重组酶介导等温扩增技术检测鰤鱼诺卡氏菌方法的建立  

Establishment of a recombinase-aided amplification(RAA)assay for detection of Nocardia seriolae

作　　者：杨惠源 陈国权 温依铭 夏洪丽[1,2] 夏立群 YANG Huiyuan;CHEN Guoquan;WEN Yiming;XIA Hongli;XIA Liqun(Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy Culture,College of Fisheries,Guangdong Ocean University,Zhanjiang 524088,China;Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment,Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedlings,Shenzhen Institute of Guangdong Ocean University,Shenzhen 518120,China)

机构地区：[1]广东海洋大学水产学院,广东省水产动物病害防控与健康养殖重点实验室,广东湛江524088 [2]广东海洋大学深圳研究院,广东省水生动物健康评估工程技术研究中心,深圳海水经济动物种苗健康评价公共技术服务平台,广东深圳518120

出　　处：《大连海洋大学学报》2023年第1期104-111,共8页Journal of Dalian Ocean University

基　　金：国家重点研发计划“蓝色粮仓科技创新”项目(2020YFD0900201);广东省自然科学基金(2021A1515011222);广东省重点领域研发计划项目(2020B0202010009);深圳大鹏新区科技创新和产业发展专项(KJYF202001-08)。

摘　　要：为建立一种基于重组酶介导等温扩增技术(recombinase-aided amplification,RAA)的快速检测鰤鱼诺卡氏菌(Nocardia seriolae)的方法,针对鰤鱼诺卡氏菌的特异性基因组片段,设计重组酶介导扩增引物,通过检测7株鰤鱼诺卡氏菌近缘菌和9种水产养殖过程中常见病原菌,筛选出特异性RAA检测引物,并通过将含有目的片段的重组质粒进行梯度稀释以分析该检测方法的灵敏度,最后采用RAA方法检测患病鱼、健康鱼及11种不同来源的鰤鱼诺卡氏菌菌株,以评价该检测方法的应用性和覆盖度。结果表明:本研究中设计的引物仅针对鰤鱼诺卡氏菌可以扩增出目的条带,检测灵敏度为100 pg/μL,人工感染鰤鱼诺卡氏菌的杂交鳢(Channa maculate♀×C.argus♂)肝脏、体肾和脾脏等组织基因组DNA能够扩增出阳性条带,不同来源的鰤鱼诺卡氏菌菌株可以扩增出阳性条带。研究表明,本研究中所建立的鰤鱼诺卡氏菌重组酶介导等温检测方法具有准确、简便的特点,可对鰤鱼诺卡氏菌进行快速检测。To establish a recombinase-aided amplification(RAA)assay for the detection of Nocardia seriolae,the primers for RAA were designed according to the specific genome fragments of N.seriolae with intraspecific conservation and interspecific variation.The specific primer of RAA was verified by detecting 7 related Nocardia spp.and 9 common pathogens in aquaculture,and the sensitivity of the detection method was analyzed by gradient dilution of the recombinant plasmid containing the target fragment.The reliability and coverage of the RAA assay were assessed in diseased and healthy fish as well as several N.seriolae strains from different sources.Results showed the RAA assay established in this study specifically detected N.seriolae,and the detection sensitivity was 100 pg/μL.The positive bands were amplified in the tissues including liver,kidney,and spleen of artificially infected hybrid snakehead(Channa maculate♀×C.argus♂),and the N.seriolae strains of different sources.The finding indicates that the RAA assay established in this study is rapid and convenient and can meet the needs for the rapid detection of N.seriolae.

关 键 词：鰤鱼诺卡氏菌 重组酶介导等温扩增(RAA) 快速检测 

分 类 号：S943[农业科学—水产养殖]