环介导等温扩增技术在牛病毒性腹泻检测中的应用  

Application of loop mediated isothermal amplification in the detection of bovine viral diarrhea

作　　者：周娴[1] 陈文钦[1] 王雯熙 孙智武 张苗苗[1] 郭妮妮 宋先荣 吴修竹 ZHOU Xian;CHEN Wenqin;WANG Wenxi;SUN Zhiwu;ZHANG Miaomiao;GUO Nini;SONG Xianrong;WU Xiuzhu(Hubei Vocational College of Bio-Technology,Wuhan 430070,China)

机构地区：[1]湖北生物科技职业学院,武汉430070

出　　处：《养殖与饲料》2025年第5期41-46,共6页Animals Breeding and Feed

基　　金：第二批国家级职业教育教师教学创新团队专业领域课题研究项目(ZI2021100108);2020年度湖北省职业技术教育学会科学研究重点课题(ZJGA202045)。

摘　　要：[目的]基于环介导等温扩增(loop mediated isothermal amplification,LAMP)技术,研究1种快速、灵敏检测牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)检测方法,为牛病毒性腹泻的快速诊治提供参考。[方法]参考GenBank上BVDV序列,依据BVDV的结构蛋白5'-UTR保守序列设计出多组引物探针,从中优选出1组扩增效果最佳的引物探针,经过优化反应体系和退火温度,建立BVDV的环介导等温扩增技术(LAMP)检测方法。随后对该方法的敏感性、特异性进行了评估,并应用该检测方法进行临床样品检测。[结果]所建立的LAMP方法展现出高度特异性,仅对BVDV产生扩增信号,与牛常见病原(如口蹄疫、沙门氏菌等)无交叉反应;通过梯度稀释重组质粒验证,其检测灵敏度为102 copies/μL,较常规PCR提升10倍;临床样本检测结果与PCR法完全一致(符合率100%)。[结论]本研究建立的BVDV LAMP检测方法敏感性高、特异性强,适用于牛病毒性腹泻病的临床诊治。[Objectives]A rapid and sensitive method for detecting bovine viral diarrhea virus(BVDV)based on the loop-mediated isothermal amplification(LAMP)technology was developed to provide reference for the diagnosis of bovine viral diarrhea.[Methods]Multiple sets of primers and probes were designed based on the conserved sequence of 5'-UTR gene of BVDV in GenBank,and a set of primers and probes with the best effect of amplification were screened.A method for detecting BVDV with LAMP was established by optimizing the reaction system and annealing temperature.The sensitivity and specificity of the method established were evaluated,and the method was used to detect clinical samples.[Results]The LAMP method established had high specificity,producing amplification signals only for BVDV and showing no cross-reaction with common bovine pathogens including foot-and-mouth disease and Salmonella.Its sensitivity of detection verified by gradient dilution of recombinant plasmids was 102 copies/μL,10 times higher than that of conventional PCR.The results of detecting clinical samples were completely consistent with that of PCR method,with a concordance rate of 100%.[Conclusions]The established method of detecting BVDV with LAMP has high sensitivity and strong specificity.It will provide technical support for the clinical diagnosis and treatment of bovine viral diarrhea.

关 键 词：牛病毒性腹泻病毒 环介导等温扩增技术 快速检测 临床诊治 应用 

分 类 号：S852.65[农业科学—基础兽医学]