中国兽医科学2025，Vol.55Issue(6)：757-763,7.DOI:10.16656/j.issn.1673-4696.2025.0111
羊肠道病毒TaqMan实时荧光定量RT-PCR检测方法的建立及初步应用
Establishment and preliminary application of TaqMan real-time fluorescence quantitative RT-PCR for detection of caprine enterovirus
摘要
Abstract
To establish a sensitive,specific and accurate real-time fluorescent quantitative RT-PCR detection method for caprine enterovirus(CEV),this study designed specific primers and probes targeting the conserved region of CEV gene sequences,and prepared standard products.A TaqMan real-time fluorescent quantitative RT-PCR detection method was established by optimizing the reaction system and reaction con-ditions.Meanwhile,its sensitivity,specificity and repeatability were evaluated,and its performance was compared with conventional RT-PCR for detecting clinical samples.The results demonstrated that the standard curve of the established method had an R2 value of 0.995,indicating a good linear relationship between copy number and Ct value.This method exhibited high specificity and no cross-reactivity with foot-and-mouth disease virus(FMDV),peste des petits ruminants virus(PPRV),sheep pox virus(SPV),orf virus(ORFV)and bovine viral diarrhea virus(BVDV).The minimum detection limit of sensitivity was 23 copies/μL and the in-tra-and inter-assay coefficients of variation were less than 2.5％,indicating excellent sensitivity and reproducibility.By using 598 clinical samples,the positive rate of this method(83/598)was higher than that of conventional RT-PCR(30/598).In summary,a sensitive and specific TaqMan real-time fluorescence quanti-tative RT-PCR assay for CEV has been successfully established in this study,it is recommended to utilize sheep feces or anal swab for CEV epidemiological investigation.This significant achievement provides crucial technical support for the diagnosis and epidemiological investigation of CEV.关键词
羊肠道病毒/TaqMan实时荧光定量RT-PCR/诊断Key words
caprine enterovirus/TaqMan real-time fluorescence quantitative RT-PCR/diagnosis分类
畜牧业引用本文复制引用
马雪青,张雨星,李平花,王松泰,魏达礼,赵志荀,朵红,卢曾军,孙普..羊肠道病毒TaqMan实时荧光定量RT-PCR检测方法的建立及初步应用[J].中国兽医科学,2025,55(6):757-763,7.基金项目
兰州市科技计划项目(2022-2-46) （2022-2-46）
重点研发与转化计划科技援青合作专项(2022-QY-207) （2022-QY-207）
