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

在这项研究中，我们开发了一种新的mtdna高变区PCR鉴定物种的方法。从人类、狗、猫、山羊、绵羊、牛、兔、猪、马、大鼠、小鼠、雪貂、貉、日本猴、大猩猩和黑猩猩等动物的血液、毛发和肌肉中获得了DNA样本。制备两组引物扩增mtDNA D-loop。其中一组mt-U1/mt-U2扩增高变区1 （HV1）。PCR产物的大小从雪貂的370倍到山羊的823倍不等，表明动物之间是可区分的。人类DNA的大小为545 bp，与牛的大小（543 bp）相似。为了解决这一问题，我们制备了人类特异性的第二组引物mt-HV2F/mt-HV2R。仅在人类DNA （600 bp）中观察到HV2/3产物。用两组引物进行双链PCR时，人类显示两条条带，而其他动物显示一条条带。因此，物种被成功地鉴定出来。当未知样本与作为对照的DNA样本大小不相同时，PCR产物测序和BLAST使我们能够识别物种。事实上，当在实际案例中遇到未知样本时，我们在应用此类技术后从鼬鼠（Mustela itatsi）中识别出核苷酸序列。综上所述，该方法具有简单、简便、快速、准确等特点，在法医生物学领域具有广阔的应用前景。

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.