PCR结合斑点杂交技术检测鹦鹉喙羽病病毒方法的建立及应用  

Establishment and Application of PCR Combined with Nucleic Acid Dot Blot for Detection of PBFDV

作　　者：于晓慧 田似报 王琳 崔治中[3] 常爽[3] 赵鹏[3] 李阳 Yu Xiaohui;Tian Sibao;Wang Lin;Cui Zhizhong;Chang Shuang;Zhao Peng;Li Yang(China Animal Health and Epidemiology Center, Qingdao, Shandong 266114, China;Sino-scienci Gene Technology Co., Ltd, Beijing 100190, China;College of Veterinary Medicine, ShandongAgricul~ral University, Tai'an, Shandong 271018, China)

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266114 [2]北京中科基因技术有限公司,北京100190 [3]山东农业大学动物医学院,山东泰安271018

出　　处：《中国动物检疫》2018年第11期66-70,共5页China Animal Health Inspection

基　　金：国家重点研发计划项目(2016YFC1201304)

摘　　要：鹦鹉喙羽病(PBFD)是鹦鹉目前最常见的疾病,对鹦鹉养殖业危害极其严重。根据鹦鹉喙羽病毒(PBFDV)基因片段的克隆和序列分析,设计合成1对特异性引物,以CP基因为模板,经PCR扩增获得830 bp的核苷酸DNA,并用DIG标记DNA,制备用于检测PBFDV的特异性核酸探针。用该核酸探针对疑似感染PBFDV的鹦鹉病料进行斑点杂交检测,并对鉴定为阳性的PBFDV进行全基因组扩增和测序分析。结果显示,利用PCR结合斑点杂交技术检测PBFDV,特异性强、敏感度高,具有可重复性。鉴定为阳性的2株PBFDV全基因组序列之间同源性为100%,与已报道序列的同源性为81.5%～98.9%。本研究为我国开展PBFDV感染的分子流行病学调查和临床诊断提供了一种敏感、特异的检测方法。Psittacine beak and feather disease (PBFD) is the most common disease in parrots and seriously threatens the parrot farming industry. In this article, according to the analysis of PBFDV gene fragment, a pair of specific primers were designed and synthesized. Taking the CP gene as the template, a DNA band with the length of 830 bp was amplified by PCR, and the obtained DNA samples were labeled with DIG and used to prepare a specific probe for detecting PBFDV. Using the nucleic acid probe, the suspicious parrot samples were tested by dot blot hybridization, then whole genome amplification and sequence analysis were carried out towards virus isolated from samples which were identified as PBFDV-positive. Results showed that, the PCR combined with dot blot hybridization method was specific, sensitive and repeatable in PBFDV detection. In addition, the homology of whole genome sequences of two PBFDV isolates were 100%, compared with other reported PBFDV strains, the homology rang was 81.5% to 98.9%. In conclusion, this study would provide a sensitive and specific method for molecular epidemiological investigation and clinical diagnosis of PBFDV infection in China.

关 键 词：鹦鹉喙羽病 核酸斑点杂交 全基因组 遗传进化 

分 类 号：S852.65[农业科学—基础兽医学]