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

在这项研究中，我们已经发展了一种新的方法，物种鉴定的聚合酶链反应的线粒体DNA高变区。从人、狗、猫、山羊、绵羊、牛、兔、猪、马、大鼠、小鼠、雪貂、浣熊、日本猴、大猩猩和黑猩猩的血液、毛发和肌肉中提取DNA样品。制备两对引物用于扩增mtDNA D环。一组mt-U1/mt-U2扩增高变区1（HV 1）。PCR产物的大小范围从雪貂的370 ×到山羊的823 ×，表明动物之间是可以区分的。人DNA的大小为545 bp，与牛的大小（543 bp）相似。为了解决这个问题，我们制备了第二对引物，mt-HV 2F/mt-HV 2 R，它们对人是特异性的。仅在人DNA中观察到HV 2/3产物（600 bp）。当使用两个引物组进行双重PCR时，人显示两条带，而其他动物显示一条带。因此，成功地识别了物种。当未知样品与DNA样品大小不一致时，PCR产物测序和BLAST允许我们鉴定物种。实际上，当在实际情况中遇到未知样品时，我们在应用这些技术后鉴定了来自黄鼠狼（Mustela itatsi）的核苷酸序列。总之，由于目前的物种鉴定方法是非常简单，容易，快速，准确，有望应用于法医学领域。

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.