基于基因组特异性SSR序列的实蝇PCR鉴定方法  

PCR-based identification of fruit-flies using specific SSR sequences

作　　者：丁思敏[1] 王书平[2] 贺康[1] 李飞[1] 蒋明星[1] DING Si-Min;WANG Shu-Ping;HE Kang;LI Fei;JIANG Ming-Xing(Institute of Insect Sciences,Zhejiang University,Hangzhou 310058,China;Technical Center for Animal Plant and Food Inspection and Quarantine,Shanghai Entry-Exit Inspection and Quarantine Bureau,Shanghai 200135,China)

机构地区：[1]浙江大学昆虫科学研究所,杭州310058 [2]上海出入境检验检疫局动植物与食品检验检疫技术中心,上海200135

出　　处：《应用昆虫学报》2018年第4期759-765,共7页Chinese Journal of Applied Entomology

基　　金：重大/新发农业入侵生物风险评估及防控关键技术研究(2017YFC1200602);国家自然科学青年基金(31701785)

摘　　要：【目的】实蝇科昆虫是全球范围内重要的检疫性害虫,其幼虫取食寄主果实,引起果实腐烂、变质,造成巨大经济损失。由于口岸截获多为实蝇卵、幼虫、蛹等非成虫虫态,需饲养至成虫才能根据形态准确鉴定,因此快速可靠的分子鉴定技术体系亟需完善。【方法】利用公开发表的实蝇基因组序列,通过生物信息学方法从4种检疫性实蝇即地中海实蝇Ceratitis capitata、瓜实蝇Bactrocera cucurbitae、橘小实蝇Bactrocera dorsalis、昆士兰实蝇Bactrocera tryoni中挖掘物种特异性的简单重复序列(Simple sequence repeats,SSR),针对各物种设计含有其特异性简单重复序列的引物,提取实蝇样品基因组DNA,采用常规PCR方法优化并筛选各物种特异性引物。【结果】在4种实蝇基因组中共发现20条物种特异性的SSR,基于这些序列设计的3对特异性引物能有效区分地中海实蝇、瓜实蝇和橘小实蝇,扩增片段大小分别为1 251、1 307、823 bp,而在其他物种中检测不到条带。【结论】基因组水平的序列分析发现了物种特异性的SSR,通过筛选获得物种特异性的PCR引物,可应用于3种实蝇的分子鉴定,为口岸检疫人员快速鉴定区分非成虫状态的实蝇提供了实用性技术。[Objectives] Fruitflies（Diptera, Drosophilidae） are important globally distributed quarantinable pests. They generally lay eggs in fruits and vegetables, and their larvae feed on these after hatching, causing serious economic loss to fruit and vegetable growers. The eggs, larvae, and pupae of fruitflies are frequently discovered in quarantine ports, however, these life-stages are difficult to identify because fruitfly species are traditionally distinguished on the basis of adult morphology. Morphological identification of eggs, larvae, and pupae therefore requires these to be reared to the adult stage, which takes a long time. It is therefore useful to develop rapid and accurate molecular methods that can identify fruitfly eggs, larvae, and pupae. [Methods] Species specific, simple sequence repeats（SSR） were discovered in the genomes of four quarantined fruit fly species; Ceratitis capitate, Bactrocera cucurbitae, Bactrocera dorsalis and Bactrocera tryoni, using a well-designed bioinformatics pipeline. Species-specific primers were designed from these SSRs and screened using genomic DNA extracted from specimens of different fruitfly species as templates. The polymerase chain reaction（PCR） was then used to amplify and detect SSRs in DNA samples obtained from fruitfly eggs, larvae and pupae. [Results] Twenty species-specific SSRs were obtained from the four quarantined fruitfly species. Three species-specific primers were designed which produced single bands of 1 251, 1 307 and 823 bp, respectively and were confirmed to reliably distinguish C. capitata, B. cucurbitae and B. dorsalis. [Conclusion] Bioinformatics analysis of genomic sequences allows us to identify species-specific SSR. After the optimization of species-specific primers designed from these SSR sequences, PCR can be used to accurately identify fruitflies. This work provides a theoretical basis and technical support for inspectors to reliably identify fruitfly eggs, larvae and pupae intercepted at ports.

关 键 词：实蝇 简单重复序列 物种特异性引物 PCR 检疫 

分 类 号：S433[农业科学—农业昆虫与害虫防治]