Positive control sequence for HF183.

Molecular source tracking (MST) can improve community health by enabling the identification of the source species of fecal bacteria contamination in waterways. However, widespread adoption of this method at a large scale is hindered by the cost of commercial extraction kits and the technical expertise required to use them. We developed a simpler, efficient, scalable, accessible, and semi-quantitative method to extract DNA from environmental water samples via heat lysis. After filtering water samples onto a polycarbonate membrane, the membrane is suspended in AE buffer, heated, and then centrifuged. The liquid supernatant is then used directly in quantitative PCR (qPCR) analysis. Our filter-heat-spin (FHS) extraction method was compared to a commercial DNA extraction kit (Qiagen DNeasy). In qPCR the traditional kit extraction yielded 2.4 times more copies/μL (on average) of the target sequence, but the FHS extraction yielded 5.1 times more DNA (on average) than the traditional extraction kit due to the higher volume of buffer used in the FHS extraction. Additionally, FHS maintained identical long-term stability as compared to the kit extraction over a five-week period. Further, the cost per FHS extraction is roughly $0.05 per sample and requires 15 minutes to complete, while the typical kit extraction is roughly $4.48 per sample and requires 50 minutes to complete. The proposed FHS extraction method allows for efficient and inexpensive water sample processing, which in turn, reduces barriers for the implementation of MST.