染料法实时荧光PCR检测牛嵴病毒

A real-time PCR suitable for detection of bovine kobuvirus based on the fluorescent dye

牛嵴病毒(BKoV)可能是与犊牛腹泻相关的潜在病原,为建立快速检测BKoV的方法,本研究根据BKoV 3D基因的保守序列,设计特异性引物对,通过优化反应条件和体系,建立了基于EvaGreen染料检测BKoV的实时定量PCR方法。该方法Ct值与模板在1.08×10^(2)~1.08×10^(8) copies/μL范围内线性关系良好,扩增效率为106.4%;该方法仅检测BKoV时结果呈阳性,检测其他犊牛腹泻相关病原时结果呈阴性;能检出BKoV的最低检出限为10.8 copies/μL;批内和批间的变异系数均小于2%,重复性好。用该方法对311份采自河南、山东和四川省的犊牛腹泻样本进行检测,河南省BKoV的检出率为7.38%(20/271),其他两省的样本均未检测出BKoV。本研究建立的方法灵敏度高、特异性强、稳定性好,为BKoV的检测和流行病学调查提供了有力手段。

Bovine kobuvirus(BKoV)is a potential pathogen associated with calf diarrhea.By designing primers targeted to conserved sequence of 3D gene of BKoV and optimizing the reaction conditions and system,an EvaGreen real-time PCR method was successfully established.Ct value showed a good linear relationship with the standard in the range of 1.08×10^(2)—1.08×10^(8) copies/μL and the amplification efficiency was 106.4%.Only BKoV gene was specifically amplified,no amplification curve for other calf diarrhea pathogens.The detection minimum limit for BKoV was 10.8 copies/μL.The coefficients of variation between intra-assay and the inter-assay were less than 2%,indicating a high repeatability.Total 311 diarrheic samples collected from Henan,Shandong and Sichuan province,the BKoV detection rate in Henan region was 7.38%(20/271).The real-time PCR assay for detecting BKoV in this study has good sensitivity,specificity and repeatability,which can be provide an effective means for detection and epidemiological investigation of BKoV.