Species identification and individualization by PCR of mtDNA-HV1 region with a universal primer set

使用通用引物组对 mtDNA-HV1 区域进行 PCR 进行物种鉴定和个体化

基本信息

批准号：
16590539
负责人：
YUASA Isao
金额：
$ 2.24万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-16590539/
关键词：
mtDNA HV1 region species identification individualization PCR D-loop 動物種の同定

项目摘要

In this study, we have developed a new method for species identification by PCR of mtDNA-hypervariable regions. DNA samples were obtained fom blood, hair, and muscle of several animals including human, dog, cat, goat, sheep, ox, rabbit, pig, horse, rat, mouse, ferret, racoon dog, Japnese monkey, gorilla and chimpanzee. Two primer sets were prepared to amplify mtDNA D-loop. One set, mt-U1/mt-U2, amplified the hypervariable region 1 (HV1). The size of PCR products ranged from about 370 by in ferret to 823 by in goat, indicating that animals were distinguishable from each other. Human DNA showed a 545 bp-size, which was similar to the size of ox (543 bp). To dissolve this problem, we prepared the second primer set, mt-HV2F/mt-HV2R, which were specific for human. The HV2/3 product was observed in DNA from human only (600 bp). When duplex PCR was done using the two primer sets, human showed two bands, whereas the other animals, one band. Thus, species was succesfully identified. When unknown samples were not identical in size to DNA samples as a contol, sequencing of PCR poducts and BLAST permitted us to identify species. In fact, when encountering unknown samples in a pratical case, we identified a nucleotide sequence from a weasel (Mustela itatsi) after application of such techniques. In conclusion, since the present species identification method is very simple, easy, rapid, and exact, it is expected to be applied to a field of forensic biology.

在这项研究中，我们开发了一种通过 mtDNA 高变区 PCR 进行物种鉴定的新方法。 DNA 样本取自多种动物的血液、毛发和肌肉，包括人类、狗、猫、山羊、绵羊、牛、兔、猪、马、大鼠、小鼠、雪貂、貉、日本猴、大猩猩和黑猩猩。准备两个引物组来扩增 mtDNA D 环。一组 mt-U1/mt-U2 扩增了高变区 1 (HV1)。 PCR 产物的大小范围从雪貂的约 370 by 到山羊的 823 by，表明动物之间是可以区分的。人类 DNA 的大小为 545 bp，与牛的大小 (543 bp) 相似。为了解决这个问题，我们制备了第二对人类特异性引物mt-HV2F/mt-HV2R。仅在人类 DNA（600 bp）中观察到 HV2/3 产物。当使用两个引物组进行双重 PCR 时，人类显示出两条带，而其他动物则显示出一条带。至此，物种鉴定成功。当未知样本的大小与作为对照的 DNA 样本不同时，PCR 产品和 BLAST 的测序使我们能够识别物种。事实上，当我们在实际案例中遇到未知样本时，应用此类技术后，我们识别出了来自黄鼠狼（Mustela itatsi）的核苷酸序列。综上所述，由于本物种鉴定方法非常简单、容易、快速、准确，因此有望应用于法医生物学领域。