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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 dual-fluorescence RT-PCR rapid detection method for the simultaneous detection and differential diagnosis of influenza A and H1N1 influenza viruses. Methods Specific primers and Taqman probes were designed according to the conserved regions of influenza virus A (M) gene and NA gene of influenza A (H1N1) virus, and the optimized double-fluorescence RT-PCR reaction system was established. The double RT-PCR reaction system Specificity, sensitivity and stability, and applied to the detection of suspected influenza or H1N1 flu gargle samples. Results The method was highly specific to the detection of Influenza A and H1N1 viruses. The detection limits were 0.01 TCID50 and 0.1 TCID50 respectively, which showed good stability. Flu virus nucleic acid can be detected directly from the patient’s mouthwash solution for suspected influenza or swine flu. Conclusion The double-fluorescence quantitative RT-PCR established in this study can detect both Influenza A and H1N1 influenza virus simultaneously with high sensitivity and good stability. It is a new method for rapid detection of influenza virus.