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.分子溯源技术（Molecular Source Tracking, MST）可通过识别水环境中粪便源性细菌污染的来源菌种，改善社区公共健康状况。然而，该方法的大规模推广应用却受限于商用提取试剂盒的高昂成本与操作所需的专业技术门槛。为此，我们开发了一种通过热裂解方式从环境水样中提取DNA的方法，该方法具备操作简便、高效可扩展、易于获取且可实现半定量的优势。具体流程为：将水样过滤至聚碳酸酯膜（polycarbonate membrane）后，将该膜悬浮于AE缓冲液（AE buffer）中进行加热处理，随后离心，所得液体上清液可直接用于定量聚合酶链式反应（quantitative PCR, qPCR）分析。我们将所开发的滤膜-热裂解-离心（filter-heat-spin, FHS）提取方法与商用DNA提取试剂盒（Qiagen DNeasy）进行了对照实验。qPCR检测结果显示，传统试剂盒提取法的目标序列拷贝数均值可达2.4 copies/μL，但得益于更大的缓冲液使用体积，FHS法提取得到的DNA总量均值较传统试剂盒法高出5.1倍。此外，在为期五周的检测周期内，FHS法与试剂盒提取法的长期稳定性表现一致。进一步对比表明，FHS法的单样本提取成本仅约0.05美元，耗时15分钟即可完成；而常规试剂盒提取法的单样本提取成本约为4.48美元，耗时需50分钟。本研究提出的FHS提取法可实现高效且低成本的水样处理，进而降低了分子溯源技术（MST）的应用门槛。