Department of Forensic Sciences, Graduate School of Sungkyunkwan University, 16419, Suwon, Republic of Korea.
Int J Legal Med. 2024 May;138(3):751-756. doi: 10.1007/s00414-023-03144-5. Epub 2023 Dec 13.
Analyzing all biological evidence at a crime scene presents serious time, budget, and labor constraints. Therefore, selecting valid evidence is crucial for efficient screening. The ABO blood group is a marker that can serve as valid evidence for identifying investigative leads in criminal case. Conventional identification of ABO blood groups using serological methods has only been for blood and is difficult to apply to other body fluids. ABO genotyping was conducted by analyzing single nucleotide polymorphisms (SNP) representative of each blood group. However, this method is time-consuming, expensive, and requires sophisticated instruments. In this study, we developed rapid ABO genotyping method using loop-mediated isothermal amplification (LAMP) and multiplex real-time polymerase chain reaction (PCR). Three SNP sites in the ABO gene (nt 261, 526, and 803) were selected to classify the ABO genotypes. For the specificity test, we performed sequencing of 60 saliva samples to confirm that the genotyping. We conducted experiments to apply ABO genotyping using two amplification methods to mock forensic sample using cotton swab and filter paper. As a result, using LAMP, we successfully identified six ABO genotypes within 30 min at a constant temperature (65 ℃). Moreover, by using multiple real-time PCR, it was possible to detect not only the major group but also the subgroup of the ABO genotype (ex. cis-AB). The amplification results using the new methods were in concordance with the sequencing results. Therefore, these ABO genotyping methods are expected to select valid evidence successfully and efficiently at the crime scene.
在犯罪现场分析所有生物证据都存在时间、预算和劳动力方面的严重限制。因此，选择有效的证据对于高效筛选至关重要。ABO 血型是一种可以作为犯罪案件中调查线索的有效证据的标志物。传统的 ABO 血型血清学鉴定方法仅适用于血液，难以应用于其他体液。ABO 基因分型是通过分析代表每个血型的单核苷酸多态性（SNP）来进行的。然而，这种方法耗时、昂贵，并且需要复杂的仪器。在这项研究中，我们使用环介导等温扩增（LAMP）和多重实时聚合酶链反应（PCR）开发了一种快速 ABO 基因分型方法。选择 ABO 基因中的三个 SNP 位点（nt 261、526 和 803）来对 ABO 基因型进行分类。为了进行特异性测试，我们对 60 个唾液样本进行了测序，以确认基因分型结果。我们使用两种扩增方法对模拟法医样本（棉签和滤纸）进行了 ABO 基因分型实验。结果表明，使用 LAMP 在 30 分钟内可在恒定温度（65℃）下成功鉴定出六种 ABO 基因型。此外，通过使用多重实时 PCR，可以检测到 ABO 基因型的主要组，也可以检测到亚组（例如 cis-AB）。新方法的扩增结果与测序结果一致。因此，这些 ABO 基因分型方法有望在犯罪现场成功、高效地选择有效证据。
