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

摘要

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.%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.