应用DNA条形码技术鉴定未知双翅目蛹

    岳巧云1，邱德义1，黄艺文1，侯捷2，刘国雄1，胡龙飞2

    1.中山出入境检验检疫局技术中心，广东 中山528403；2.云浮出入境检验检疫局

    摘要：目的建立应用DNA条形码技术鉴定未知病媒种类的方法，尤其是非成虫态虫体或者肢体残缺个体的DNA条形码鉴定的方法和技术，以缩短鉴定周期。方法提取从英国进口废纸中截获的双翅目单只蛹基因组DNA，利用多细胞无脊椎动物线粒体细胞色素氧化酶I（COI）的通用引物进行DNA扩增。PCR产物直接从两端测序，测序引物同扩增引物，序列经拼接后得到清晰可读的709bpCOI基因片段碱基序列。同时将截获的同批蛹的其他个体在实验室内孵化成成虫进行形态学鉴定，并抽提孵化的成虫的基因组DNA，扩增其COI基因片段，测序并与蛹的序列进行比较。与GenBank中的序列进行同源性比对，并用ClustalW2在线建立AD系统发育树。结果 未知种类的蛹和Genbank中红头丽蝇Calliphora vicina 的COIDNA序列（包括引物序列）99.4%相同。蛹化成虫形态鉴定为红头丽蝇，孵化后的成虫的DNA条形码数据与蛹的数据完全相符。结论根据结果可以判断所截获的未知种类的蛹为红头丽蝇Calliphoravicina。本文的研究证明了DNA条形码技术是一种快捷准确的种类鉴定方法，尤其是运用在非成虫态的种类鉴定上。

    关键词：DNA条形码技术；未知种类蛹；红头丽蝇；种类鉴定

    中图分类号：R384.2　文献标识码：B

    Application of DNA Barcoding techniques tothe species

    identification of unknown Diptera pupa

    YUE Qiao-yun*, QIU De-yi, HUANG Yi-wen, HOUJie, LIU Guo-xiong，HU Long-fei

    *Zhongshan Entry-Exit Inspection andQuarantine Bureau, Zhongshan，Guangdong 528403, China

    Abstract:   Objective  To establish a method of application DNA Barcoding techniques tothe species identification of unknown disease-borne insect，especially to the non-adult or morphological incompletespecimen,  so the species identification time could beshortened.   Methods   Genomic DNA wasextracted from the unknown single pupa found in the waste paperimported from England. PCR was performed with the metazoaninvertebrates degenerate Mitochondrial Cytochrome Oxidase I (COI)primers. Sequencing was performed from both ends with the sameprimers of the PCR ones. 709bp COI sequences were obtained. Otherpupae were incubated into adults; morphology identification and DNABarcoding were performed to the incubated adult as well. Sequencesblasted in the GenBank, and a AD-tree was built with Clustal W2 online.   Results   Sequence of the COI fragmentfrom the unknown pupa （including primers） were 99.4% identical tothe published sequence of Calliphora vicina in the GenBank,Incubated adult was identified as Calliphora vicinamorphologically, COI DNA sequence of the incubated adult was 100%identical to the pupa.  Conclusion   Based on theseresults, we could conclude that the intercepted pupa is Calliphoravicina. In this study we confirmed again that DNA Barcoding is arapid and exact tool for species identification, especially for thenon-adult specimen.

    Key words:    DNA barcoding;unknown Diptera pupa; Calliphora vicina；Species identification

    《中国国境卫生检疫杂志》