嗜尸性苍蝇mtDNA中COⅡ基因序列检测

目的利用线粒体DNA(m tDNA)上细胞色素氧化酶辅酶Ⅱ(COⅡ)中635bp基因序列,解决嗜尸性苍蝇及其卵和幼虫种类鉴定的难题。方法随机采集放置在呼和浩特地区室外草地家兔尸体上的嗜尸性苍蝇、幼虫、苍蝇腹中的卵。利用Chelex方法提取上述苍蝇m tDNA;通过Perk in-E lm er 9600扩增仪进行PCR扩增;琼脂糖水平电泳和银染显色技术进行扩增结果检测;PCR胶回收试剂盒纯化;AB I 377测序仪测序;DNAMAN 4.0序列分析软件,进行序列比对,截取等长度片段;MEGA2.1软件包进行序列分析和构建系统发育树。结果上述嗜尸性苍蝇m tDNA上COⅡ基因序列在双翅目嗜尸性苍蝇的种内差异均数小于1%,种间差异均数大于3%,成虫与幼虫、卵无明显差异。以此能够根据COⅡ序列差异判断两个个体是否同种。然而,对于亲缘关系非常接近的铜绿蝇和丝光绿蝇来说,由于二者的种内、种间进化分歧均数非常接近,运用上述两个片段则很难区别。结论m tDNA上COⅡ序列分析能有效地对绝大多数嗜尸性苍蝇进行种类鉴定。该检测方法快速、简便和精确,能作为法医鉴别嗜尸性苍蝇种类的依据。

Sequencing of mitochondrial DNA COⅡ gene in sarcosaphagous flies

Objective To identify species of sarcosaphagous flies and their larvae and eggs by analysis based on each 635bp region of the gene for cytochrome oxidase subunitⅡ(COⅡ) encoding region of mtDNA. Methods Specimens of sarcosaphagous flies and their larvae and eggs were collected from rabbit corpses placed on the grassland in the Hohhot district. The mtDNA was extracted from the specimens using the Chelex technique and were amplified using a Perkin-Elmer 9600 thermal cycler. PCR products were electrophoresized with horizontal gelose followed by silver stain and purified using Nucleic Acid Purification Kit. Sequences of both strands were obtained by direct sequence of the double-stranded PCR product using one of the PCR primers and the ABI PRISM Big Dye Terminator Cycle Sequencing Kit. Sequence reactions were electrophoresized on ABI Model 377 DNA Sequencers. Using multiple-alignment program DNAMAN(version 4.0), analyses of the sequence alignments were carried out. A phylogenetic tree was constructed by the MEGA2.1 package by neighbor-joining method. Results A 635bp region of the gene for cytochrome oxidase subunitⅡ(COⅡ) encoding region of mtDNA in sarcosaphagous flies collected from Hohhot district was detected to be less than 1% in sequence divergence within species and larger than 3% between species. In contrast, due to very similar sequence divergence in sister species, the data obtained from the method could not be used for identification in the same species, i.e. the species within the Lucilia sericata (Meigen) and Lucilia cuprina (Wiedemann). Conclusion Sequencing a 635bp region of cytochrome oxidase subunitⅡ(COⅡ) encoding region of mtDNA in sarcosaphagous flies can be effectively used for species identification in most of sarcosaphagous flies. Owing to the rapid, easy and accurate nucleotide sequencing technology, identification of sarcosaphagous flies is applicable in forensic practice.