摘要
为了建立一种基于牛白血病病毒(BLV)env结构基因的高效、便捷和特异的重组酶介导等温扩增(RAA)快速检测方法,本试验利用DNAMAN进行序列比对,构建重组质粒pESI-env,设计并筛选RAA引物和探针,建立反应体系,进一步优化反应程序和引物探针用量,建立检测方法,并评价所建立方法的特异性、灵敏性和重复性,将该方法对临床样本的检测结果与酶联免疫吸附试验(ELISA)和荧光定量PCR(qPCR)进行比对。结果显示,最佳引物探针组合为F4/R1/P;该方法在20 min即可完成反应;与牛病毒性腹泻病毒(BVDV)、牛传染性鼻气管炎病毒(IBRV)和牛轮状病毒(BRV)无交叉反应,最低检出限可达1.62×10^(1) copies/μL,且重复性良好;建立方法的临床样本阳性检出率略高于ELISA和qPCR两种标准检测方法。本试验成功建立了一种高效、便捷且特异性强的BLV RAA荧光检测方法,摆脱了传统方法对昂贵设备、专业人员和检测技术的依赖,为BLV的临床检测提供了技术支持。
To establish an efficient,convenient,and specific recombinase-mediated isothermal amplification(RAA)rapid detection method based on the env structural gene of bovine leukemia virus(BLV),this study used DNAMAN for sequence alignment,constructed the recombinant plasmid pESI-env,designed and screened RAA primers and probes,established the reaction system,and further optimized the reaction protocol and primer-probe usage.The method's specificity,sensitivity,and reproducibility were evaluated,and the results were compared with enzyme-linked immunosorbent assay(ELISA)and quantitative PCR(qPCR)in clinical samples.The results showed that the optimal primer-probe combination was F4/R1/P.The method completed the reaction in 20 minutes.There was no cross-reaction with bovine viral diarrhea virus(BVDV),infectious bovine rhinotracheitis virus(IBRV),and bovine rotavirus(BRV).The minimum detection limit was 1.62×10^(1)copies/μL,and the method showed good repeatability.The positive detection rate of clinical samples using this method was slightly higher than that of both ELISA and qPCR standard methods.This study successfully established an efficient,convenient,and highly specific BLV RAA fluorescent detection method,which eliminates the reliance on expensive equipment,specialized personnel,and complex detection techniques,providing technical support for BLV clinical detection.
作者
侯欣怡
王建发
连帅
耿子健
武瑞
HOU Xinyi;WANG Jianfa;LIAN Shuai;GENG Zijian;WU Rui(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China;College of Biology and Agriculture,Jiamusi University,Jiamusi 154007,China)
出处
《中国兽医杂志》
北大核心
2025年第8期46-52,共7页
Chinese Journal of Veterinary Medicine
基金
国家自然科学基金项目(31972747)。
