The oral microbiome has become an emerging focus of oral cancer research, with growing evidence linking microbial communities to disease development, progression, and prognosis. However, there is limited consensus on optimal sampling strategies, storage methods, and analytical approaches. This narrative review critically evaluates current strategies for sampling, preservation, DNA extraction, sequencing, and data analysis in oral microbiome research related to oral cancer. We compared commonly used sampling methods, including saliva, oral rinse, swab, brush, and tissue biopsy, and reviewed preservation conditions, extraction kits, sequencing platforms, and analytical pipelines reported in recent oral microbiome studies. Sampling approaches affect microbial yield and site specificity. Saliva and oral rinse samples are convenient and noninvasive but may dilute lesion-specific microbial signals, whereas lesion-directed swabbing or brushing yields greater microbial biomass and biological relevance. Preservation media and storage temperature significantly influence microbial stability, and DNA extraction methods vary in their ability to remove host DNA. Although 16S rRNA gene sequencing remains the most common approach, shotgun metagenomics offers higher resolution and function insights but is still limited by clinical applicability. Differences in data pre- and post-processing models and normalization strategies further contribute to inconsistent microbial profiles. Given that oral mucosal sites differ markedly in structure and microenvironment, careful consideration is required to ensure that collected samples accurately represent the biological question being addressed. Methodological consistency across all workflow stages—from collection to analysis—is essential to generate reproducible, high-quality data and to enable reliable translation of oral microbiome research into clinical applications for cancer detection and risk assessment. Together, these insights provide a framework to guide future study design and support the development of clinically applicable microbiome-based biomarkers.
口腔微生物组已成为口腔癌研究的新兴焦点，越来越多的证据表明微生物群落与疾病的发生、发展和预后相关。然而，关于最佳采样策略、储存方法和分析手段的共识仍然有限。本文通过叙述性综述，对当前口腔癌相关微生物组研究中的采样、保存、DNA提取、测序及数据分析策略进行了批判性评估。我们比较了常用的采样方法，包括唾液、口腔漱洗液、拭子、刷取及组织活检，并回顾了近期口腔微生物组研究中报告的保存条件、提取试剂盒、测序平台和分析流程。采样方式影响微生物的获取量和部位特异性：唾液和口腔漱洗液样本便捷且无创，但可能稀释病灶特异性微生物信号；而针对病灶的拭子或刷取样本则能获得更高的微生物生物量及生物学相关性。保存介质和储存温度显著影响微生物稳定性，DNA提取方法在去除宿主DNA的能力上存在差异。尽管16S rRNA基因测序仍是最常用的方法，鸟枪法宏基因组测序能提供更高分辨率和功能信息，但其临床适用性仍受限。数据预处理与后处理模型以及标准化策略的差异进一步导致了微生物谱的不一致。鉴于口腔黏膜部位在结构和微环境上存在显著差异，需要仔细考量以确保采集的样本能准确反映所研究的生物学问题。从采集到分析的全流程方法学一致性，对于产生可重复的高质量数据、推动口腔微生物组研究可靠地转化为癌症检测和风险评估的临床应用至关重要。综上，这些见解为未来研究设计提供了指导框架，并支持开发具有临床适用性的微生物组生物标志物。
肿瘤(癌症)患者之家