摘要
【目的】建立牛病毒性腹泻病毒(BVDV)、牛冠状病毒(BCoV)、牛轮状病毒(BRV)、牛恶性卡他热病毒(MCFV)4种致牛腹泻病毒多重荧光定量PCR检测方法,为BVDV、BCoV、BRV和MCFV的鉴别诊断提供技术支持。【方法】根据BVDV的5′UTR基因、BCoV的N基因、BRV的VP6基因和MCFV的ORF9基因的保守区域设计引物,克隆目的基因构建标准阳性重组质粒,建立可同时检测4种致牛腹泻病毒的多重荧光定量PCR检测方法,并对该方法的特异性、敏感性和重复性进行评价。用建立的多重荧光定量PCR检测方法对76份临床样品进行检测,并与常规PCR检测方法的结果进行比较。【结果】建立了BVDV、BCoV、BRV和MCFV多重荧光定量PCR检测方法,该方法建立的标准曲线线性关系良好,仅可特异性扩增出4种目标基因片段;对BVDV、MCFV、BRV和BCoV的最低检出限分别为9.4×10^(1),1.4×10^(3),9.1×10^(1)和1.2×10^(2)拷贝/μL,与普通PCR相比,灵敏性高出10~1000倍;批内、批间差异均小于5%。76份临床样品检测结果显示,多重荧光定量PCR检测结果与普通PCR检测结果的符合率分别为98.7%(BCoV)、97.4%(BRV)、100.0%(BVDV)和100.0%(MCFV)。【结论】建立了BVDV、BCoV、BRV和MCFV多重荧光定量PCR检测方法,该方法特异性强、灵敏度高、重复性好,可用于牛腹泻病毒病原的实验室检测。
【Objective】With the vigorous development of cattle industry,the problem of bovine diarrhea caused by viruses is becoming more and more serious and seriously restricts the healthy development of cattle industry.In this study,a multiplex fluorescent quantitative PCR detection method for bovine diarrhea viruses of BVDV,BCoV,BRV and MCFV was established.【Method】Primers were designed according to the conserved regions of the 5′UTR gene of BVDV,the N gene of BCoV,the VP6 gene of BRV and the ORF9 gene of MCFV,and the target gene was cloned to construct a standard positive recombinant plasmid.A multiplex fluorescent quantitative PCR detection method was established to simultaneously detect 4 bovine diarrhea viruses.The specificity,sensitivity and repeatability of the method were evaluated.A total of 76 clinical samples were detected with the established method,and the results were compared with those of conventional PCR.【Result】A multiplex fluorescent quantitative PCR assay for BVDV,BCoV,BRV and MCFV was established.The standard curve established by this method had a good linear relationship and only 4 target gene fragments could be amplified specifically.The lowest detection limits for BVDV,MCFV,BRV and BCoV were 9.4×10^(1),1.4×10^(3),9.1×10^(1) and 1.2×10^(2) copies/μL,respectively.The sensitivity was 10-1000 times higher than that of ordinary PCR.The intra batch and inter batch differences were less than 5%.The results of 76 clinical samples showed that the coincidence rates of multiplex fluorescent quantitative PCR and common PCR were 98.7%(BCoV),97.4%(BRV),100.0%(BVDV)and 100.0%(MCFV),respectively.【Conclusion】A multiplex fluorescence quantitative PCR for detection of BVDV,BCoV,BRV and MCFV was established,the method has strong specificity,high sensitivity and good repeatability,and can be used for laboratory detection of bovine diarrhea virus.
作者
高睿
徐伟
罗艳
谢晓刚
李梦磊
张琪
GAO Rui;XU Wei;LUO Yan;XIE Xiaogang;LI Menglei;ZHANG Qi(Department of Animal Engineering,Yangling Vocational&Technical College/Engineering Research Center of Animal Disease Prevention and Control Universities of Shaanxi Province,Yangling,Shaanxi 712100,China;Animal Epidemic Control Center of Binzhou City,Shaanxi Province,Binzhou,Shaanxi 713500,China;College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China)
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2023年第10期20-28,共9页
Journal of Northwest A&F University(Natural Science Edition)
基金
陕西省重点研发计划一般项目(2022NY-098)。
关键词
牛腹泻病
牛病毒性腹泻病毒
牛冠状病毒
牛恶性卡他热病毒
牛轮状病毒
多重荧光定量PCR检测
bovine diarrheal disease
bovine viral diarrhea virus
bovine coronavirus
bovine malignant catarrhal fever virus
bovine rotavirus
multiple fluorescence quantification PCR
