鱼类类立克次氏体PCR检测方法的建立及初步应用  

Development and Primary Application of a PCR Assay for Detection of Piscirickettsia-like Organisms

作　　者：雷燕 张会军 王娟 张文文 唐绍林 肖洋 王雪鹏[3] 

机构地区：[1]广州利洋水产科技股份有限公司,广东广州510515 [2]广州金水动物保健品有限公司,广东广州510515 [3]山东农业大学动物科技学院,山东泰安271018

出　　处：《广东海洋大学学报》2015年第6期30-34,共5页Journal of Guangdong Ocean University

基　　金：山东省现代农业产业技术体系(SDAIT-19);泰安市科技发展计划项目(201440774);浙江省近岸水域生物资源开发与保护重点实验室开放基金项目(J2013006)

摘　　要：根据Gen Bank中类立克次氏体基因的保守序列,设计一对针对鱼类类立克次氏体PCR检测的特异性通用引物,通过对PCR扩增条件的优化,建立快速检测鱼类类立克次氏体的PCR方法,并用该方法对类立克次氏体阳性罗非鱼(Oreochromis nilotica)进行PCR扩增,结果得到与实验设计相符的390 bp的特异性扩增条带,而对健康罗非鱼、乌鳢(Ophiocephalus argus)、海鲈(Dicentrarchus labrax)、中华绒螯蟹(Eriocheir sinensis)及其他对照组的扩增结果为阴性。测序比对结果证实,该PCR方法检测结果准确,最低可检测出约1 pg的类立克次氏体质粒DNA;利用建立的PCR方法,对来自广东、云南、海南、天津、江苏、安徽、湖北等地的252份临床样品进行检测,共检出阳性样品30份,提示该PCR方法可用于类立克次氏体的临床快速检测。According to the published gene sequences of the piscirickettsia-like organisms in Gen Bank, a pair of specific primers was designed, and a rapid PCR method was established for detecting fish piscirickettsia-like organisms. The reaction parameters were optimized to develop the PCR method for detecting fish piscirickettsia-like organisms. The specific band of 390 bp were amplified from the positive samples, but no specific band was amplified from healthy Oreochromis nilotica, Ophiocephalus argus, Dicentrarchus labrax, Eriocheir sinensis and other control group. Sequencing analysis indicated that this method was accurate, and the minimal amount of DNA plasmid of piscirickettsia-like organisms that could be detected was 1 pg. 252 clinical fish samples from Guangdong, Yunnan, Hainan, Tianjin, Jiangsu, Anhui, Hubei were tested by the PCR, and 30 of which were positive. The results indicated that the PCR assay could be used for the detection of clinical fish samples.

关 键 词：鱼类类立克次氏体 PCR检测 临床应用 

分 类 号：S941.4[农业科学—水产养殖]