多重实时PCR检测三种呼吸道病毒

Development of a multiplex real-time PCR to deect three respiratory viruses

目的建立多重实时PCR检测体系可同时快速检测引起人呼吸道感染的甲型流感病毒(IAV)、呼吸道合胞病毒(RSV)和SAPS冠状病毒(SARS-CoV)。方法通过对PCR反应条件的优化,建立了同时检测IAV、RSV和SARS-CoV的多重实时PCR方法。结果经熔解曲线分析,证实了该法可同时或分别检测3种重要呼吸道病毒。检测灵敏度分别为10^(0.7)TCID_(50)/ml、1 TCID_(50)/ml和10^(-0.5) TCID_(50)/ml。采用相同的反应体系和条件,分别对其他7种呼吸道病毒(包括乙型流感病毒,副流感病毒2型,柯萨奇病毒B3和B5血清型及腺病毒2、3和7血清型)进行扩增,均未检测出扩增产物。此种方法可在4h内完成。结论建立的多重实时PCR检测方法适用于3种重要呼吸道病毒的快速检测。

Objective To develop multiplex real-rime polymerase chain reaction（PCR） method that simultaneously and rapidly detect three important viral pathogens. Methods Under optimized PCR conditions, a multiplex real-time PCR method was developed to simultaneously detect influenza A virus（IAV）, respiratory syncytial virus（RSV）and SARS coronavims（SARS-coV）. Results Confirmation of detecting three respiratory viruses respectively and simultaneously was based on the analysis of melting curve. Cultured IAV, RSV and SARS-CoV were detectable and the sensitivities were 10^0.7 TCID50/ml, 1 TCID50/ml and 10^-0.5 TCID50/ml. The specificity of the technique was verified by using other 7 respiratory viruses including influenza B virus, human parainfluenza virus type 2, coxsackievirus serotypes B3, B5 and human adenovirus serotypes 2, 3 and 7, and none of these viruses were found to be positive by the same multiplex real-time PCR condition. It took less than 4 hours in the whole process. Conclusion The multiplex RT-PCR assay can be used as a rapid, sensitive and specific method for detection of IAV, RSV and SARS-COV.