快速诊断小鹅瘟PCR方法的优化建立  

Establishment and Optimization of PCR for Rapid Detection of Gosling Plague

作　　者：赵磊[1] 王旋[1] 龚争[1] 吴章青 张训海[1] 

机构地区：[1]安徽科技学院家禽疫病防控监测安徽省重点实验室,安徽凤阳233100

出　　处：《安徽科技学院学报》2014年第6期5-9,共5页Journal of Anhui Science and Technology University

基　　金：安徽省农业产业技术体系家禽产业;安徽科技学院自然科学一般项目(ZRC2013381)

摘　　要：为建立快速诊断小鹅瘟的PCR方法,根据已发表的GPV主要结构蛋白VP3基因序列,经多序列比对和Oligo 6.0软件分析设计特异性PCR引物。以微量法提取病鹅肝组织DNA,优化PCR反应引物退火温度、循环次数和鉴定其特异性,并与酚氯仿法和煮沸法进行同步对比研究。结果显示,建立的PCR检测方法能够特异性检测病鹅肝组织中GPV的核酸,其扩增片段大小为343bp,最佳退火温度为58℃,最佳循环次数为35次。微量法提取的病毒核酸PCR检测灵敏度分别是酚氯仿法的50倍和煮沸法的2500倍。该PCR检测方法不与鹅副黏病毒、禽流感病毒、传染性支气管炎病毒、鸭瘟病毒以及健康鹅肝组织发生交叉反应。本研究建立的GPV PCR检测方法能够快速诊断小鹅瘟。To establish PCR method for rapid detection of the pathogen of gosling plague （GP）, according to the published major structural protein VP3 gene sequence of goose parvovirus, specific PCR primers for detection of GPV were designed by multiple sequence alignment analysis and Oligo 6.0. Viral DNA in the liver of goose was extracted by method of mini - extraction. And annealing temperature and the number of cycles of PCR were been optimized. Specificity of the PCR method was been identified. The study was synchronized and compared with phenol - chloroform method and boiling method. The results showed that the established PCR assays could ampli- fy fragment size of 343bp from the pathogen of GPV in the liver of goose, and its optimal annealing temperature was 58~C, its optimum number of PCR cycles was 35 times. The PCR sensitivity of viral nucleic acid extracted by mini - extraction method was 50 times of phenol - chloroform method and 2,500 of times. The rapid PCR as- say didn＇t react with Newcastle Disease virus, Avian Influenza virus, Infectious Bronchitis virus, Duck Plague virus and liver of health goose. This study established PCR method for rapid diagnosis of clinical gosling plague.

关 键 词：小鹅瘟 PCR 快速诊断 

分 类 号：S852.659.2[农业科学—基础兽医学]