直接多重TaqMan qPCR方法快速鉴定褐飞虱属3种飞虱  

Direct Multiplex TaqMan qPCR Assay for Rapid Detection of Three Sibling Species from Nilaparvata Distant

作　　者：罗举[1] 杨素文 贝文勇 余军伟 唐健[1] 刘淑华 LUO Ju;YANG Suwen;BEI Wenyong;YU Junwei;TANG Jian;LIU Shuhua(China National Rice Research Institute,Hangzhou 311401,China;Agricultural Comprehensive Service Center,Niumashi Town,Shaodong City,Hunan Province,Shaoyang 422803,China;Plant Protection and Inspection Station of Zhaoping County,Guangxi Zhuang Autonomous Region,Hezhou 546800,China;Hangzhou Ustar Biotechnology Co.,Ltd.,Hangzhou 310015,China)

机构地区：[1]中国水稻研究所,杭州311401 [2]湖南省邵东市牛马司镇农业综合服务中心,湖南邵阳422803 [3]广西昭平县植保植检站,广西贺州546800 [4]杭州优思达生物技术有限公司,杭州310015

出　　处：《中国水稻科学》2023年第3期329-336,共8页Chinese Journal of Rice Science

基　　金：浙江省自然科学基金资助项目(LGN21C140002);十四五重点研发计划资助项目(2021YFD1401100);水稻生物学国家重点实验室开放课题(20210303);中央级公益性科研院所基本科研业务费专项(CPSIBRF-CNRRI-202123)。

摘　　要：【目的】褐飞虱(Nilaparvata lugens)是水稻重大害虫,灯光诱捕一直是褐飞虱监测的重要方法。然而,褐飞虱的两个同属近似种伪褐飞虱(N.muiri)和拟褐飞虱(N.bakeri)也具有扑灯行为,常被误判为褐飞虱。针对测报灯下褐飞虱属近似种形态鉴定费时费力、专业技能要求高,伪褐飞虱和拟褐飞虱常被误判为褐飞虱这一问题,拟建立一种褐飞虱属3种飞虱的快速分类鉴定方法。【方法】以条形码基因ITS1为靶标,筛选褐飞虱属通用引物及物种特异性探针,建立并优化直接多重Taqman qPCR检测体系(Direct Multiplex TaqMan quantitative PCR,dmTqPCR),并分析其特异性、灵敏度及实用性。【结果】本研究建立的3种飞虱dmTqPCR检测方法特异性强,可准确区分褐飞虱、伪褐飞虱和拟褐飞虱;灵敏度高,检出限可达10拷贝/反应;大样本检测结果表明,382个样本从样本获取到结果输出的整个过程可在3 h内完成,检出率及准确率均为100%。【结论】本研究建立的褐飞虱属近似种的dmTqPCR鉴定方法可以用于褐飞虱、伪褐飞虱和拟褐飞虱的快速分类鉴定,有利于灯下褐飞虱的精准测报。【Objective】Nilaparvata lugens is a destructive insect pest on rice,and light trapping has always been an important monitoring method.However,its two sibling species,N.muiri and N.bakeri,can also be trapped by light and are easily mistaken as N.lugens.Morphological identification of N.lugens and its sibling species is time-consuming and labor-intensive,with the requirement for expertise and experience.To address this problem for identification of N.lugens and its two sibling species,the present study is aimed to develop a rapid and accurate identification method.【Method】At first,design and select the interspecies general primes and species-specific probes based on the barcoding gene ITS1.Then,establish and optimize the Direct Multiple Taqman qPCR assay(dmTqPCR).Finally,analyze and evaluate the specificity,sensitivity and practicability.【Results】The dmTqPCR assay developed in the present study has high specificity and high sensitivity,which could accurately identify N.lugens,N.bakeri and N.muiri,and the LOD(limit of detection)are up to 10 copies/reaction.The entire detection process for 382 specimens,including the crude tissue liquid preparation,amplification and detection,can be completed in 180 min with an accuracy of 100%.【Conclusion】The established dmTqPCR assay is suitable for rapid and accurate identification of N.lugens,N.muiri and N.bakeri,and it is expected to promote the development of precision prediction and forecasting of N.lugens.

关 键 词：褐飞虱 伪褐飞虱 拟褐飞虱 ITS1 直接多重Taqman qPCR 

分 类 号：S435.112.3[农业科学—农业昆虫与害虫防治]