Princess Margaret Cancer Centre, University Health Network, Toronto, ON, Canada.
Department of Medical Biophysics, Temerty Faculty of Medicine, University of Toronto, Toronto, ON, Canada.
PLoS One. 2023 May 2;18(5):e0285214. doi: 10.1371/journal.pone.0285214. eCollection 2023.
Saliva is an emerging source of disease biomarkers, particularly for cancers of the head and neck. Although analysis of cell-free DNA (cfDNA) in saliva holds promise as a liquid biopsy for cancer detection, currently there are no standardized methodologies for the collection and isolation of saliva for the purposes of studying DNA. Here, we evaluated various saliva collection receptacles and DNA purification techniques, comparing DNA quantity, fragment size, source, and stability. Then, using our optimized techniques, we tested the ability to detect human papillomavirus (HPV) DNA- a bona fide cancer biomarker in a subset of head and neck cancers- from patient saliva samples. For saliva collection, we found that the Oragene OG-600 receptacle yielded the highest concentration of total salivary DNA as well as short fragments <300 bp corresponding to mononucleosomal cell-free DNA. Moreover, these short fragments were stabilized beyond 48 hours after collection in contrast to other saliva collection receptacles. For DNA purification from saliva, the QIAamp Circulating Nucleic Acid kit yielded the highest concentration of mononucleosome-sized DNA fragments. Freeze-thaw of saliva samples did not affect DNA yield or fragment size distribution. Salivary DNA isolated from the OG-600 receptacle was found to be composed of both single and double-stranded DNA, including mitochondrial and microbial sources. While levels of nuclear DNA were consistent over time, levels of mitochondrial and microbial DNA were more variable and increased 48 hours after collection. Finally, we found that HPV DNA was stable in OG-600 receptacles, was reliably detected within the saliva of patients with HPV-positive head and neck cancer, and was abundant among mononucleosome-sized cell-free DNA fragments. Our studies have defined optimal techniques for isolating DNA from saliva that will contribute to future applications in liquid biopsy-based cancer detection.
唾液是疾病生物标志物的新兴来源，特别是对头颈癌。虽然游离 DNA（cfDNA）在唾液中的分析有望成为癌症检测的液体活检，但目前尚无用于收集和分离唾液以研究 DNA 的标准化方法。在这里，我们评估了各种唾液收集容器和 DNA 纯化技术，比较了 DNA 数量、片段大小、来源和稳定性。然后，使用我们优化的技术，从患者的唾液样本中测试了检测人乳头瘤病毒（HPV）DNA 的能力-这是头颈部癌症中真正的癌症生物标志物。对于唾液收集，我们发现 Oragene OG-600 容器产生了最高浓度的总唾液 DNA 以及对应于单核小体游离 DNA 的<300bp 的短片段。此外，与其他唾液收集容器相比，这些短片段在收集后 48 小时以上得到稳定。对于从唾液中纯化 DNA，QIAamp 循环核酸试剂盒产生了最高浓度的单核小体大小的 DNA 片段。唾液样本的冻融处理不影响 DNA 产量或片段大小分布。从 OG-600 容器中分离出的唾液 DNA 由单链和双链 DNA 组成，包括线粒体和微生物来源。虽然核 DNA 的水平随时间保持一致，但线粒体和微生物 DNA 的水平更具可变性，并且在收集后 48 小时增加。最后，我们发现 HPV DNA 在 OG-600 容器中稳定，在 HPV 阳性头颈部癌症患者的唾液中可靠地检测到，并且在单核小体大小的游离 DNA 片段中丰富。我们的研究定义了从唾液中分离 DNA 的最佳技术，这将有助于未来在基于液体活检的癌症检测中的应用。
