双重荧光定量RT-PCR在快速检测A、B型流感病毒上的应用

摘要

目的 建立双重荧光定量RT-PCR技术同时快速检测A、B型流感病毒,并应用于临床样本的检测.方法 在A型流感病毒M基因和B型流感病毒HA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,并应用于疑似流感含漱液标本检测.结果 该方法对A、B型流感病毒检测具有高度特异性,检出限分别为0.1TCID50和0.01TCID50,具有较好的稳定性.可从疑似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可以同时准确分型A、B型流感病毒,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 detection of influenza viruses A and B, and the constructed method was primarily applied to clinical samples test.Methods The specific primers and probes were designed in the conserved region of the M and HA gene of influenza viruses A and B, respectively. The reaction conditions were optimized and the sensitivity, specificity and the stab ility of the assay were evaluated. The clinical specimens collected from the patients were detected by this assay. Results For specific detecting the influenza A and B viruses,the detection limits of the assay were 0.1TCID50and 0.01 TCID50. The viral RNA was directly detected from the clinical specimens by this assay. Only three hours were needed for viral RNA extraction and real-time PCR amplification.Conclusion This assay is a rapid, sensitive and specific one for simultaneous detection of influenza viruses A and B.