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重组酶聚合酶扩增技术快速检测鳗利斯顿氏菌 被引量:2

Rapid Detection of Listonella anguillarum by Recombinase Polymerase Amplification Technique
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摘要 鳗利斯顿氏菌(Listonella anguillarum)是一种常见的病原体,能感染多种养殖动物,每年给水产养殖业带来巨大的经济损失。随着集约化养殖模式在水产养殖业的大规模应用,水产病原体的传播速度也大幅度提高。为此,迫切需要建立一种快速、准确的检测技术。本研究以鳗利斯顿氏菌的金属蛋白酶基因(zinc metalloprotease gene,emp A)为靶基因,设计多对引物和1条exo探针,通过优化反应体系和条件,建立了一种基于重组酶聚合酶扩增(recombinase polymerase amplification,RPA)技术的快速实时检测鳗利斯顿氏菌的方法。结果表明,建立的Real-time RPA检测技术能够在20 min内特异地检测出鳗利斯顿氏菌,与其他7种常见水产致病菌均不发生交叉反应。该检测方法最低能够检测出1 pg/μL鳗利斯顿氏菌基因组模板,对人工污染的样品最低检测限为3.4×10~2CFU/m L,且重复性良好。因此,利用本研究建立的Real-time RPA检测技术,能特异、准确、高效地检测出鳗利斯顿氏菌,而且操作简单、耗时短,为水产养殖中鳗利斯顿氏菌的快速诊断及商业试剂盒的开发提供科学依据和技术参考。 Listonella anguillarum has emerged as a serious pathogen causing significant mortalities in various fish and shellfish species over a wide geographical area.With the large-scale application of intensive farming model in aquaculture,the speed of the transmission of aquatic pathogens has been greatly improved.Cultivation of Listonella anguillarum is seen as the gold standard for detection,although it is very time consuming and labour intensive.Accordingly,the establishment of practical and effective diagnostic methods has long been desired.In this study,several pairs of primers and an exo-probe targeting the zinc metalloprotease gene(empA) of Listonella anguillarum was designed and a rapid real-time detection method for Listonella anguillarum based on recombinase polymerase amplification(RPA) was established by optimizing the reaction system and conditions.The results showed that the established Real-time RPA technique could detect Listonella anguillarum directly within 20 min and did not cross-react with other seven common aquatic pathogenic bacteria.The detection limit of Real-time RPA for gDNA of Listonella anguillarum was 1 pg/μL,and reproducibility was good.However,the lowest detection limit of artificial pollution sample was 3.4×10~2 CFU/mL.It is expected to be the routine which can provide scientific basis and technical reference for the development of rapid diagnostic kit and commercial aquaculture Listonella anguillarum.
作者 庞建虎 乔龙亮 黄海龙 党晨阳 朱鹏 徐继林 PANG Jian-Hu1, QIAO Long-Liang1, HUANG Hai-Long1, DANG Chen-Yang1, ZHU Peng1,2 ,XU Ji-Lin1( 1 School of Marine Sciences, Ningbo University, Ningbo 315211, China; 2 Ningbo Institute of Oceanography, Ningbo 315832, China)
出处 《农业生物技术学报》 CSCD 北大核心 2018年第6期1056-1063,共8页 Journal of Agricultural Biotechnology
基金 国家现代农业产业技术体系建设专项资金(No.CARS-47) 宁波市科技攻关项目(No.2017C110003) 浙江省公益技术应用研究(No.2017C33133) 宁波大学添路职业发展基金学生培育项目
关键词 鳗利斯顿氏菌 RPA 检测 金属蛋白酶基因(empA) Listonella anguillarum RPA Detection Zinc metalloprotease gene (empA)
作者简介 通讯作者,zhupeng@nbu.edu.cn
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