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，我们开发了一种新方法，用于通过mtDNA-HYPERVARAIBLE区域的PCR鉴定物种。获得了几种动物的FOM血液，头发和肌肉的DNA样品，包括人，狗，猫，羊，羊，牛，兔子，猪，马，老鼠，老鼠，小鼠，雪貂，浣熊狗，Japnese猴子，大猩猩和黑猩猩。准备两个底漆集以扩增mtDNA d-loop。一组MT-U1/MT-U2放大了高变量区域1（HV1）。 PCR产品的尺寸范围从大约370个在雪貂上到823，表明动物可以彼此区分。人DNA显示出545 bp大小，类似于OX的大小（543 bp）。为了解决这个问题，我们准备了第二个底漆集MT-HV2F/MT-HV2R，该集合对人类是特定的。 HV2/3的产物仅来自人类的DNA（600 bp）。当使用两个引物组进行双链PCR时，人类显示了两个频段，而另一只动物则显示了一个条带。因此，物种被成功地鉴定出来。如果未知样品的大小与DNA样品不一样，则PCR拟合和爆炸的测序使我们能够识别物种。实际上，在使用此类技术后，在遇到未知的样品时，我们从黄鼠狼（Mustela itatsi）中鉴定出核苷酸序列。总之，由于当前的物种鉴定方法非常简单，简单，快速且精确，因此预计将应用于法医生物学领域。