呼吸道合胞病毒TaqMan探针实时定量RT-PCR检测方法的建立及应用

摘要

目的:建立呼吸道合胞病毒(RSV)核酸特异、快速、敏感的TaqMan探针实时荧光定量PCR检测方法,并对临床样本进行检测.方法:比对编码RSV非编码蛋白的基因序列,选取其保守片段设计引物和探针,建立实时荧光定量RT-PCR检测方法,并与传统RT-PCR方法进行比较,分别对两者的灵敏性、特异性、重复性及临床样本检验的适用性进行评价.结果:所建立的实时荧光定量RT-PCR检测方法可用于RSV的特异性检测.相对于传统RT-PCR方法100拷贝/反应的检测灵敏度,实时荧光定量RT-PCR的检测灵敏度达到10拷贝/反应,检测范围为1010～101拷贝/反应,且具有良好的特异性和重复性.从169份临床呼吸道标本中检出RSV阳性40例,高于普通PCR方法(31/169).结论:建立了RSV的TaqMan探针实时定量PCR检测方法,并可用于临床鼻咽拭子样本的检测,在临床上具有较好的应用前景.%Objective: To develop a specific, rapid, sensitive TaqMan based real-time quantitative PCR assay for detection and quantitation of respiratory syncytial virus (RSV). Methods: The specific primers and fluorescence-labeled probe were designed according to the conservative gene sequence of RSV. Absolute viral copy was achieved through the standard curve. Subsequently, experiments were undertaken to assess specificity, sensitivity and reproducibility, then compared with conventional PCR using clinic specimen. Results: Compared with conventional RT-PCR 100 copies per reaction mixture, the sensitivity of this real-time RT-PCR assay was 10 copies per reaction and the detection limit was ranging from 1010 -101 copies per reaction. Moreover, this real-time RT-PCR assay showed a good specificity and reproducibility. Among 169 nasopharyngeal swab specimens, 40 specimens were identified positive for RSV using real-time RT-PCR, higher than that by conventional RT-PCR (31/ 169). Conclusion: A real-time RT-PCR assay for detection of RSV has been developed. This assay maybe applied for surveillance and clinical diagnosis of RSV.