非典型性瘟病毒SYBR Green Ⅱ荧光定量RT-PCR检测方法的建立与应用  

Development and application of a SYBR Green Ⅱ real-time RT-PCR for detection of swine atypical pestivirus

作　　者：杨晓宇[1] 徐雷 殷鑫欢 张继宗 鲁令华 冯宇 朱玲[1,2] YANG Xiao-yu;XU Lei;YIN Xin-huan;ZHANG Ji-zong;LU Ling-hua;FENG Yu;ZHU Ling(College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China;Key Laboratory of Animal Disease and Human Health of Sichuan Province,Sichuan Agricultural University,Chengdu 611130,China)

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]四川农业大学动物疫病与人类健康四川省重点实验室,四川成都611130

出　　处：《中国预防兽医学报》2018年第12期1133-1136,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：四川省科技支撑计划项目(2017NZ0038);四川省"十三五"育种攻关项目(2016NYZ0052);国家"十二五"科技支撑计划(2015BAD12B04-2.3)

摘　　要：为建立一种快速检测猪非典型性瘟病毒(APPV)的方法,本实验建立了APPV SYBR Green Ⅱ荧光定量RT-PCR检测方法。该方法特异性试验结果显示,除APPV外,猪繁殖与呼吸综合征病毒、猪瘟病毒、猪流行性腹泻病毒和猪轮状病毒等常见猪病病原检测均为阴性,表明其特异性良好;该方法最低检测限为2.8×10~3拷贝/μL,敏感性较高;重复性试验的组内、组间变异系数均小于1%。利用该方法对临床56份来自四川各地的临床病料样品进行检测,检出5份阳性样品,且与常规PCR检测方法的阳性符合率为100%。本研究建立的APPV荧光定量RT-PCR检测方法为APPV临床检出以及后期并发症的研究奠定了基础。In order to establish a rapid detection method for porcine atypical pestivirus (APPV), the APPV SYBR Green II real-time RT-PCR detection method was established with a pair of specific primers for APPV. The assay was specific for APPV detection. The method showed a linear relationship from 2.8×103 copies/μL to 2.8×108 copies/μL of APPV cDNA. The intra- and inter-group coefficients of variation were both less than 1%. In addition, 56 clinical samples from various areas of Sichuan were tested and 5 positive samples were detected. The positive coincidence rate with the conventional PCR detection method was 100%. The results showed that the APPV real-time RT-PCR detection method established in this study had high specificity and sensitivity for the detection of APPV, which laid the foundation for the clinical detection of APPV and the study of late stage of the disease.

关 键 词：非典型性瘟病毒 荧光定量RT-PCR SYBR Green Ⅱ 

分 类 号：S852.65[农业科学—基础兽医学]