仔猪先天性震颤瘟病毒RT-PCR检测方法的建立  

Establishment of RT-PCR for Detection of Atypical Porcine Pestivirus

作　　者：吴松松[1] 周荷田 张文波[1] 邓舜洲[1] 张志庆 杨丹凤 WU Song-song;ZHOU He-tian;ZHANG Wen-bo;DENG Shun-zhou;ZHANG Zhi-qing;YANG Dan-feng(College of Animal Science and Technology,Jiangxi Agricultural University Nanchang,Jiangxi,330045,China;Jiangxi JIN YI BO Biological Technology Co.Ltd.,Nanchang Jiangxi,330013,China)

机构地区：[1]江西农业大学动物科学技术学院,江西南昌330045 [2]江西金伊博生物科技有限公司,江西南昌330013

出　　处：《动物医学进展》2019年第4期28-31,共4页Progress In Veterinary Medicine

基　　金：江西省科技支撑计划项目(20121BBF60031);江西省生猪产业技术体系-疫病岗位项目(JXARS-03)

摘　　要：旨在建立检测仔猪先天性震颤瘟病毒(APPV)的逆转录-聚合酶链反应(RT-PCR)方法。根据GenBank上登录的APPV基因组序列,设计1对引物,以临床上表现典型震颤仔猪的淋巴结为材料,提取RNA,建立了RT-PCR方法,并进行敏感性、重复性和特异性试验,对其扩增产物测序比对分析;利用该方法,对临床病料进行检测。结果显示,从典型震颤仔猪的淋巴结中均扩增到与预期大小有一致的片段,经测序比对,扩增片段序列与APPV相应片段的核苷酸同源性在89%以上;该方法只能扩增出APPV片段,而其他几种常见猪病毒均为阴性,3次重复检测结果基本一致,可检测最低cDNA浓度为184.11ng/μL;对临床病料检测,典型病例的检出率达100%。试验结果表明,建立的RT-PCR方法具有良好的特异性、重复性、敏感性等优点,可用于临床APPV引起的仔猪先天性震颤早期检测、病毒鉴定和分子流行病学调查。The study aimed at establishing a RT-PCR method for the detection of piglet congenital tremor pestivirus(APPV).According to the APPV genome sequence in GenBand,one pair of primers were designed and RNA was extracted from the lymph nodes of clinically typical tremor piglets.The RT-PCR method was established and the sensitivity,repeatability and specificity were tested.The amplified products were sequenced and compared with the method used to detect the clinical materials.As a result,the amplified fragment was identical to the predicted fragment from the lymph nodes of typical tremor piglets,and the nucleotide sequence homology between the amplified fragment and APPV fragment was over 89%.APPV fragments were only amplified,and several other common swine viruses were negative.The results of three replicates were basically the same,the lowest detectable cDNA concentration was 184.11 ng/μL.The detection rate of clinical materials in typical cases was 100%.The experimental results showed that established RT-PCR method has the advantages of good specificity,repeatability and sensitivity,and can be used for early detection of congenital tremor,virus identification and molecular epidemiology of piglets infected with clinical APPV.

关 键 词：仔猪先天性震颤 瘟病毒 逆转录-聚合酶链反应 

分 类 号：S852.659.5[农业科学—基础兽医学] Q789[农业科学—兽医学]