Assessing eDNA capture method from aquatic environment to optimise recovery of human mt-eDNA

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.