猴副流感病毒5型实时荧光定量pcr方法的建立与-中国试验动物学报.docVIP

研究报告 猴副流感病毒5型实时荧光定量PCR方法的建立与初步应用[1] 冯育芳，李晓波，邢进，付瑞，王吉，卫礼，岳秉飞，贺争鸣(中国食品药品检定研究院，国家实验动物微生物、遗传检测中心，北京100050) ? 【摘 要】 目的 建立猴副流感病毒5型（SV5）实时荧光定量PCR方法，并初步应用于猴样本检测。方法 根据SV5病毒NP基因保守片段设计特异引物和TaqMan探针，使用分子生物方法制备质粒标准品，建立SV5实时荧光定量PCR方法；对该方法的线性、敏感性、特异性及稳定性进行测定；并使用该方法对24个猴样品进行检测。结果 成功建立SV5实时荧光定量PCR方法；该方法线性范围为6.8×109～6.8×105copies/μL，灵敏度为6.8 copies/μL，批内变异系数(CV)为0.63%~8.71%，批间CV为1.52%~12.38%，而且方法特异性好；在24个猴肺样品中未检测出阳性。结论 该方法的建立为实验动物和动物源性生物制品中SV5的定量检测奠定了基础。 【关键词】 猴副流感病毒5型；实时荧光定量PCR；定量检测 【中图分类号】R33【文献标识码】A【文章编号】1671-7856（201）-0000-00 Doi:10.3969/j.issn.1671-7856.2013.010.000 ? Establishment of a real time RT-PCR method for detection of simian parainfluenza virus 5 (SV5) and its preliminary application FENG Yu-fang, LI Xiao-bo, XING Jin, FU Rui, WANG Ji, WEI Li,YUE Bing-fei, HE zheng-ming (National Institutes for Food and Drug Control, Products, National Center for Microbiological and Genetic Monitoring of Laboratory Animals, Beijing 100050, China) [Abstract] Objective To establish a real-time RT-PCR method for detection of simian parainfluenza virus type 5 (SV5) in biological materials from monkeys and explore its application．Methods According SV5 virus NP gene fragments, a pair of specific primers and TaqMan probe were designed and synthesized. SV5 RNA standards were prepared using molecular biological techniques. Then the linearity, sensitivity, specificity and stability of the method were determined. Last, this method was used for testing lung samples of 24 monkeys. Results A real-time PCR method for detection of SV5 was successfully established. The linear range was 6.8×109 copies/μL to 6.8×105 copies/μL, sensitivity was 6.8 copies/μL, coefficient of variation (CV) was 0.63% to 8.71%, inter-assay CV was 1.52% to 12.38%, and the method was highly specific. No positive reaction was detected in the lung samples of 24 monkeys. Conclusions The establishment of this method provides a useful basis for quantitative detection of SV5 in