School of Forensic Medicine, Shanxi Medical University, Jinzhong, 030600, Shanxi, China.
Shanxi Key Laboratory of Forensic Medicine, Jinzhong, 030600, Shanxi, China.
Int J Legal Med. 2024 May;138(3):1205-1219. doi: 10.1007/s00414-023-03105-y. Epub 2023 Oct 18.
Blood-containing mixtures often appear in murder and robbery cases, and their identification plays a significant role in solving crimes. In recent years, the co-detection of DNA methylation markers (CpG) and single nucleotide polymorphism (SNP) markers has been shown to be a promising tool for the identification of semen and its donor. However, similar research on blood stains that are frequently found at crime scenes has not yet been reported. In this study, we employed blood-specific CpG-linked SNP markers (CpG-SNP) for blood-specific genotyping and the linking of blood and its donor. The tissue-specific CpG markers were screened from the literature and further verified by combining bisulfite conversion with amplification-refractory mutation system (ARMS) technology. Meanwhile, adjacent SNP markers with a minor allele frequency (MAF) greater than 0.1 were selected within 400 bp upstream and downstream of the CpG markers. SNP genotyping was performed using SNaPshot technology on a capillary electrophoresis (CE) platform. Finally, a multiplex panel, including 19 blood-specific CpG linked to 23 SNP markers, as well as 1 semen-specific CpG, 1 vaginal secretion-specific CpG, and 1 saliva-specific CpG marker, was constructed successfully. The panel showed good tissue specificity and blood stains stored at room temperature for up to nine months and moderately degraded (4 < DI < 10) could be effectively identified. Moreover, it could also be detected when blood content in the mixed stains was as low as 1%. In addition, 15 ng of DNA used for bisulfite conversion was required for obtaining a complete profile. The cumulative discrimination power of the panel among the Han population of northern China could reach 0.999983. This is the first investigation conducted for the simultaneous identification of blood and its donor regardless of other body fluids included in mixed stains. The successful construction of the panel will play a vital role in the comprehensive analysis of blood-containing mixtures in forensic practice.
含血混合物经常出现在谋杀和抢劫案件中，其鉴定在解决犯罪方面起着重要作用。近年来，DNA 甲基化标记物（CpG）和单核苷酸多态性（SNP）标记物的联合检测已被证明是鉴定精液及其供体的一种有前途的工具。然而，在犯罪现场经常发现的血斑的类似研究尚未报道。在这项研究中，我们使用了血液特异性 CpG 连接 SNP 标记物（CpG-SNP）进行血液特异性基因分型和血液及其供体的关联。组织特异性 CpG 标记物是从文献中筛选出来的，并通过结合亚硫酸氢盐转化和扩增不可阻挡突变系统（ARMS）技术进一步验证。同时，在 CpG 标记物上下游 400bp 范围内选择了次要等位基因频率（MAF）大于 0.1 的相邻 SNP 标记物。SNP 基因分型在毛细管电泳（CE）平台上使用 SNaPshot 技术进行。最后，成功构建了一个包含 19 个与 23 个 SNP 标记物相连的血液特异性 CpG、1 个精液特异性 CpG、1 个阴道分泌物特异性 CpG 和 1 个唾液特异性 CpG 标记物的多重面板。该面板显示出良好的组织特异性，室温下储存长达九个月且中度降解（4 < DI < 10）的血斑可以有效识别。此外，即使混合污渍中的血液含量低至 1%，也可以检测到。此外，获得完整图谱需要用于亚硫酸氢盐转化的 15ngDNA。该面板在中国北方汉族人群中的累积鉴别力可达到 0.999983。这是首次进行的无论混合污渍中包含其他体液如何都能同时鉴定血液及其供体的研究。该面板的成功构建将在法医实践中对含血混合物的综合分析中发挥重要作用。
