线粒体16srRNA和ND4基因在种属鉴定中的应用研究

A study of 16srRNA and ND4 genes in mitochondrial DNA for forensic species identification

目的 构建一种用于种属鉴定的线粒体DNA（mtDNA）16srRNA和ND4基因荧光标记复合扩增检测体系。方法 利用引物设计软件（Primer 5）对两个mtDNA序列ND4基因和16srRNA基因设计两对引物，每对引物中的一条在5’端标记荧光素（6-FAM）。按传统复合扩增技术建立复合扩增体系，用ABIPRISM 310基因分析仪对产物进行分析。结果 人类DNA扩增产物出现两个峰，片段大小分别为110bp的人类特异片段和149bp的人与动物共有片段，而动物DNA扩增产物出现一个峰，片段大小为149bp。对30个实验室存放5～15年的陈旧人血痕也能明确判断其种属来源。结论 该体系可以明确区分人源性生物检材与其它常见动物样本，对实验室长期存放的陈旧检材也具有较好的检测能力。

Objective To establish a fluorescent multiplex amplification of 16srRNA and ND4 genes in mitochondrial DNA in order to provide a method for forensic species identification. Methods The mitochondrial DNA sequences of human and different kinds of animals, including monkey, pig, dog, fish, rat, guinea pig, rabbit, horse, eel, frog, chicken, duck, goat and cow, were obtained from GenBank. The difference of mitochondrial DNA sequences between human and animals were analyzed with software. With software Primer 5 ,two pairs of primers were designed for 16srRNA and ND4 genes chosen from mtDNA sequence. One primer of each pair was labeled with 6-FAM in 5＇ end. Multiplex amplification was carried out with the primers. The amplified products were analyzed by ABIPRISM 310 Genetic Analyzer. DNA samples from Human and 14 kinds of different animals were analyzed. Results The fluorescent multiplex amplification of 16srRNA and ND4 genes was successfully carried out. The amplified products of human DNA had two peaks, one of which was human-specific 110-bp-length ment. The products of animals DNA had only one peak, the nt and another was 149-bp-length fragsize of which was 149bp. Therefore, the 149- bp-length fragment belonged to both Human and animals. Conclusion A system of fluorescent multiplex amplification of mitochondrial DNA for forensic species identification was successfully established, and it could distinguish Human samples from familiar animal samples.