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. 2021 Dec 25;50(6):748-754.
doi: 10.3724/zdxbyxb-2021-0158.

Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19

Xiaofang Jia  1 , Xiaonan Zhang  1 , Yun Ling  2 , Xinyu Zhang  1 , Di Tian  1 , Yixin Liao  1 , Zhigang Yi  1 , Hongzhou Lu  3
Affiliations

Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19

Xiaofang Jia et al. Zhejiang Da Xue Xue Bao Yi Xue Ban. .

Abstract

To explore the application value of nanopore sequencing technique in the diagnosis and treatment of secondary infections in patients with severe coronavirus disease 2019 (COVID-19). A total of 77 clinical specimens from 3 patients with severe COVID-19 were collected. After heat inactivation, all samples were subjected to total nucleic acid extraction based on magnetic bead enrichment. The extracted DNA was used for DNA library construction, then nanopore real-time sequencing detection was performed. The sequencing data were subjected to Centrifuge software database species matching and R program differential analysis to obtain potential pathogen identification. Nanopore sequencing results were compared with respiratory pathogen qPCR panel screening and conventional microbiological testing results to verify the effectiveness of nanopore sequencing detection. Nanopore sequencing results showed that positive pathogen were obtained in 44 specimens (57.1%). The potential pathogens identified by nanopore sequencing included , , and , et al. , , were also detected in clinical microbiological culture-based detection; was detected in respiratory pathogen screening qPCR panel; was only detected by the nanopore sequencing technique. Comprehensive considerations with the clinical symptoms, the patient was treated with antibiotics against , and the infection was controlled. Nanopore sequencing may assist the diagnosis and treatment of severe COVID-19 patients through rapid identification of potential pathogens.

Keywords: Coronavirus disease 2019; Metagenomics next-generation sequencing; Nanopore sequencing; Pathogen detection; Severe acute respiratory syndrome coronavirus 2.

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Conflict of interest statement

所有作者均声明不存在利益冲突

Figures

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图 1
基于纳米孔测序的宏基因组测序流程图临床标本首先通过55 ℃热灭活处理，再用玻璃珠匀浆破碎和基于异硫氰酸胍盐裂解的方法抽提标本中的总核酸，获得的总核酸进行DNA文库构建，进一步用纳米孔测序进行分析. 测序数据的分析流程：首先去除reads中的接头序列，删除低质量和低复杂性的序列；随后用Centrifuge软件分析，对候选病原体的特定序列进行比对，将微生物reads分类为科、属或种进行鉴定；最后进行R语言分析，通过比较标本和对照中检测到相应物种的reads数比值，最终找出标本中可能存在的病原体. 整个测序分析检测流程的总时长约为32 h.
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图 2
2019冠状病毒病重型患者P2肺泡灌洗液标本多重定量PCR检测和纳米孔测序结果A：肺炎克雷伯菌多重定量PCR检测结果（Ct值为32.1）；B：肺炎克雷伯菌的纳米孔测序基因组覆盖率图.
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