基于种特异性标记鉴定不同虫态草地贪夜蛾的技术研究  

Identification of different developmental stages of Spodoptera frugiperda using species-specific markers

作　　者：李清文 张静 粟智平 战一迪 刘勇[1] LI Qing-Wen;ZHANG Jing;SU Zhi-Ping;ZHAN Yi-Di;LIU Yong(College of Plant Protection,Shandong Agricultural University,Tai’an 271018,China;Technology Center of Yantai Customs District,Yantai 264000,China;Technology Center of Jinan Customs District,Jinan 250014,China;Agriculture Rural Affairs and Water Conservancy Bureau of Huangshan District,Huangshan 245700,China)

机构地区：[1]山东农业大学植物保护学院,泰安271018 [2]烟台海关技术中心,烟台264000 [3]济南海关技术中心,济南250014 [4]黄山区农业农村水利局,黄山245700

出　　处：《应用昆虫学报》2023年第4期1307-1313,共7页Chinese Journal of Applied Entomology

基　　金：国家重点研发计划(2021YFE0115600)。

摘　　要：【目的】草地贪夜蛾Spodoptera frugiperda的卵、低龄幼虫、蛹甚至成虫和其他鳞翅目害虫混合发生时,常难以准确识别;植保监测站以及海关等获取的可能包括草地贪夜蛾等大量昆虫残片的混合样品,也需要快速准确的物种鉴定。本文构建了基于种特异性标记的草地贪夜蛾的快速准确鉴定方法。【方法】利用线粒体DNA COⅠ基因通用引物LCO1490/HCO2198扩增11种供试鳞翅目昆虫的COⅠ序列,然后根据草地贪夜蛾的保守序列片段设计6对PCR特异性引物、1对qPCR特异性引物和探针,提取供试昆虫的DNA,并以此为模板对引物进行种特异性检测。【结果】PCR引物SF_F3/SF_R3和qPCR引物60F/154R可特异性扩增不同虫态(卵、幼虫、蛹和成虫)的草地贪夜蛾基因组模板;灵敏度检测表明,PCR和qPCR引物对草地贪夜蛾DNA模板的最低检测浓度分别为1ng/μL和10pg/μL。【结论】本研究建立的基于种特异性标记的物种快速鉴定方法,提高了不同虫态的草地贪夜蛾的鉴定效率和准确度。[Objectives]To develop a rapid,and accurate,method of identifying all developmental stages of Spodoptera frugiperda based on a species-specific marker in order to enable the detection of this important invasive pest in the mixed samples of insect fragments commonly obtained by customs and plant protection monitoring stations.[Methods]The mtDNA COⅠgene universal primer LCO1490/HCO2198 was used to amplify COⅠgene sequences of 11 lepidopteran species.Six pairs of PCR specific primers,and one pair of qPCR specific primers and a probe,were designed according to a conserved sequence fragment of S.frugiperda.The DNA of the tested insect species were extracted as templates to determine the specificity of the primers.[Results]The PCR primers SF3F/SF3R,and the qPCR primers 60F/154R,had specific amplification capabilities for the genome template of different developmental stages(egg,larva,pupa and adult)of S.frugiperda.Sensitivity tests indicate that the minimum PCR and qPCR detection concentrations of the primers for the DNA template of S.frugiperda were 1 ng/μL and 10 pg/μL,respectively.[Conclusion]A rapid identification protocol developed based on species-specific markers can improve the efficiency and accuracy of identifying different developmental stages of S.frugiperda.

关 键 词：草地贪夜蛾 线粒体DNA COⅠ基因 PCR 实时荧光定量PCR 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]