Zheng Chuang-Ming, Kang Ho Won, Moon Seongmin, Byun Young Joon, Kim Won Tae, Choi Yung Hyun, Moon Sung-Kwon, Piao Xuan-Mei, Yun Seok Joong
Key Laboratory of Cellular Function and Pharmacology of Jilin Province, Yanbian University, Yanji, China.
Department of Urology, Chungbuk National University College of Medicine, Cheongju, Korea.
Investig Clin Urol. 2025 May;66(3):272-280. doi: 10.4111/icu.20240454.
This study aimed to evaluate and optimize microbial DNA extraction methods from urine, a non-invasive sample source, to enhance DNA quality, purity, and reliability for urinary microbiome research and biomarker discovery in bladder cancer.
A total of 302 individuals (258 with genitourinary cancers and 44 with benign urologic diseases) participated in this study. Urine samples were collected via sterile catheterization, resulting in 445 vials for microbial analysis. DNA extraction was performed using three protocols: the standard protocol (SP), water dilution protocol (WDP), and chelation-assisted protocol (CAP). DNA quality (concentration, purity, and contamination levels) was assessed using NanoDrop spectrophotometry. Microbial analysis was conducted on 138 samples (108 cancerous and 30 benign) using 16S rRNA sequencing. Prior to sequencing on the Illumina MiSeq platform, Victor 3 fluorometry was used for validation.
WDP outperformed other methods, achieving significantly higher 260/280 and 260/230 ratios, indicating superior DNA purity and reduced contamination, while maintaining reliable DNA yields. CAP was excluded due to poor performance across all metrics. Microbial abundance was significantly higher in WDP-extracted samples (p<0.0001), whereas SP demonstrated higher alpha diversity indices (p<0.01), likely due to improved detection of low-abundance taxa. Beta diversity analysis showed no significant compositional differences between SP and WDP (p=1.0), supporting the reliability of WDP for microbiome research.
WDP is a highly effective and reliable method for microbial DNA extraction from urine, ensuring high-quality and reproducible results. Future research should address sample variability and crystal precipitation to further refine microbiome-based diagnostics and therapeutics.
本研究旨在评估和优化从尿液（一种非侵入性样本来源）中提取微生物DNA的方法，以提高DNA质量、纯度和可靠性，用于膀胱癌尿液微生物组研究和生物标志物发现。
共有302名个体（258例泌尿生殖系统癌症患者和44例良性泌尿系统疾病患者）参与本研究。通过无菌导尿收集尿液样本，共获得445瓶用于微生物分析的样本。使用三种方案进行DNA提取：标准方案（SP）、水稀释方案（WDP）和螯合辅助方案（CAP）。使用NanoDrop分光光度法评估DNA质量（浓度、纯度和污染水平）。对138份样本（108份癌性样本和30份良性样本）进行16S rRNA测序以进行微生物分析。在Illumina MiSeq平台上测序之前，使用Victor 3荧光测定法进行验证。
WDP的表现优于其他方法，260/280和260/230比值显著更高，表明DNA纯度更高且污染减少，同时保持了可靠的DNA产量。由于在所有指标上表现不佳，CAP被排除。WDP提取的样本中微生物丰度显著更高（p<0.0001），而SP显示出更高的α多样性指数（p<0.01），这可能是由于对低丰度分类群的检测得到了改善。β多样性分析表明SP和WDP之间没有显著的组成差异（p=1.0），支持了WDP在微生物组研究中的可靠性。
WDP是一种从尿液中提取微生物DNA的高效可靠方法，可确保高质量和可重复的结果。未来的研究应解决样本变异性和晶体沉淀问题，以进一步完善基于微生物组的诊断和治疗方法。
