树鼩腺病毒TaqMan实时荧光定量PCR检测方法的建立及初步应用  

Establishment and application of a TaqMan real-time fluorescence quantitative PCR for detection of tree shrew adenovirus(TAV)

作　　者：宋庆凯 李晓飞[1] 苗雨润[1] 张志成 王璇 袁圆 代解杰[1] 孙晓梅[1] SONG Qingkai;LI Xiaofei;MIAO Yurun;ZHANG Zhicheng;WANG Xua;YUAN Yuan;DAI Jiejie;SUN Xiaomei(Center of Tree Shrew Germplasm Resources,Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College;Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases;Yunnan Innovation Team of Standardization and Application Resear)

机构地区：[1]中国医学科学院/北京协和医学院医学生物学研究所树鼩种质资源中心,云南省重大传染病疫苗研发重点实验室,中国医学科学院医学生物学研究所实验树鼩标准化与应用研究省创新团队,昆明650118

出　　处：《中国比较医学杂志》2018年第3期72-77,共6页Chinese Journal of Comparative Medicine

基　　金：国家科技支撑计划(编号:2014BAI01B00);云南省联合支持国家计划项目(编号:2015GA009);云南省科技人才和平台计划项目(编号:2017HC019)。

摘　　要：目的通过建立树鼩腺病毒(tree shrew adenovirus,TAV)TaqMan实时荧光定量PCR检测方法,快速准确地检测树鼩腺病毒。方法根据TAV全基因组3’端保守序列设计合成特异性引物;用含目的基因片段的重组质粒作为标准品进行标准曲线绘制,建立TAV TaqMan实时荧光定量PCR检测方法。结果建立的TAV实时荧光定量PCR检测方法特异性好,与树鼩源及其他物种常见病毒无交叉反应;方法的最低检测限度为每微升3.7拷贝,灵敏性强;相关检测系数R2为0.998,扩增效率为95.7%;扩增结果具有良好的线性关系,且重复性检测效果好。结论成功建立了TAV TaqMan实时荧光定量PCR检测方法,灵敏度高,重复性好,可用于TAV感染的早期快速检测。Objective To establish a quick and accurate method for detection of tree shrew adenovirus(TAV)using TaqMan real-time fluorescence quantitative PCR.Methods Based on the published TAV genome sequence,a 3’conserved sequence was used to design specific probe primers.A standard curve was prepared using a recombinant plasmid containing the target gene fragment.A real-time fluorescence quantitative PCR method was established for detecting TAV based on TaqMan probe.Results The detection method was specific and was not cross-reactive with other common pathogens.The detection limit of the method was 3.7 copies/μL,showing a high sensitivity.The correlation coefficient was 0.998,and the efficiency was 95.7%.The amplification result showed a fine linear relationship,and the repeatability test effect was good.Conclusions The TAV real-time quantitative PCR detection method based on TaqMan probe has been successfully established.It has high sensitivity and reproducibility and can be applied to early detection of TAV infection.

关 键 词：树鼩腺病毒 3’保守区域 TAQMAN 实时荧光定量PCR 

分 类 号：R-33[医药卫生]