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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。
Objective To solve the problem of sarcophagous flies and identification of eggs and larvae by using the sequence of 635bp in cytochrome oxidase coenzyme Ⅱ (CO Ⅱ) in mitochondrial DNA (m tDNA). Methods Randomly collected the eggs of the dead flies, larvae and flies in the belly of rabbits in the outdoor grassland of Hohhot. The flies mtDNA was extracted by Chelex method; PCR amplification was performed by Perkin Elmer 9600; agarose electrophoresis and silver staining were used to detect the amplification results; PCR gel recovery kit was purified; AB I 377 sequencer sequencing; DNAMAN 4.0 sequence analysis software, sequence alignment, interception and other length fragments; MEGA2.1 software package sequence analysis and construction of phylogenetic tree. Results The average number of COⅡ gene sequences in the mtDNA of the zombie flies was less than 1% in the species of Dipterous zombie flies, and the mean number of species differences was more than 3%. There was no significant difference between adults and larvae. In this way, we can judge whether two individuals are the same species according to the difference of COⅡ sequence. However, for the closely related species Lucilia sericata and Lucilia sericata, the use of these two fragments is very difficult to distinguish because of the very close divergence between species within and between species. Conclusion CO 2 sequence analysis on m tDNA can effectively identify most of the sarcastic flies. This test method is fast, easy and accurate and can be used as a forensic basis to identify the type of sarcastic flies.