鉴别RHDV与RHDV2荧光定量RT-PCR检测方法的建立与应用  

Establishment and application of a real-time RT-PCR assay for the differential detection of RHDV and RHDV2

作　　者：肖跃强[1] 孙培姣 周迎春[4] 陈萌萌[3] 崔平 杨慧 于雪 于新友 王芳[3] 沈志强[1,2] XIAO Yue-qiang;SUN Pei-jiao;ZHOU Ying-chun;CHEN Meng-meng;CUI Ping;YANG Hui;YU Xue;YU Xin-you;WANG Fang;SHEN Zhi-qiang(Shandong Binzhou Animal Science&Veterinary Medicine Academy,Binzhou 256601,China;Shandong Lvdu Bio-science&Technology Co.Ltd,Binzhou 256600,China;Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China;Anhui Provincial Center for Animal Diseases Control and Prevention,Hefei 230061,China;Binzhou Animal Husbandry and Veterinary Management Service Center,Binzhou 256600,China)

机构地区：[1]山东省滨州畜牧兽医研究院,山东滨州256600 [2]山东绿都生物科技有限公司,山东滨州256600 [3]江苏省农业科学院兽医研究所,江苏南京210014 [4]安徽省动物疫病预防与控制中心,安徽合肥230061 [5]滨州市畜牧兽医管理服务中心,山东滨州256600

出　　处：《中国预防兽医学报》2021年第5期512-520,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：滨州市科技发展计划项目(2015ZC0108);山东省自然科学青年基金项目(ZR2014CQ045)。

摘　　要：为建立一种敏感、特异、快速、操作简便、易于判定的鉴别检测兔出血症病毒(RHDV)与兔出血症病毒2型(RHDV2)荧光定量RT-PCR检测方法,本研究选择RHDV与RHDV2 VP60基因序列高度同源的保守区域设计了3对引物,经荧光定量PCR扩增比较,筛选出1对引物,对各反应条件进行优化,建立了RHDV与RHDV2荧光定量PCR检测方法,且基于RHDV较RHDV2扩增产物熔解曲线Tm值高1.57℃~2.74℃,能够达到鉴别检测的目的。特异性试验显示,该方法仅对RHDV和RHDV2检测为阳性,对猪瘟兔化弱毒、兔多杀性巴氏杆菌、A型魏氏梭菌、波氏杆菌检测均为阴性,特异性强。敏感性试验显示,该方法对RHDV与RHDV2 VP60基因重组质粒标准品的最低检测限均为1×10^(1)拷贝/μL,敏感性高;重复性试验显示,RHDV VP60基因质粒标准品批内和批间Cp值最大变异系数分别为1.91、3.60,Tm值最大变异系数分别小于0.72、0.48;RHDV2 VP60基因质粒标准品批内和批间Cp值最大变异系数分别为1.90、3.93,Tm值最大变异系数分别为0.29、0.17,均小于5%,重复性好。采集RHDV AV34株感染病死兔的肝脏、肺脏、脾脏、肾脏、心肌组织、内皮组织、口鼻粘液、肌肉组织、肛门排泄物、尿液等组织样品,进行病毒组织分布与排毒的检测,结果显示,各组织样品均为阳性,表明病毒在体内分布广泛,病毒可通过口鼻分泌物及粪尿排泄物排出。利用该方法检测临床肝组织样品,结果显示,RHDV AV34标准株阳性肝组织与14份RHDV临床肝组织样品、RHDV2分离株SC2020/04感染样品均为阳性,而常规RT-PCR方法对其中1份RHDV临床肝组织样品检测为阴性,两种方法符合率为93.3%。本研究建立了一种能够鉴别检测RHDV与RHDV2的荧光定量RT-PCR方法,为开展兔病毒性出血症的临床诊断、流行病学调查与防控等提供了有效的技术保障。In order to establish a sensitive,specific,rapid and easy operating real-time RT-PCR method that can detect RHDV and RHDV2 discriminately,3 pairs of primers were designed based on the highly homologous and conserved sequences of RHDV and RHDV2 VP60 genes.One pair of them was selected after comparative tests,and a real-time RT-PCR method was developed for RHDV and RHDV2 detection with the reaction conditions optimization.In this assay,RHDV and RHDV2 can be differentiated caused by the Tm values in melting curves of RHDV were higher than that of RHDV2 at a range of 1.57℃-2.74℃.Specificity tests showed that only RHDV and RHDV2 were detected to be positive,but hog cholera lapinization virus(HCLV),rabbit Pasteurella multocida,type A Clostridium wednerii and Bordetella bronchiseptica were all negative in this assay.Sensitivity tests showed a good result that the limit detection of this assay was 1×10^(1) copies/μL of recombinant plasmid.Repeatability and reproducibility test of this method were good,the maximum coefficients of variation of intra-assay and inter-assay Cp and Tm values of RHDV VP60 gene plasmid were 1.91 and 3.60,0.72 and 0.48,respectively,and that of RHDV2 VP60 gene plasmid were 1.90 and 3.93,0.29 and 0.17,respectively.The viral distribution and shedding were detected for the collected samples of liver,lung,spleen,kidney,myocardial tissue,endothelial tissue,mouth and nose mucus,muscle tissue,anal excrement,urine in the RHDV infected rabbits,and all of them were positive,it is indicated that the virus is widely distributed in the rabbit and the virus can be excreted through oral and nasal secretions,feces and urine excrement.Using this method,the RHDV AV34 strain and 14 RHDV field samples and RHDV2 field isolate SC2020/04 were detected to be positive,while 1 of 14 RHDV field samples were detected to be negative with the conventional RT-PCR method,the coincidence rate of these two methods was 93.3%.In this study,a real-time RT-PCR method for RHDV and RHDV2 universal and differential diagnosis was es

关 键 词：兔出血症病毒 兔出血症病毒2型 荧光定量RT-PCR 鉴别 

分 类 号：S852.65[农业科学—基础兽医学]