摘要
水牛匈爱病毒(BufHuV)是2021年在广西水牛中新发现的一种小核糖核酸病毒,本研究旨在建立一种可用于临床快速且特异检测牛匈爱病毒的方法。基于GenBank上公布的bovine hunnivirus、ovine hunnivirus和BufHuV的5′UTR保守区设计了1对特异性引物,首次建立了检测牛匈爱病毒的SYBRGreenⅠ实时荧光定量PCR方法。结果表明,标准曲线在1.1×10^(8)copies/μL~1.1×10^(3)copies/μL质粒标准品稀释浓度范围内呈线性关系,斜率为-3.2657,相关系数为0.9998,熔解曲线为单峰,检测下限为1.1×10^(1)copies/μL,比常规PCR敏感性高100倍;特异性高,与多种常见的牛病病毒均无交叉反应;重复性好,批内和批间变异系数均小于1.0%。收集了257份有腹泻迹象的牛粪样品,用于对所建立平台的性能评估,结果,建立的实时荧光定量PCR的阳性检出率为3.89%,高于普通PCR的阳性检出率(1.56%)。上述结果表明,该方法将为牛匈爱病毒的临床样品检测、流行病学监测提供一种快速、可靠、低成本的替代方案,也为后续研究奠定了基础。
Water buffalo hunnivirus(Buf Hu V)is a picornavirus newly discovered in Guangxi buffalo in 2021,the aim of this study was to develop a rapid and specific method for clinical detection of Buf Hu V.Based on the 5′UTR conserved regions of bovine hunnivirus,ovine hunnivirus and Buf Hu V published on GenBank,a pair of specific primers were designed,and the SYBR GreenⅠreal-time quantitative PCR for the detection of Buf Hu V was first established.The results showed that the standard curve was linear in the range of dilution concentration from 1.1×10^(8)copies/μL to 1.1×10^(3)copies/μL,and the slope was-3.2657with the correlation coefficient of 0.9998,the dissolution curve was a single peak,and the lower limit of detection was 1.1×10^(1)copies/μL,which is 100 times more sensitive than ordinary PCR.The method is high specificity which had no cross-reaction with many common bovine disease viruses.The variation coefficients of intra-assay and inter-assay were both less than 1.0%,indicating good repeatability.A total of 257 fecal samples with signs of diarrhea were collected and used to evaluate the performance of the platform.The positive rate of real-time quantitative PCR was 3.89%,which was higher than that of common PCR(1.56%).This method will provide a rapid,reliable and low-cost alternative for clinical sample detection and epidemiological surveillance of hunnivirus,and lay a theoretical foundation for follow-up studies.
作者
朱鑫玥
董覃婷
罗宇航
张广欣
乔成鹏
黄稳妃
邹延林
陈樱
欧阳康
韦祖樟
苏亚权
潘艳
黄伟坚
ZHU Xin-yue;DONG Qin-ting;LUO Yu-hang;ZHANG Guang-xin;QIAO Cheng-peng;HUANG Wen-fei;ZOU Yan-lin;CHEN Ying;OUYANG Kang;WEI Zu-zhang;SU Ya-quan;PAN Yan;HUANG Wei-jian(College of Animal Science and Technology,Guangxi University,Nanning 530005,China;Guangxi Agricultural Vocational University,Nanning 530007,China)
出处
《中国兽医科学》
CAS
CSCD
北大核心
2022年第10期1252-1258,共7页
Chinese Veterinary Science
基金
国家重点研发计划项目(2021YFD1100100)
国家现代农业产业体系广西牛羊产业创新团队建设项目(nycytxgxcxtd-2021-09)
