鉴别猪流行性腹泻病毒ORF3基因截短株常规PCR及双重荧光PCR方法的建立及初步应用  

Development and preliminary application of regular PCR and duplex fluorescent PCR method for detection of porcine epidemic diarrhea virus truncated ORF3 gene

作　　者：杜琛 廖梦娟 曾德平 唐桂浩 闭云贵 柏家果 陆颖 速雪丽 陈樱[1] 韦祖樟[1] 黄伟坚[1] 欧阳康[1] DU Chen;LIAO Mengjuan;ZENG Deping;TANG Guihao;BI Yungui;BAI Jiaguo;LU Ying;SU Xueli;CHEN Ying;WEI Zuzhang;HUANG Weijian;OUYANG Kang(Key Laboratory of Animal Infectious Diseases and Molecular Immunology,College of Animal Science and Technology,Guangxi University,Nanning 530005,China)

机构地区：[1]广西大学,动物科学技术学院动,物传染病与分子免疫学实验室,广西南宁530005

出　　处：《中国兽医学报》2022年第9期1737-1743,共7页Chinese Journal of Veterinary Science

基　　金：广西自然科学基金资助项目(2021GXNSFAA196067)。

摘　　要：猪流行性腹泻(PED)是由猪流行性腹泻病毒(PEDV)引起的一种以水样腹泻、呕吐和脱水为主要特征的猪急性肠道传染病。PEDV细胞适应性毒株与其他毒株之间的区别主要发生在ORF3基因,该基因的缺失可作为病毒适应细胞的标志。本实验室前期分离获得了1株广西自然截短的PEDV毒株17GXCZ-1ORF3d,为更好地鉴别不同类型毒株,本研究建立了可鉴别PEDV ORF3基因自然截短株的常规PCR以及双重荧光PCR检测方法。结果显示,2种方法均能鉴别PEDV ORF3基因截短株,且特异性好。另外,双重荧光PCR的检测下限为10^(0.59) PFU/mL,比所建立的常规PCR方法灵敏100倍,敏感性高。对收集的221份临床样品进行初步应用,常规PCR检出126份阳性样品,其中鉴别出14份ORF3基因截短株单独感染样品,而双重荧光PCR检出阳性样品145份,同样鉴别出14份ORF3基因截短株单独感染样品。本研究建立的2种PCR方法特异性强、灵敏度高、重复性好,对PEDV ORF3基因截短株的快速鉴别与检测具有重要意义。Porcine epidemic diarrhea(PED)caused by porcine epidemic diarrhea virus(PEDV)is an acute intestinal infectious disease of pigs that is characterized by watery diarrhea,vomiting and dehydration.The difference between the cell adapted strains and other strains mainly occurred in ORF3 gene,the deletion of which could be the marker of adaptation to cell culture.PEDV Guangxi naturally truncated strains,17GXCZ-1ORF3d,were isolated in previous study.To better identify the different types of PEDV strains,the regular PCR and duplex fluorescence PCR detection meth-ods were established based on this strain in this study.The results showed that the methods were specific for detecting PEDV truncated strains.The lower limit of duplex fluorescence PCR was 10^(0.67) PFU/mL,which was 100 times more sensitive than regular PCR.The detection results of 221 clinical samples showed that 145 positive samples including 14 truncated ORF3 gene samples alone were detected by duplex fluorescence PCR,while 126 positive samples were detected by regular PCR,with a consistent detection about truncated ORF3 gene samples alone.The duplex fluorescence PCR method established in this study is considered to be a high specificity,sensitivity and repeatability tool for the rapid identification and detection of PEDV naturally truncated strains.

关 键 词：猪流行性腹泻病毒 双重荧光PCR ORF3基因 自然截短毒株 鉴别诊断 

分 类 号：S855.3[农业科学—临床兽医学]