目的: 研究基于探针捕获法的病原靶向高通量测序（tNGS）检测流程的建立和优化,以及自动化的实现,并对分析性能进行验证研究。方法: 分析tNGS的影响因素,包括探针设计、生产质控、数据量和测序读长等,通过采用多种细菌、真菌、病毒和非典型病原体等62种微生物构建的参考盘进行分析性能评价,同时比较手工操作与自动化流程在检出限、精密度和交叉污染等性能指标的差异。结果: 62种微生物不同病原浓度的准确性、检出限、精密度、特异性及线性的结果显示随着探针覆盖度的提高,目标病原的检出限性能随之提高,且仍保持较高的特异性;经优化后的tNGS检测流程在准确性、检出限、精密度、特异性、线性等方面均表现出良好的分析性能;自动化流程在检出限性能保持一致的前提下,精密度和抗交叉污染性能均优于手工操作流程。结论: 研究建立的探针捕获法tNGS检测流程具有良好的分析性能,为临床病原检测提供了新策略。未来进一步研究应着重于临床样本确认、自动化流程的全面验证,以推动该技术在临床实际工作中更好的应用。
Objective: To study the establishment and optimization of the pathogen targeted next-generation sequencing (tNGS) process based on the probe capture method, as well as the automated implementation of it, and to verify the analytical performance. Methods: The factors influencing the tNGS methodology were analyzed, including the probe design and production quality control, data volume and sequencing read length. The analytical performance was evaluated by using a reference panel constructed with 62 types of microbes, including various bacteria, fungi, viruses, and atypical pathogens. As well as, the differences in limit of detection, precision and cross-contamination between manual operations and automated were compared. Results: The study showed that as the probe coverage increased, the limit of detection performance increases accordingly, and the probe still maintained high specificity. The optimized tNGS demonstrated good analyze performance in terms of accuracy, limit of detection, precision, specificity linearity and so on. Under the premise of the same limit of detection performance, the automated method was superior to manual operation in precision and cross-contamination resistance performance. Conclusion: The tNGS process established in this study has good analytical performance and provides a new strategy for clinical pathogen detection. Future studies should focus on clinical sample confirmation and automated process validation to promote better application of the technology in practical clinical work.
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