先前的研究表明,人类来源的环境DNA(eDNA)可以从天然水体中回收,从这些环境样本中生成DNA谱可能有助于法医调查。然而,影响检测人类eDNA概率的因素和最佳采样方案的设计存在基础知识空白。其中之一是理解eDNA信号与有效捕获eDNA颗粒所需的最适当的过滤器尺寸最强烈相关的颗粒尺寸。这项研究评估了与从淡水样品中回收的人血液和皮肤细胞的不同粒径相关的线粒体eDNA的量。样品(300mL)取自实验10L的淡水罐,其中掺有50μL的人血液或皮肤细胞,通过在淡水中用力摩擦手2分钟而沉积。通过使250mL实验水样品通过六个不同的过滤器孔径(0.1至8μm)来收集子样品。在72小时内加标之后以四个时间间隔重复该过程,以评估回收的eDNA的量的粒度是否随着eDNA降解而改变。使用针对HV1线粒体基因区域的人类特异性定量聚合酶链反应(qPCR)测定,确定了与不同粒径分数相关的线粒体eDNA的总量。在人类血液的情况下,在0h,0.45微米的过滤器孔径捕获了最大量的线粒体eDNA,捕获42%的检测到的eDNA。然后在48小时后改变了模式,5μm过滤器孔径捕获最大量的eDNA(67%),和81%的eDNA在72小时。值得注意的是,十倍稀释被证明是在所有时间点增强从8μm过滤器中回收eDNA的有价值的策略,主要是由于在血红蛋白中观察到的PCR抑制。对于人类皮肤细胞,从8μm的过滤器孔径中回收了最大量的eDNA,并且在时间上是一致的(捕获37%,56%,和88%的eDNA在0小时,48小时,和分别为72小时)。不同细胞类型之间回收的eDNA数量存在明显差异,在一些法医场景中,可能存在多种细胞类型。这些结果表明,最好使用5μm的过滤器孔径来捕获人体血液,使用8μm的过滤器孔径来捕获人体皮肤细胞,以最大程度地从淡水样品中回收DNA。根据对eDNA有贡献的细胞类型,可以采用不同过滤器孔径的组合来优化从水样中回收人DNA。这项研究为优化从水生环境中有效回收人类eDNA的策略提供了基础,为其在法医和环境科学中的广泛应用铺平了道路。
Previous studies have shown that environmental DNA (
eDNA) from human sources can be recovered from natural bodies of water, and the generation of DNA profiles from such environmental samples may assist in forensic investigations. However, fundamental knowledge gaps exist around the factors influencing the probability of detecting human eDNA and the design of optimal sampling protocols. One of these is understanding the particle sizes eDNA signals are most strongly associated with and the most appropriate filter size needed for efficiently capturing
eDNA particles. This study assessed the amount of mitochondrial eDNA associated with different particle sizes from human blood and skin cells recovered from freshwater samples. Samples (300 mL) were taken from experimental 10 L tanks of freshwater spiked with 50 µL of human blood or skin cells deposited by vigorously rubbing hands together for two minutes in freshwater. Subsamples were collected by passing 250 mL of experimental water sample through six different filter pore sizes (from 0.1 to 8 µm). This process was repeated at four time intervals after spiking over 72 hours to assess if the particle size of the amount of eDNA recovered changes as the
eDNA degrades. Using a human-specific quantitative polymerase chain reaction (qPCR) assay targeting the HV1 mitochondrial gene region, the total amount of mitochondrial eDNA associated with different particle size fractions was determined. In the case of human blood, at 0 h, the 0.45 µm filter pore size captured the greatest amount of mitochondrial eDNA, capturing 42 % of the
eDNA detected. The pattern then changed after 48 h, with the 5 µm filter pore size capturing the greatest amount of eDNA (67 %), and 81 % of
eDNA at 72 h. Notably, a ten-fold dilution proved to be a valuable strategy for enhancing eDNA recovery from the 8 µm filter at all time points, primarily due to the PCR inhibition observed in hemoglobin. For human skin cells, the greatest amounts of eDNA were recovered from the 8 µm filter pore size and were consistent through time (capturing 37 %, 56 %, and 88 % of eDNA at 0 hours, 48 hours, and 72 hours respectively). There is a clear variation in the amount of eDNA recovered between different cell types, and in some forensic scenarios, there is likely to be a mix of cell types present. These results suggest it would be best to use a 5 µm filter pore size to capture human blood and an 8 µm filter pore size to capture human skin cells to maximize DNA recovery from freshwater samples. Depending on the cell type contributing to the eDNA, a combination of different filter pore sizes may be employed to optimize the recovery of human DNA from water samples. This study provides the groundwork for optimizing a strategy for the efficient recovery of human eDNA from aquatic environments, paving the way for its broader application in forensic and environmental sciences.