College of Science & Engineering, Flinders University, Adelaide, Australia.
College of Science & Engineering, Flinders University, Adelaide, Australia.
Forensic Sci Int Genet. 2018 Sep;36:20-25. doi: 10.1016/j.fsigen.2018.06.004. Epub 2018 Jun 6.
All previous examinations of the shedder status of individuals have been based on conclusions inferred from the amount of DNA deposited by donors after they have held an object for a fixed period of time. In all interpretations of shedder status experiments have involved a range uncertainties, especially in regards to results arising from studies carried out in different laboratories. These apply to the efficiency of the swab collecting DNA from the item touched, the amount of DNA left on the swab after attempts to recover it, and the percentage loss of DNA during the lysis and extraction processes. No previous study has attempted to mitigate these uncertainties or verify how much of the DNA deposited was collected through swabbing, how much DNA present on the swab was recovered or how much DNA is lost during the extraction process. We present a study that accurately measures the deposition, collection and amplification of DNA deposited by a range of donors allowing for an accurate determination of the shedder status of individuals. Eleven donors were asked to wash their hands and then deposit a thumbprint onto glass slides by making pressure for 15 seconds 0, 15, 60 and 180 minutes after handwashing. Both left and right thumbs were used and all testing was performed in triplicate. Measurement of the quantity of cellular material deposited on the slides was carried out using DiamondTM Nucleic Acid Dye and fluorescence microscopy on each of 264 thumbprints. Fluorescence microscopy was then used to demonstrate that all the DNA present on the slides was recovered by the swabbing operations and then direct PCR, using the Identifiler™ Plus kit, was used to ensure that none of the DNA present on swabs was lost during DNA profiling. The combination of using a DNA binding dye and direct PCR allowed an accurate means of measuring the extent to which individuals exhibit different extents of shedding. This small study, 11 donors, showed that individuals fell into one of three distinct groups: heavy, intermediate, and light shedders, regardless of the hand used.
之前对个体脱落物状态的所有检查都是基于这样的结论，即捐献者在固定时间持有物品后，所遗留的 DNA 量。在对脱落物状态实验的所有解释中，都涉及到一系列的不确定性，尤其是在涉及到在不同实验室进行的研究结果时。这些不确定性适用于从被触摸物品上采集 DNA 的拭子的效率、试图回收后留在拭子上的 DNA 量，以及在裂解和提取过程中 DNA 的损失百分比。以前没有研究试图减轻这些不确定性或验证通过拭子收集的沉积 DNA 量、拭子上存在的 DNA 量有多少被回收，以及在提取过程中丢失了多少 DNA。我们进行了一项研究，该研究准确测量了一系列捐献者的 DNA 沉积、收集和扩增情况，从而可以准确确定个体的脱落物状态。要求 11 名捐献者洗手，然后通过在洗手后 0、15、60 和 180 分钟施加 15 秒的压力将指纹印在载玻片上。左右拇指都被使用，所有测试均重复进行三次。使用 DiamondTM 核酸染料和荧光显微镜对 264 个指纹中的每一个进行了载玻片上细胞物质沉积量的测量。然后使用荧光显微镜证明，通过拭子操作回收了载玻片上的所有 DNA，然后直接使用 PCR，使用 Identifiler™ Plus 试剂盒，确保在 DNA 图谱制作过程中不会丢失拭子上存在的任何 DNA。使用 DNA 结合染料和直接 PCR 的组合允许一种准确的方法来测量个体表现出不同脱落程度的程度。这项小型研究，涉及 11 名捐献者，表明个体分为三个明显的组：重度、中度和轻度脱落者，无论使用哪只手。
