纳米孔测序技术在腺病毒疫情中实时检测应用研究

摘要

Objective To study the application of nanopore sequencing technology in pathogen detection for an adenovirus outbreak in Tangshan in February,2019,and to report the results of sequencing and analysis of samples processed with different methods.Methods Nasopharyngeal swabs from 16 severe cases and 16 mild cases were randomly selected.These samples were mixed and enriched for direct sequencing on the MinION sequencer.Real-time PCR was performed to detect adenovirus.The positive sample with the lowest Ct value was further sequenced directly using MnION.Results Eight severe cases and 15 mild cases were positive for adenovirus.The nanopore sequencing identified the first reads of adenovirus type 7 in 14 and 22 minutes on the mixed sample and single sample,respectively.Within 3 h of sequencing,99％ of the reference genome were covered.Phylogenetie analysis revealed that the strains in the outbreak had a close genetic relation with a strain isolated in Beijing.Conclusions Nanopore sequencing technology can be used for rapid adenovirus identification in epidemics and obtaining genome sequences.The results proved the potential value of MiniON in real-time detection of novel emerging or unknown or mutated pathogens.%目的 研究纳米孔测序技术在2019年2月河北省某市一起腺病毒疫情检测中的应用情况,并对不同样本处理结果进行描述和分析.方法 随机选择重症、轻症病例鼻咽拭子各16份,混合富集后通过MinION测序仪直接测序.采用实时荧光PCR方法检测腺病毒,进一步应用MinION对Ct值最低的单个腺病毒阳性样本进行直接测序.结果 8例重症、15例轻症病例荧光PCR检测为腺病毒阳性.MinION测序分别在14 min和22 min检测到混合样本和单个样本中7型腺病毒序列,两者3h内产生测序数据基本覆盖腺病毒参考基因组.系统发育分析发现疫情毒株与北京一株毒株具有共同遗传来源.结论 纳米孔测序技术可以快速检测疫情感染腺病毒并获得基因组序列,为MinION在新突发、未知或变异病原的检测应用提供了重要参考.