Cotton-based nucleic acid extraction

<strong>Abstract</strong>: Nucleic acid amplification technique (NAAT)-assisted detection is the primary intervention for pathogen molecular diagnostics. However, NAATs such as quantitative real-time polymerase chain reaction (qPCR) require prior purification or extraction of target nucleic acid from the sample of interest since the latter often contains polymerase inhibitors. Similarly, genetic disease screening is also reliant on the successful extraction of pure patient genomic DNA from the clinical sample. However, such extraction techniques traditionally utilize spin-column techniques that in turn require centralized high-speed centrifuges. This hinders any potential deployment of qPCR- or PCR-like NAAT methods in resource-constrained settings. The development of instrument-free nucleic acid extraction methods, especially those utilizing readily available materials would be of great interest and benefit to NAAT-mediated molecular diagnosis workflows in resource-constrained settings. In this report, we screened medical-grade cotton, a readily available over-the-counter biomaterial to extract <em>E. coli</em> genomic DNA (gDNA) spiked in 30%, 45%, and 60% serum. The extraction was carried out in a completely instrument-free manner using cotton and a sterilized toothpick and was completed in 30 min (with using chaotropic salt) or 10 min (without using chaotropic salt). The quality of the extracted DNA was then probed using PCR followed by agarose gel analysis, qPCR, and sequencing. Our method demonstrated that the high-quality DNA extraction could be performed both with or without using a chaotropic salt, albeit with a difference in the quality of the extracted DNA**摘要**：核酸扩增技术（Nucleic acid amplification technique, NAAT）辅助检测是病原体分子诊断的主流干预手段。然而，诸如实时定量聚合酶链式反应（quantitative real-time polymerase chain reaction, qPCR）这类NAAT技术，由于待测样本中往往含有聚合酶抑制剂，因此需要预先从样本中纯化或提取目标核酸。同理，遗传病筛查也依赖于从临床样本中成功获取高纯度的患者基因组DNA。传统核酸提取方法多采用离心柱技术，而该技术需依赖大型高速离心机，这极大限制了qPCR或类PCR型NAAT技术在资源匮乏地区的推广应用。开发无需专用仪器的核酸提取方案，尤其是依托易得材料的方法，对资源匮乏地区依托NAAT的分子诊断工作流程具有重要的应用价值与社会意义。本研究以市售易得的医用级棉作为生物材料，对掺入30%、45%及60%血清的大肠杆菌（E. coli）基因组DNA（genomic DNA, gDNA）开展提取筛选实验。整个提取过程完全无需专用仪器，仅使用医用棉与灭菌牙签即可完成，且可通过两种路径实现：添加离液盐时耗时30分钟，不添加离液盐时仅需10分钟。随后通过PCR结合琼脂糖凝胶分析、qPCR及测序对提取所得DNA的质量进行验证。实验结果表明，无论是否添加离液盐，本方法均可实现高质量的基因组DNA提取，仅在提取产物的质量上存在小幅差异。