, N2 subtype-specific RT-PCR, and further the H5/N1, H9N2, A/H5/N1 specific multiplex RT-PCR assays, based on the matrix, hemagglutinin and neuraminidase genes of known avian influenza virus, were developed for detection or detection-and-differentiation of H5N1 and H9N2 subtypes. The entire nucleic acid isolation, amplification, and detection procedure was completed within 3-4h. A total of 36 (isolates) of avian influenza and clinically relevant diseases were detected by the RT-PCR and the multiplex RT-PCR assays respectively. The results showed the assays were sensitive and high specific, with no cross-reaction to other subtype or clinically relevant viruses, and gave an excellent (100%) correlation with the conventional virus isolation method. The multiplex RT-PCR and the conventional virus isolation agreed on the H5N1, H9N2 subtypes determination of 80 swab samples. These methods therefore provide rapid, simple, cheap, safe, sensitive, type-specific and subtype-specific identification and diagnosis of H5N1 and H9N2 avian influenza virus in clinical samples.