应用多重TaqMan MGB探针实时聚合酶链反应检测A型和B型流感病毒的研究

目的 应用多重real-time PCR方法检测A型和B型流感病毒,探讨其在快速诊断流感病毒感染中的优越性.方法 根据A型和B型流感病毒M基因的相对保守序列,设计两套特异性引物和TaqMan MGB探针,进行多重real-time PCR检测A型和B型流感病毒.将该方法与病毒分离、免疫层析、传统RT-PCR进行比较,并对多重real-time PCR的特异性进行评估.并应用multiplex real-time PCR方法和常规的病毒分离方法对广东省流感样病例334份咽拭标本进行检测,比较其灵敏性和特异性.结果 多重real-time PCR反应可以检测到A、B型流感病毒的最低病毒量分别为10-1和10-3TCID50/反应.该方法在对流感病毒株以及其他呼吸道病毒株样品的检测中,未发现假阳性和假阴性现象,特异性为100%.该方法对334份临床标本进行检测,结果与经典检测方法(病毒分离鉴定结合血清学检测)的符合率达100%,且灵敏度明显高于病毒分离.结论 该研究建立的多重TaqMan MGB real-time PCR是一种快速、灵敏性和特异性高的流感病毒检测方法,对于流感的早期诊断及指导临床有重要意义.

Application of a TaqMan MGB multiplex real-time PCR assay for rapid diagnosis of influenza virus A and B infection

Objective To develop a rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza A and influenza B viruses.Methods Two sets of specific primers and TaqMan MGB probes used in the multiplex real-time polymerase chain reaction was designed according to the conservative M1gene of influenza A and B.Following Reed and Muench method, the TCID50 of clinically isolated influenza virus was calculated.The tenfold tissue culture dilutions were then used for evaluation of the sensitivity of the multiplex real-time PCR.Thereafter,the multiplex real-time PCR sensitivity for influenza A and B virus were compared with cell culture,rapid immunological tests and PCR.Specificity of the method was determined by testing specimens containing RSV,measles and parainfluenza virus,all of which yielded negative results with no discernible background.A total of 334 respiratory samples from influenza-like-illnesses cases were analyzed by the multiplex real-time PCR.Results The lowest dilutions of virus detected by the assay were 10-1 and10-3 TCID50/reaction for the influenza virus A and B respectively.The specificity was 100%.237 of 334(70.96%) samples were detected the influenza virus by the method,which was consistent with those of currently used methods of virus isolation in MDCK,sera diagnosis by hemagglutination inhibition(HI) assay and real-time PCR.All of these culture-positive samples and an additional 164 influenza virus were detected by the multiplex real-time PCR,which observed more sensitive than conventional cell culture with corresponding high specificity.Conclusion The TaqMan MGB multiplex real-time PCR assay indicated optimal specificity and sensitivity compared to conventional culture and rapid immunological methods and deserved to application in rapid diagnosis and surveillance of flu.