Ivens Marco J R, Kamminga Sergio, Benchamach Kawtar, Akile Chaimae, Wessels Els, Claas Eric C J, Boers Stefan A
Center of Infectious Diseases, Medical Microbiology and Infection Control, Leiden University Medical Centre, Albinusdreef 2, Leiden, 2333 ZA, The Netherlands.
Sci Rep. 2025 Aug 5;15(1):28606. doi: 10.1038/s41598-025-14071-3.
The detection and identification of bacterial species in clinical samples are crucial for patient management and antibiotic treatment. When culture-based identification methods fail, 16 S rRNA gene next-generation sequencing (NGS) serves as a valuable alternative. However, its clinical utility is often limited by prolonged time to results (TtR) and limited species-level resolution. This study aimed to develop and validate a faster, more discriminative 16 S rRNA gene NGS workflow. Our current 16 S rRNA gene NGS protocol uses micelle-based PCR (micPCR) targeting the V4 region, followed by Illumina sequencing. This method ensures accurate quantification of 16 S rRNA gene copies in (low biomass) clinical samples by reducing PCR artefacts and correcting for background DNA contamination. To shorten the TtR and improve species-level determination, the micPCR protocol was adapted to amplify full-length 16s rRNA genes, followed by nanopore sequencing using the Flongle Flow Cell with automated data analysis using the Genome Detective platform. Testing with a synthetic microbial community and six clinical samples showed that the 16 S rRNA gene micPCR/nanopore sequencing protocol maintains good accuracy and sensitivity, reducing TtR to 24 h and enhancing species-level resolution. This optimized workflow improves clinical diagnostics, making it a valuable tool for guiding patient treatment decisions.
临床样本中细菌种类的检测和鉴定对于患者管理和抗生素治疗至关重要。当基于培养的鉴定方法失败时，16S rRNA基因下一代测序（NGS）是一种有价值的替代方法。然而，其临床应用通常受到结果报告时间（TtR）延长和物种水平分辨率有限的限制。本研究旨在开发和验证一种更快、更具区分性的16S rRNA基因NGS工作流程。我们目前的16S rRNA基因NGS方案使用基于胶束的PCR（micPCR）靶向V4区域，随后进行Illumina测序。该方法通过减少PCR假象并校正背景DNA污染，确保对（低生物量）临床样本中的16S rRNA基因拷贝进行准确定量。为了缩短TtR并改善物种水平的测定，对micPCR方案进行了调整，以扩增全长16s rRNA基因，随后使用Flongle流动槽进行纳米孔测序，并使用Genome Detective平台进行自动化数据分析。对合成微生物群落和六个临床样本的测试表明，16S rRNA基因micPCR/纳米孔测序方案保持了良好的准确性和灵敏度，将TtR缩短至24小时，并提高了物种水平分辨率。这种优化的工作流程改善了临床诊断，使其成为指导患者治疗决策的有价值工具。
