重大检疫性害虫玉米根萤叶甲的种特异性SS-CO Ⅰ快速检测技术研究  

Rapid identification of the important quarantine pest Diabrotica virgifera virgifera by SS-COⅠ

作　　者：张桂芬[1,2] 王玉生 郭建洋[1,2] 冼晓青[1,2] 万方浩 张金良[3] 王福莲 张亚宁[4] ZHANG Guifen;WANG Yusheng;GUO Jianyang;XIAN Xiaoqing;WAN Fanghao;ZHANG Jinliang;WANG Fulian;ZHANG Yaning(State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection,Chinese Academy of Agricultural Sciences, Beijing 100193, China;Center for Management of InvasiveAlien Species, Ministry of Agriculture, Beijing 100193, China;Beijing Plant Protection Station,Beijing 100029, China;College of Agronomy, Yangtze University, Jingzhou 434100, China)

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100193 [2]农业部外来入侵生物预防与控制研究中心,北京100193 [3]北京市植物保护站,北京100029 [4]长江大学农学院,荆州434100

出　　处：《植物保护》2019年第1期109-115,134,共8页Plant Protection

基　　金：国家重点研发计划(2017YFC1200600;2016YFC1201200);农业部"948"项目(2016-X48)

摘　　要：玉米根萤叶甲在欧洲和美国是一种严重为害玉米的入侵性害虫,传播速度快,侵入我国的可能性极高。本文针对其难以快速准确进行形态鉴别的问题,以玉米根萤叶甲为研究对象,以其他9种/生物型叶甲总科常见害虫为参照,采用基于COⅠ基因的种特异性PCR方法(species-specific COⅠ, SS-COⅠ),研究其快速分子检测鉴定技术。通过提取10种/生物型叶甲DNA和通用型引物扩增测序,获得其基因片段的碱基序列,并比对分析设计1对玉米根萤叶甲特异性引物(DvvZCE1/DvvZCF1),其扩增片段为462 bp。种特异性检验结果显示,该对引物只对玉米根萤叶甲的COⅠ基因具有扩增能力,对其他常见叶甲类害虫,包括玉米双斑长跗萤叶甲、榆黄毛萤叶甲、黄曲条跳甲、油菜蚤跳甲、黄点直缘跳甲、莲草直胸跳甲、枸杞负泥虫以及褐足角胸叶甲的棕黄型和蓝绿型没有扩增能力,该对引物不仅对成虫有很好的扩增效果,对单粒卵、2龄幼虫以及成虫残体(包括触角、头部、胸部、腹部、前足、后足)也具有同样的扩增效果,其最低检出阈值为102.73 pg/μL(相当于1/122 880头雌性成虫)。玉米根萤叶甲特异性SS-COⅠ检测技术在其口岸检疫,以及有效阻截中具有重要意义。The western corn rootworm Diabrotica virgifera virgifera LeConte, one of the most devastating and invasive pest insect of field maize in Europe and USA, has most likely invaded China due to its rapid propagation. Identification based on the external morphology of D. virgifera virgifera is blocked by small body size, high degree of similarity and pleomorphism. In this study, a molecular method based on species-specific cytochrome c oxidase subunit I (SS-COⅠ) PCR technique was developed for rapidly identifying the western corn rootworm by using nine other common leaf beetle species as reference. The mitochondrial DNA COⅠ(mtDNA COⅠ) gene from D. virgifera virgifera and nine other leaf beetle species common in China, including Monolepta hieroglyphica (Motschulsky), Pyrrhalta maculicollis (Motschulsky), Phyllotreta striolata (Fabricius), Psylliodes punctifrons Baly, Ophrida xanthospilota Baly, Agasicles hygrophila Selman & Vogt, Lema decempunctata Gebler, and Basilepta fulvipes (Motschulsky) biotype brown-yellow and biotype blue-green, were amplified by using the universal primer pair LCO1490/HCO2198 and sequenced. Moreover, one pair of SS-COⅠ primers (DvvZCE1/DvvZCF1) amplifying a single band of 462 bp from D. virgifera virgifera was designed. The specificity test of the primer pair against the other nine leaf beetle species indicated that it was specific to D. virgifera virgifera and no cross reactions were observed. The method was tested with egg, 2nd-instar larva, male and female adults as well as adult debris (including antenna, head, thorax, abdomen, foreleg and posterior leg), showing that it was applicable. In addition, the 462 bp DNA fragment could be clearly detected even at the concentration of 102.73 pg/μL, equal to 1/122 880 of a whole female adult of D. virgifera virgifera . The SS-COⅠ marker developed here for rapid identification of D. virgifera virgifera could be used in port quarantine and interception in international transportation of agricultural products.

关 键 词：玉米根萤叶甲 SS-COⅠ标记 种特异性引物 快速鉴定 分子检测 

分 类 号：S435.132[农业科学—农业昆虫与害虫防治]