宏基因组Nanopore测序在儿童呼吸道病原微生物快速检测中的应用价值

The application value of Nanopore metagenomics sequencing in the rapid detection of pediatric respiratory tract pathogens

目的探索宏基因组Nanopore(三代)测序在儿童呼吸道病原微生物快速检测中的应用价值。方法对收集的1例重症肺炎患儿的肺泡灌洗液标本,传统方法检测为腺病毒抗原阳性,肺炎支原体和EB病毒PCR检测阳性,患儿经治疗痊愈出院。以此肺泡灌洗液标本提取DNA后,分别行宏基因组三代和二代测序。建立微生物参考基因组数据库,建立三代测序数据实时分析流程,分别将三代和二代测序数据和参考数据库比对,检测并鉴定病原微生物。结果三代、二代测序和传统方法检测从收到样本至检出腺病毒用时分别为3.2 h、55.5 h和48 h,检测费用人民币4200、3600和1790元。三代测序在开始后的12 min即检出1093条腺病毒序列,测序完成后,共得到4920000条序列,检出腺病毒99284条序列,占总序列数的2.02%;检出肺炎支原体和EB病毒序列各1条。二代测序检出的微生物组成和三代类似。结论宏基因组三代测序能实现对所有潜在儿童呼吸道病原微生物的快速实时检测。

Objective To explore the clinical application value of Nanopore metagenomics sequencing in the rapid detection of pediatric respiratory tract pathogens.Methods We collected a bronchoalveolar lavage fluid sample from a severe pneumonia patient,which was positive for adenovirus antigen by conventional tests and Mycoplasma pneumoniae and Epstein⁃Barr virus by PCR.The patient was discharged after recovery.After DNA extraction,Nanopore and Illumina sequencing were performed respectively.We constructed microbe reference genome database and developed real⁃time analysis pipeline for Nanopore sequencing data.Nanopore and Illumina sequencing data were mapped to the reference database to detect the pathogens.Results From receipt of sample to the detection of adenovirus,Nanopore,Illumina and conventional methods took 3.2 hours,55.5 hours and 48 hours respectively,and cost 4,200,3,600 and 1,790 RMB respectively.We detected 1,093 adenovirus reads at 12 minutes after the start of Nanopore sequencing.A total of 4,920,000 reads were sequenced.Among them,we detected 99,284 adenovirus reads,accounting for 2.02%of all the sequencing reads.In addition to adenovirus,Mycoplasma pneumoniae and Epstein⁃Barr virus reads were also detected.The same sample was validated by Illumina sequencing and the detected microbial composition was similar to Nanopore.Conclusion Nanopore metagenomics sequencing could rapidly and real⁃timely detect all potential pediatric respiratory track pathogens.