宏基因组二代测序技术在外源病毒污染检测应用中的挑战

Challenges and breakthroughs in the application of metagenomic next-generation sequencing technology in the detection of exogenous virus

关键词:  二代测序  外源病毒  生物制品  病毒检测

Key words:  next-generation sequencing  exogenous virus  biologicals  virus detection

摘要:

外源病毒污染是威胁生物制品安全的核心风险，传统检测方法因灵敏度差异、广谱性有限及依赖动物实验等问题，难以应对低丰度或未知病原体的筛查需求。宏基因组二代测序（mNGS）技术凭借其高灵敏性与无偏性，为外源病毒检测提供了全新解决方案，并被ICH Q5A(R2)列为推荐方法。然而，宏基因组二代测序技术在实际应用中面临多重技术挑战：宿主核酸干扰（占比>99%）导致目标信号被掩盖，试剂污染与标签跳跃（Index Hopping）现象引发假阳性风险，数据库缺失与分析工具算法阈值设置争议加剧未知病原体误判，标准化流程缺失致使实验室间结果不可比等。本文结合典型案例，全面剖析宏基因组二代测序技术在实际应用中面对的挑战性问题，并针对性总结出多项优化方案，包括靶向宿主核酸剔除、双端UMI标记以及多中心标准化验证等。实现宏基因组二代测序技术向“监管级方法”发展，需通过技术创新驱动、数据库统一与监管协同，为生物制品安全评价提供坚实技术支撑。

Abstract:

Exogenous virus contamination represents a critical risk to biopharmaceutical safety. Conventional detection methods, constrained by insufficient sensitivity, limited broad-spectrum capability, and reliance on animal experimentation, struggle to address the screening demands for low-abundance or unknown pathogens. Metagenomic next-generation sequencing (mNGS), with its high sensitivity and unbiased nature, offers a novel solution for exogenous virus detection and has been endorsed as a recommended method in ICH Q5A(R2). However, mNGS faces multiple technical challenges in practical applications: host-derived nucleic acid interference (>99% of total content) obscures target signals, reagent contamination and index hopping introduce false-positive risks, incomplete databases and controversies over analytical algorithm thresholds exacerbate misidentification of unknown pathogens, and the lack of standardized protocols compromises inter-laboratory comparability. Through case studies, this paper comprehensively analyzes the challenges encountered in mNGS implementation and proposes targeted optimization strategies, including host nucleic acid depletion, dual-end unique molecular identifier (UMI) tagging, and multicenter standardization validation. To advance mNGS as a "regulatory-grade method," collaborative efforts in technological innovation, unified pathogen databases, and regulatory harmonization are essential to establish robust technical frameworks for biopharmaceutical safety evaluation.