双重荧光定量RT-PCR快速检测甲型和甲型H1N1流感病毒

Simultaneous detection of influenza A viruses and 2009 pandemic influenza A(H1N1) virus by multiplex real-time RT-PCR

目的建立双重荧光RT-PCR快速检测方法,用于甲型和甲型H1N1流感病毒的同时检测和鉴别诊断。方法针对甲型流感病毒M基因和甲型H1N1流感病毒NA基因的保守区序列分别设计特异性引物和Taqman探针,建立优化双重荧光RT-PCR反应体系,评价所建双重RT-PCR反应体系的特异性、敏感性和稳定性,并应用于疑似流感或甲型H1N1流感含漱液标本检测。结果该方法对甲型、甲型H1N1流感病毒检测具有高度特异性,检出限分别为0.01 TCID50和0.1 TCID50,具有较好的稳定性。可从疑似流感或甲型H1N1流感患者含漱液中直接检测到流感病毒核酸。结论本研究建立的双重荧光定量RT-PCR可以同时准确检测甲型和甲型H1N1流感病毒,灵敏度高,稳定性好,是一种快速检测流感病毒的新方法。

Objective To establish a TaqMan-based multiplex real-time PCR assay for the rapid and simultaneous detection of influenza A viruses and 2009 pandemic influenza A （ H1N1 ） virus. Methods The specific primers and probes were designed in the conserved region of the M gene for influenza A viruses and the NA gene for 2009 pandemic influenza A （H1N1） virus, respectively. The reaction conditions were optimized and the sensitivity, specificity and stability of the assay were evaluated. The clinical specimens collected from the patients were detected by this assay. Results The results showed that the assay possessed high specificity for influenza A viruses and 2009 pandemic influenza A （H1N1） virus detection and the detection limit was up to 0.01 TCID50 and 0. 1 TCID50 respectively. The viral RNA could be directly detected from the clinical specimens by this assay. Conclusion The multiplex real-time RT- PCR assay can provide rapid, sensitive and reliable detection of influenza A viruses and 2009 pandemic influenza A （ H1 N1 ） virus and is useful in the detection of influenza virus.