B型呼吸道合胞病毒荧光定量RT-PCR检测

Development of a real-time fluorescence quantitative RT-PCR assay to detect respiratory syncytial virus B

目的建立B型人呼吸道合胞病毒(respiratory syncytial virus,RSV)的快速荧光定量RT-PCR检测方法,用于B型RSV实验室早期诊断与病毒核酸的定量分析。方法利用B型RSV N基因保守区设计引物和TaqMan-MGB(minor groove binder,DNA小沟结合物)探针,优化反应体系和反应条件;并对方法进行特异性、灵敏度和重复性评价;同时以10倍梯度稀释的质粒为标准品建立荧光定量RT-PCR的标准曲线,构建定量分析模型。并用该方法对100份临床呼吸道样本进行检测。结果该方法与其他呼吸道病毒均无交叉反应,检测灵敏度达1TCID50/ml50%组织培养物感染剂量病毒滴度,临床样本中B型RSV的检测阳性率达18%。结论B型RSV荧光定量RT-PCR检测方法快速、敏感、特异,适用于B型RSV的早期诊断和核酸定量分析。

Objective To develop a rapid real- time RT- PCR assay for early detection of human respiratory syncytial virus（RSV） B and quantification of the virus. Methods One pair of primer and one TaqMan - MGB（minor groove binder） fluorogenic probe were designed from the conservative region of N gene of RSV B. Optimized reactive system and thermal cycle conditions were set up. Specificity, sensitivity and repeatability of the method were evaluated. The standard curve and virus quantitative andlysis model were constructed using the standard plasmids in series dilution, One hundred clinical respiratory specimens were detected by this method, Results This method had no cross reaction with other common respiratory virus. The sensitivity was 1 Tissue Culture Infectious Doseso/ml（TCID50/ml）. 18 % of the clinical specimens were positive of RSV B. Conclusion A rapid, sensitive an specific real - time RT PCR assay to detect RSV B type is established which is suitable for early diagnosis and virus quantification.