中国媒介生物学及控制杂志 ›› 2021, Vol. 32 ›› Issue (4): 390-397.DOI: 10.11853/j.issn.1003.8280.2021.04.002

• 媒介生物病原学监测专题 • 上一篇    下一篇

纳米孔测序实时检测鼠传巴尔通体

栗冬梅1, 周若冰1, 李寿江2, 鲁亮1, 饶华祥3, 宋秀平1, 李庆多1, 刘起勇1   

  1. 1. 中国疾病预防控制中心传染病预防控制所媒介生物控制室, 传染病预防控制国家重点实验室, 感染性疾病诊治协同创新中心, 北京 102206;
    2. 青海省疾病预防控制中心, 青海 西宁 810007;
    3. 长治医学院, 山西 长治 046000
  • 收稿日期:2021-03-02 出版日期:2021-08-20 发布日期:2021-08-20
  • 通讯作者: 刘起勇,E-mail:liuqiyong@icdc.cn
  • 作者简介:栗冬梅,女,博士,研究员,主要从事病原生物学与疾病控制研究工作,E-mail:lidongmei@icdc.cn
  • 基金资助:
    国家科技重大专项(2017ZX10303404,2018ZX10712001)

Real-time detection of rodent-borne Bartonella by nanopore sequencing

LI Dong-mei1, ZHOU Ruo-bing1, LI Shou-jiang2, LU Liang1, RAO Hua-xiang3, SONG Xiu-ping1, LI Qing-duo1, LIU Qi-yong1   

  1. 1. State Key Laboratory of Infectious Disease Prevention and Control, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Department of Vector Biology and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China;
    2. Qinghai Center for Disease Control and Prevention, Xining, Qinghai 810007, China;
    3. Changzhi Medical College, Changzhi, Shanxi 046000, China
  • Received:2021-03-02 Online:2021-08-20 Published:2021-08-20
  • Supported by:
    Supported by the National Science and Technology Major Project of China (No. 2017ZX10303404, 2018ZX10712001)

摘要: 目的 以巴尔通体(Bartonella spp.)作为指示菌基于MinION纳米孔高通量测序平台评估一种针对鼠传病原体的实时检测技术。方法 选取巴尔通体培养阳性的野外鼠脾和肺组织样品,提取全基因组核酸,应用16S核糖体RNA(16S ribosomal RNA,16S rRNA)基因的通用引物进行常规PCR扩增后的桑格测序(Sanger sequencing)和纳米孔测序,比较一代测序和三代纳米孔测序的结果,检验纳米孔测序技术对鼠传病原体检测的可行性和准确性。结果 16S rRNA基因常规扩增后经桑格测序有12份样品未鉴定出巴尔通体,纳米孔测序全部鉴定出巴尔通体。每份样品用于分类分析的reads数为4~609 424个,reads长度以1 500 bp为主,平均准确率为79.2%~92.0%,以巴尔通体检出为主,其reads数为1~77 833个。应用云端实时分析软件EPI2ME在测序开始后30 min内即产生鉴定结果。此外,多个样品还鉴定到布鲁氏菌。原始浓度大小对测序数据产出有影响,低浓度总数据量和reads数量明显减少。清洗步骤可明显降低测序芯片中残留核酸片段,但是不能完全去除,残留比平均值为1.4%。结论 16S扩增子纳米孔测序,可用于啮齿动物脏器组织病原体的直接、实时检出鉴定,纳米孔MinION测序便携、简单的操作以及实时数据传输和分析将为现场检测和病原监测工作提供便利。

关键词: 巴尔通体, 纳米孔测序, 16S扩增子测序, 宏基因组学, 鼠传疾病

Abstract: Objective To evaluate a real-time detection technology for rodent-borne pathogens based on the MinION high-throughput nanopore sequencing platform with Bartonella spp. as an indicator. Methods The whole-genome DNA was extracted from the spleen and lung tissues of wild rodents with positive Bartonella culture results. Sanger sequencing following conventional PCR amplification (using universal primers of 16S ribosomal RNA [16S rRNA] gene) and nanopore sequencing was performed and compared, and then evaluate the feasibility and accuracy of MinION nanopore sequencing technology in the detection of rodent-borne pathogens. Results By using the first-generation Sanger sequencing after the conventional 16S rRNA gene amplification, Bartonella was not identified in 12 samples. In contrast by using the third-generation nanopore sequencing, Bartonella was identified in all samples. The number of reads per sample used for taxonomic analysis ranged from 4 to 609 424, and the reads length was mainly 1 500 bp, with the mean accuracies ranging from 79.2% to 92.0%. Bartonella was the main pathogen detected, and its number of reads ranged from 1 to 77 833. By using cloud real-time analysis software EPI2ME, the identification results were generated within 30 minutes after the sequencing was started. In addition, Brucella spp. was also identified in some samples. The concentration of the original samples had an impact on the amount of sequencing data, with evidence showing that low-concentration samples had significantly less total data volume and number of reads. The cleaning procedure significantly reduced the residual nucleic acid fragments in the sequencing chip, but could not completely remove them, with a 1.40% residual ratio. Conclusion Nanopore sequencing of 16S rRNA gene amplicons can be used for direct and real-time detection and identification of pathogens in organs and tissues from rodents. Nanopore MinION sequencing provides convenience for on-site detection and pathogen monitoring with its portable and simple operation, and real-time data transmission and analysis.

Key words: Bartonella, Nanopore sequencing, 16S amplicon sequencing, Metagenomics, Rodent-borne diseases

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