中国寄生虫学与寄生虫病杂志 ›› 2019, Vol. 37 ›› Issue (6): 727-729.doi: 10.12140/j.issn.1000-7423.2019.06.021

• 研究简报 • 上一篇    下一篇

宏基因组学二代测序技术在输入性疟疾诊断中的应用

李仁清(), 王小梅, 孙玉兰, 吕燕宁, 窦相峰, 王全意*()   

  1. 北京市疾病预防控制中心,北京市预防医学研究中心,北京 100013
  • 收稿日期:2019-08-16 出版日期:2019-12-30 发布日期:2019-12-31
  • 通讯作者: 王全意 E-mail:lirenqingz@sina.com;bjcdcxm@126.com
  • 作者简介:

    作者简介:李仁清(1977-),男,博士,副研究员,从事传染病的实验室诊断研究。E-mail: lirenqingz@sina.com

Application of metagenomic Next-Generation Sequencing in the diagnosis of imported malaria

Ren-qing LI(), Xiao-mei WANG, Yu-lan SUN, Yan-ning LV, Xiang-feng DOU, Quan-yi WANG*()   

  1. Beijing Municipal Center for Disease Prevention and Control, Beijing Research Center for Preventive Medicine, Beijing 100013, China
  • Received:2019-08-16 Online:2019-12-30 Published:2019-12-31
  • Contact: Quan-yi WANG E-mail:lirenqingz@sina.com;bjcdcxm@126.com

摘要:

通过对1例自非洲输入的疟疾病例血液样本进行宏基因组二代测序(mNGS)检测,探讨mNGS在实验室检测疟疾上的可行性。采集患者外周血,制备厚、薄血膜,显微镜观察疟原虫感染情况。提取患者全血样本核酸,进行疟原虫核酸实时荧光PCR检测。将提取的患者全血样本核酸同步构建文库,采用Illumina Miseq二代测序仪进行mNGS检测。采用Megan6软件分析测序数据及重叠群序列的BLASTn比对结果,分配模式为对齐碱基模式。镜检结果显示,患者为恶性疟原虫和三日疟原虫混合感染,其中恶性疟原虫密度 ≥ 100 000/μl,三日疟原虫密度 ≥ 300/μl。实时荧光PCR检测结果显示,恶性疟原虫核酸阳性,间日疟、三日疟和卵形疟原虫核酸均为阴性。mNGS获得了271 526条测序数据,其中合格序列有254 674条。剔除与人类参考基因组匹配的序列后,最终得到1 833条测序数据,其中非人源数据只占总合格数据的0.7%。将上述序列拼接后,获得408条100个核苷酸以上长度的重叠群数据。Megan6分析结果显示,恶性疟原虫的测序数据分别占到总对齐碱基的44.7%(29 667/66 374),其重叠群数据占重叠群总对齐碱基的34.5%(22 885/66 376),因此恶性疟原虫是最优势的病原微生物。基于测序数据和重叠群数据,该病原可溯源到恶性疟原虫标准株3D7。

关键词: 恶性疟原虫, 混合感染, 输入性疟疾, 宏基因组学二代测序

Abstract:

The feasibility and application of metagenomic Next-Generation Sequencing (mNGS) in the detection of Plasmodium spp. was investigated by testing a blood sample of a malaria case imported from Africa. The peripheral blood was collected from a malaria patient from Africa. The thick and thin blood smears were prepared and examined under a microscope. Nucleic acid of patient’s whole blood was extracted and detected by real-time PCR. A cDNA library was constructed and used for mNGS by using an Illumina Miseq sequencer. The BLASTn alignment results of the reads and contig sequences were analyzed using Megan6 software, and the assignment mode was aligned by base mode. Microscopic examination showed that the patient was a co-infection of P. falciparum and P. malariae, with lower proportion of P. malariae. The results of real-time PCR showed that the detection of P. falciparum nucleic acid was positive with all negative for P. vivax, P. malariae and P. ovarian. A total of 271 526 reads were obtained, of which 254 674 reads qualified. After filtered by mapping to the Human reference genome, 1 833 reads were finally obtained, and the non-human reads accounted for only 0.7% of the total qualified reads. After de novo, 408 contigs with a length of more than 100 nucleotides were obtained. The results of Megan6 analysis showed that the reads of P. falciparum accounted for 44.7% (29 667/66 374) of the total reads, and 34.5% (22 885/66 376) of the total contig. The results suggest that P. falciparum is the most dominant pathogen. Based on the reads and contig, the pathogen can be traced to the P. falciparum 3D7.

Key words: Plasmodium falciparum, Co-infection, Imported malaria, Metagenomic Next-Generation Sequencing

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