基于酶介导双重指数扩增技术(EmDEA)的小火蚁快速检测方法的开发  

Development of a rapid detection method for Wasmannia auropunctata based on enzyme-mediated duplex exponential amplification(EmDEA)

作　　者：韩蕊 滕祎 李献锋 王临静 魏霜 张雍哲 乔曦 刘海军 吴尧 马骏 万方浩 刘聪辉 钱万强 HAN-Rui;TENG Yi;LI Xian-Feng;WANG Ling-Jing;WEI Shuang;ZHANG Yong-Zheng;QIAO Xi;LIU Hai-Jun;WU Yao;MA Jun;WAN Fang-Hao;LIU Cong-Hui;QIAN Wan-Qiang(College of Plant Health&Medicine,Qingdao Agricultural University,Shandong Engineering Research Center for Environment-Friendly Agricultural Pest Management,Shandong Province Laboratory for Biological invasions and Ecological Security,China-Australia Joint Institute of Agricultural and Environmental Health,Qingdao 266109,Shandong Province,China;Agricultural Genomics institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen 518120,Guangdong Province,China;Guangzhou Customs Technical Center,Guangzhou 510623,China;Suzhou Jingrui Technology Co.,LTD.,Suzhou 215000,Jiangsu Province,China)

机构地区：[1]青岛农业大学植物医学学院,山东省环境友好型农业有害生物防治工程技术研究中心,山东省生物入侵与生态安全高校特色实验室,中澳农业与环境健康联合研究院,山东青岛266109 [2]岭南现代农业科学与技术广东省实验室深圳分中心,农业农村部农业基因数据分析重点实验室,中国农业科学院(深圳)农业基因组研究所,广东深圳518120 [3]广州海关技术中心,广州510623 [4]苏州晶睿科技有限公司,江苏苏州215000

出　　处：《环境昆虫学报》2024年第5期1059-1067,共9页Journal of Environmental Entomology

基　　金：国家重点研发计划(2022YFC2601500)。

摘　　要：小火蚁Wasmannia auropunctata是危害性最严重的入侵蚂蚁之一,对动植物、生态、经济甚至是人类都容易造成严重影响,是我国的重大检疫害虫之一。本研究为实现小火蚁的现场快速精准检测,通过自研程序筛选出小火蚁全基因组中的特异性片段,具有更高的特异性,基于酶介导双重指数扩增技术(EmDEA)筛选了适用的引物、RNA探针,全部反应所需时间小于20 min,并验证了其特异性和灵敏度。结果显示这种方法可在短时间内达到极高检测灵敏度与特异性,最低检出限约为4 ng。这种方法极大程度降低对设备的依赖,简单、快速、特异,为小火蚁的基层检测与现场诊断提供技术支持。Wasmannia auropunctata is one of the most harmful invasive ants,posing severe threats to flora,fauna,ecosystems,economies,and even humans.It is a significant quarantine pest in China.To achieve rapid and precise on-site detection of the W.auropunctata,this study screened specific segments from the whole genome of the W.auropunctata through self-developed program,ensuring higher specificity.The study also selected suitable primers and RNA probes based on the enzyme-mediated duplex exponential amplification(EmDEA).The total reaction time required was less than 30 minutes,and both specificity and sensitivity were validated.Results demonstrate that this method could achieve extremely high detection sensitivity and specificity in a short time,with a minimum detection limit of approximately 4 ng.This method significantly reduces the dependence on equipment,making it simple,rapid,and specific,thereby providing technical support for grassroots detection and on-site diagnosis of the W.auropunctata.

关 键 词：小火蚁 酶介导双重指数扩增技术(EmDEA) 特异性引物 RNA探针 快速鉴定 精准检测 

分 类 号：Q968.1[生物学—昆虫学] S433[农业科学—农业昆虫与害虫防治]