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

作　　者：师志海[1] 王文佳[2] 王亚州[1] 孟红丽[1] 闫祥洲[1] 成海建[3] 施巧婷[1] 王二耀[1] 徐照学[1] SHI Zhi-hai;WANG Wen-jia;WANG Ya-zhou;MENG Hong-li;YAN Xiang-zhou;CHENG Hai-jian;SHI Qiao-ting;WANG Er-yao;XU Zhao-xue(Institute of Animal Husbandry and Veterinary Medicine,Henan Academy of Agricultural Sciences,Henan Key Laboratory of Farm Animal Breeding and Nutritional Regulation,Zhengzhou Key Laboratory of Veterinary Bioinformatics,Zhengzhou 450002,China;College of Veterinary Medicine and Pharmaceutical Engineering,Henan University of Animal Husbandry and Economy,Zhengzhou 450046,China;Institute of Animal Science and Veterinary Medicine,Shandong Academy of Agricultural Sciences,Jinan 250100,China)

机构地区：[1]河南省农业科学院,畜牧兽医研究所,河南省畜禽繁育与营养调控重点实验室,郑州市兽医生物信息重点实验室,河南郑州450002 [2]河南牧业经济学院动物医药学院,河南郑州450046 [3]山东省农业科学院畜牧兽医研究所,山东济南250100

出　　处：《中国兽医科学》2021年第12期1498-1503,共6页Chinese Veterinary Science

基　　金：河南省重点研发与推广专项项目(212102110360);河南省肉牛产业技术体系项目(S2013-08)。

摘　　要：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.

关 键 词：牛嵴病毒 犊牛腹泻 EvaGreen染料 

分 类 号：S852.659.6[农业科学—基础兽医学]