基于探针捕获法的病原靶向高通量测序检测流程的建立和验证研究  

Establishment and Validation of Pathogen Targeted Next-generation Sequencing Process Based on Probe Capture Method

作　　者：李丽莉[1] 任珊珊 王嘉平 易玉婷 田超 刘东来[1] 许四宏[1] Li Lili;Ren Shanshan;Wang Jiaping;Yi Yuting;Tian Chao;Liu Donglai;Xu Sihong(National Institutes for Food and Drug Control,NMPA Key Laboratory for Quality Research and Evaluation of Medical Devices,NMPA Key Laboratory for Quality Research and Evaluation of In Vitro Diagnostics,Beijing 100050,China;Beijing GenePlus Genomics Institute,Beijing 102206,China)

机构地区：[1]中国食品药品检定研究院国家药品监督管理局医疗器械质量研究与评价重点实验室国家药品监督管理局体外诊断试剂质量研究与评价重点实验室,北京100050 [2]北京吉因加基因研究院,北京102206

出　　处：《中国药事》2025年第4期415-429,共15页Chinese Pharmaceutical Affairs

基　　金：中国食品药品检定研究院关键技术研究基金(编号GJJS-2022-2-2)。

摘　　要：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-generationsequencing(tNGS) process based on the probe capture method,as well as the automated implementationof it,and to verify the analytical performance.Methods:The factors influencing the tNGS methodologywere 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 ofmicrobes,including various bacteria,fungi,viruses,and atypical pathogens.As well as,the differences in limitof 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 increasesaccordingly,and the probe still maintained high specificity.The optimized tNGS demonstrated good analyzeperformance in terms of accuracy,limit of detection,precision,specificity linearity and so on.Under thepremise of the same limit of detection performance,the automated method was superior to manual operationin precision and cross-contamination resistance performance.Conclusion:The tNGS process established inthis study has good analytical performance and provides a new strategy for clinical pathogen detection.Futurestudies should focus on clinical sample confirmation and automated process validation to promote betterapplication of the technology in practical clinical work.

关 键 词：病原靶向高通量测序 探针捕获法 分析性能 自动化 

分 类 号：R917[医药卫生—药物分析学]