检疫性昆虫DNA条形码检测技术的研发与应用实例
Development and application of quarantine insectDNA barcoding technology
叶兼菱1** 安榆林1*** 钱 路1 杨晓军1 徐 梅1 吴翠萍1 魏春艳2 牛 磊3 甘英俊4
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DOI：10.7679/j.issn.2095-1353.2015.042
作者单位：1. 江苏出入境检验检疫局，南京 210001；2. 吉林出入境检验检疫局，长春 130062；3. 南京贺林科技有限公司，南京 210037；4. 南京林业大学，南京 210037
中文关键词：DNA条形码，植物检疫，多态位点规律，鉴定特征
英文关键词： DNA barcoding, plant quarantine, combination of nucleotide diagnostic sites, identification characteristic
中文摘要：
【目的】 DNA条形码技术是近年来生物分类鉴定的研究热点之一，已成为植物检疫性昆虫鉴定的有力工具。为快速、准确地鉴定口岸截获的昆虫种类，实现“检得出、检得准、检得快”的要求，我们研发了昆虫DNA条形码试剂盒检测技术（Insect DNA barcoding identification kit）。【方法】 该检测技术针对出入境植物检疫性及危险性昆虫的主要类群，选择合适的基因片段、设计引物、对目标基因进行扩增测序，找出基因片段上区分每个物种的多态位点规律，作为该物种的鉴定特征并建立数据库，应用于植物检疫性及危险性昆虫的物种鉴定。【结果】 以检疫性昆虫木蠹象属Pissodes为例，确定了木蠹象属5种昆虫的多态位点规律（鉴定特征），构建了用于物种鉴定的数据库。通过比对数据库里的鉴定特征，将未知样品鉴定为榛梢木蠹象P. terminalis (相似度100%)，与形态鉴定结果一致。本文介绍了检测技术的原理、方法、技术流程及应用实例，并展望了其在有害生物检测中的推广应用前景。【结论】 昆虫DNA条形码试剂盒检测技术为建立标准化，准确性高的物种鉴定平台打下基础，有着良好的推广应用前景。
英文摘要：[Objectives] DNA barcoding has been one of the main focuses of insect taxonomic research in recent years and has become a powerful tool for the identification of plant quarantine insects. In order to accurately identify and intercept pest insects at ports of entry, we developed an insect DNA barcoding identification kit (IDK). This kit allows the quick and accurate identification of known, and potential, pest insects. [Methods] New primers for specific insect genera or groups were designed to improve amplification efficiency. A combination of nucleotide diagnostic sites from molecular sequences of closely related species from the same genera was generated and a database was built specifically for species identification. [Results] We took quarantine insects Pissodes for the example, the combination of nucleotide diagnostic sites (identification characteristics) of 5 Pissodes species were generated, and the database was bulit for species identification. One unidentified sample of Pissodes was compared to the identification characteristics stored in the library. Similarity between the query sequence and P. terminalis in database is 100%. This identification was confirmed by later morphological examination. This paper introduces the principles, technical methods, identification process and application of this new kit, and discusses its prospective benefits for plant quarantine. [Conclusion] Insect DNA barcoding identification kit will lay a basis for establishment of standard and accurate species identification platform. It will has a good prospect of application.