Payungporn Sunchai, Chutinimitkul Salin, Chaisingh Arunee, Damrongwantanapokin Sudarat, Buranathai Chantanee, Amonsin Alongkorn, Theamboonlers Apiradee, Poovorawan Yong
Center of Excellence in Viral Hepatitis Research, Faculty of Medicine, Chulalongkorn University, Pathumwan, Bangkok 10330, Thailand.
J Virol Methods. 2006 Feb;131(2):143-7. doi: 10.1016/j.jviromet.2005.08.004. Epub 2005 Sep 23.
H5N1 influenza A virus causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to mammalian species such as leopards, tigers and humans. The aim of this study was to develop a multiplex real-time RT-PCR for rapid detection of H5N1 influenza A virus. The selected primers and various labeled TaqMan MGB reporter probes corresponding to M, H5 and N1 were used in a single step multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. In order to validate the method, 75 clinical specimens infected with H5N1 isolated from both poultry and mammals, as well as various specimens of other subtypes and RNA from other viral pathogens of poultry and human were tested. The results showed that the multiplex real-time RT-PCR assays can be applied to detect virus suspensions of H5N1 influenza A virus from a wide host range and demonstrated the sensitivity of the assay amounted to approximately 10(2)-10(3)copies/mul. In conclusion, the highlights of this particular method lie in its rapidity, specificity and sensitivity thus rendering it feasible and effective for large-scale screening at times of H5N1 influenza A virus outbreaks.
H5N1甲型流感病毒在家禽中会引发一种迅速致命的全身性疾病，并直接从家禽传播至豹、老虎和人类等哺乳动物物种。本研究的目的是开发一种用于快速检测H5N1甲型流感病毒的多重实时逆转录聚合酶链反应（RT-PCR）。在一步法多重实时RT-PCR中，使用了针对M、H5和N1的选定引物以及各种标记的TaqMan MGB报告探针，以同时检测三重荧光信号。为了验证该方法，对75份从家禽和哺乳动物中分离出的感染H5N1的临床标本，以及其他亚型的各种标本和来自家禽及人类其他病毒病原体的RNA进行了检测。结果表明，多重实时RT-PCR检测法可用于检测来自广泛宿主范围的H5N1甲型流感病毒的病毒悬液，且该检测法的灵敏度约为10(2)-10(3)拷贝/微升。总之，这一特定方法的亮点在于其快速性、特异性和灵敏度，因此在H5N1甲型流感病毒爆发时，使其适用于大规模筛查且切实有效。
