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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709bp COI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Clustal W2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COI DNA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Calliphora vicina。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。
OBJECTIVE: To establish a method for identifying unknown vector types by using DNA barcode technology, especially for DNA barcode identification of non-adult worms or individuals with limb defects to shorten the identification cycle. Methods The genomic DNA of the single pupae of Diptera was extracted from the imported waste paper from the UK. DNA amplification was performed using universal primers of multicellular invertebrate mitochondrial cytochrome oxidase I (COI). PCR products were sequenced directly from both ends. Sequencing primers and amplification primers were obtained, and the nucleotide sequence of the 709bp COI gene fragment was obtained after splicing. At the same time, other individuals in the same batch of pupae intercepted were hatched into adults for morphological identification in laboratory and the genomic DNA of hatching adults was extracted. The COI gene fragment was amplified and sequenced and compared with the pupae sequence. Homology alignments with sequences in GenBank and AD phylogenetic tree online with Clustal W2. Results The pupae of unknown species were 99.4% identical to the COI DNA sequence (including primer sequence) of Calliphora vicina in Genbank. The morphology of the pupal adult was identified as the red head snapdragon, and the DNA barcode data of the hatched adult were completely consistent with the pupae data. Conclusion According to the results, it can be judged that the pupae of the unknown species are Calliphora vicina. This study shows that DNA barcoding is a fast and accurate method for the identification of species, especially for the identification of non-adult species.