应用改良的实时taqman荧光定量rt-pcr技术-virologicasinica.pdfVIP

第 21 卷 5 期 中 国 病 毒 学 21 （5 ）： 449-453 2006 年 9 月 VIROLOGICA SINICA September 2006 应用改良的实时 TaqMan 荧光定量 RT-PCR 技术 检测口蹄疫病毒及其 3D 基因转录水平 石立立，顾潮江，张 倩，张玮莹，李 勇，鲁 彬， 何 成，屈三甫，郑从义** （武汉大学生命科学学院，病毒学国家重点实验室，湖北武汉430072 ） Application of an Improved Real-time TaqMan RT-PCR in FMDV Detection SHI Li-li，GU Chao-jiang，ZHANG Qian ，ZHANG Wei-Ying ，LI Yong ，LU Bin ，HE cheng ， ** QU San-fu，ZHENG Cong-yi (State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China) Abstract: In this study, an improved real-time RT-PCR was applied to quantification of Foot-and-mouth disease virus in vivo and in vitro. The results suggested that this method had a high sensitivity with up to less than 10 copies / reaction. Furthermore, this novel method was successfully used to quantify the transcriptional level of FMDV-3D gene in transfected BHK-21 cells and the resultant concentration of RNA level was up to 4 6.9×10 copies/μL. Compared with SYBR Green ⅠRT-PCR, six of eight samples testing the FMDV RNA copy number was up to 6.7-fold higher when determined by TaqMan RT-PCR, indicating that the improved method shows a high sensitivity in virus detection. Key word: Foot-and-mouth disease virus; Real-time TaqMan RT-PCR; Quantificational detection 摘要：将改良的实时TaqMan荧光定量RT －PCR技术应用于口蹄疫病毒感染体内和体外的定量检测以及其3D基因转录水平分 析。结果表明：对样品中口蹄疫病毒基因组RNA 的检测灵敏度可达l0个基因拷贝，可同时检测病毒正负链复制水平且重复性 较好，所测口蹄疫病毒3D基因转录水平可高达6.9×104拷贝/μL ；与实时SYBR Green Ⅰ染料RT-PCR技术比较，改良的实时 TagMan荧光定量RT －PCR技术检测灵敏度高6.7倍。以上结果证实，改良的实时TaqMan荧光定量RT －PCR技术在病毒检测 和基因表达水平分析方面有更高的