牛轮状病毒和牛冠状病毒双重TaqMan荧光定量PCR检测方法的建立及应用  

Establishment and application of a duplex TaqMan real-time PCR assay for simultaneous detection of bovine rotavirus and bovine coronavirus

作　　者：彭志豪 韩荞忆 崔明江 范葶莉 刘莹[1] 王梦佳 马玉忠[1] 左玉柱[1] 任玉红[1] 范京惠[1] PENG Zhi-hao;HAN Qiao-yi;CUI Ming-jiang;FAN Yu-li;LIU Ying;WANG Meng-jia;MA Yu-zhong;ZUO Yu-zhu;REN Yu-hong;FAN Jing-hui(College of Veterinary Medicine,Agricultural University of Hebei,Baoding 071001,China;Hebei Animal Husbandry Station,Shijiazhuang 050035,China;Cangzhou Vocational and Technical College,Cangzhou 061000,China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北省畜牧总站,河北石家庄050035 [3]沧州职业技术学院,河北沧州061000

出　　处：《中国预防兽医学报》2020年第10期1009-1013,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：河北省农业产业技术体系奶牛创新团队(HBCT2018120406);河北省农业产业技术体系肉牛创新团队(HBC T2018130405);河北省重点研发及计划(19226611D)。

摘　　要：为建立一种同时检测牛轮状病毒(BRV)和牛冠状病毒(BCV)的方法,本研究根据GenBank中登录的BRV VP6基因保守序列和BCV N基因保守序列设计引物和探针,通过方阵法优化体系后,建立了一种可以同时检测BRV和BCV的双重TaqMan荧光定量PCR方法。该方法与牛病毒性腹泻病毒、牛细小病毒、牛肠道病毒、牛传染性鼻气管炎病毒均无交叉反应,特异性强;敏感性试验结果显示,该方法对BRV和BCV重组质粒标准品的最低检测限分别为41.8拷贝/μL和3.55拷贝/μL,敏感性高;组内和组间变异系数均小于2%,表明该方法重复性较好。利用建立的该方法对本实验室于2019年3月~9月在河北省内采集的72份牛腹泻粪便样品进行检测,结果显示BRV的阳性率为50.0%(36/72),BCV的阳性率为75.0%(54/72),BRV和BCV共感染率为33.3%(24/72);而常规PCR的上述检测结果分别为47.2%(34/72)、70.8%(51/72),共感染率为29.1%(22/72),二者对BRV和BCV检测结果的阳性符合率分别为97.2%、96.8%,表明该方法可以用于临床样品的检测。本研究建立的双重TaqMan荧光定量PCR方法对BRV和BCV的检测和流行病学调查具有重要意义。In order to establish a rapid simultaneous detection and quantitative method for bovine rotavirus(BRV)and bovine coronavirus(BCV),primers and probes were designed based on the VP6 gene of the BRV and the N gene of BCV in GenBank.After optimizing the system by the square matrix method,a duplex TaqMan real-time PCR method for simultaneous detection of BRV and BCV was successfully established.This method has no cross-reaction with BVDV,BPV,BEV,and IBRV indicated that the method established in this study has strong specificity.The sensitivity results showed that the minimum detection limit of BCV and BRV were 41.8 copies/μL and 3.55 copies/μL,respectively.The intra-and inter-group coefficients of variation were less than 2%,indicating that the method was reproducible.The duplex TaqMan real-time PCR assay was used to detect 72 bovine diarrhea stool samples,the positive rate of BRV was 50.0%(36/72),the positive BCV was 75.0%(54/72)and the co-infection rate was 33.3%(24/72).However,the detection results of conventional PCR method for BRV was 47.2%(34/72)and BCV was 70.8%(51/72),and the co-infection rate was 29.1%(22/72).The coincidence rates of BRV and BCV was 97.2%and 96.8%respectively,which showed that this method could be used to detect clinical samples.The established duplex TaqMan real-time PCR assay has important implications for clinical pathogen detection and epidemiological investigation of BRV and BCV.

关 键 词：牛轮状病毒 牛冠状病毒 双重TaqMan荧光定量PCR 

分 类 号：S852.4[农业科学—基础兽医学]