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浙江大学学报(医学版)  2021, Vol. 50 Issue (6): 748-754    DOI: 10.3724/zdxbyxb-2021-0158
2019冠状病毒病     
纳米孔测序技术在2019冠状病毒病重型患者继发感染诊断中的应用
贾小芳1,张小楠1,凌云2,张欣宇1,田棣1,廖怡馨1,易志刚1,卢洪洲3,*()
1.上海市公共卫生临床中心科学研究部,上海 201508
2.上海市公共卫生临床中心感染科,上海 201508
3.上海市公共卫生临床中心感染与免疫科,上海 201508
Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19
JIA Xiaofang1,ZHANG Xiaonan1,LING Yun2,ZHANG Xinyu1,TIAN Di1,LIAO Yixin1,YI Zhigang1,LU Hongzhou3,*()
1. Scientific Research Department, Shanghai Public Health Clinical Center, Shanghai 201508, China;
2. Infectious Disease Department, Shanghai Public Health Clinical Center, Shanghai 201508, China;
3. Department of Infection and Immunology, Shanghai Public Health Clinical Center, Shanghai 201508, China
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摘要:

目的:探讨纳米孔测序技术在2019冠状病毒病(COVID-19)重型患者继发感染诊疗中的应用价值。方法:共入组了来自3例COVID-19重型患者的77份临床标本,所有标本均经过热灭活后进行基于磁珠富集的总核酸抽提,构建DNA文库,通过纳米孔测序技术进行病原体检测。测序数据采用Centrifuge软件进行病原体数据库比对和R语言差异分析,以获得病原微生物的鉴定结果。比较纳米孔测序结果与呼吸道病原体筛查多重定量聚合酶链反应(PCR)检测及同一时期临床微生物检验结果,以验证纳米孔测序技术的有效性。结果:纳米孔测序结果显示,44份标本检出病原体(阳性率为57.1%),包括肺炎克雷伯菌、表皮葡萄球菌、屎肠球菌和狡诈菌等。其中,肺炎克雷伯菌、表皮葡萄球菌和屎肠球菌均在基于微生物培养的临床微生物检验中检出;肺炎克雷伯菌同时在呼吸道病原体筛查多重定量PCR检测中检出;狡诈菌仅在纳米孔测序中检出,综合考虑患者的临床症状进行抗菌药物治疗,患者的感染情况得到控制。结论:纳米孔测序可快速鉴定临床标本中的潜在病原体,辅助COVID-19重型患者的临床诊断和治疗方案的制订。

关键词: 2019冠状病毒病严重急性呼吸综合征冠状病毒2纳米孔测序宏基因组测序病原体检测    
Abstract:

Objective: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). Methods: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. Results: Nanopore sequencing results showed that positive pathogen were obtained in 44 specimens (57.1%). The potential pathogens identified by nanopore sequencing included Klebsiella pneumoniae, Staphylococcus epidermidis, Enterococcus faecium and Dolosigranulum pigrum, et al. Klebsiella pneumoniae, Staphylococcus epidermidis, Enterococcus faecium were also detected in clinical microbiological culture-based detection; Klebsiella pneumoniae was detected in respiratory pathogen screening qPCR panel; Dolosigranulum pigrum was only detected by the nanopore sequencing technique. Comprehensive considerations with the clinical symptoms, the patient was treated with antibiotics against Dolosigranulum pigrum, and the infection was controlled. Conclusion:Nanopore sequencing may assist the diagnosis and treatment of severe COVID-19 patients through rapid identification of potential pathogens.

Key words: Coronavirus disease 2019    Severe acute respiratory syndrome coronavirus 2    Nanopore sequencing    Metagenomics next-generation sequencing    Pathogen detection
收稿日期: 2021-06-05 出版日期: 2022-03-22
CLC:  R372  
基金资助: 上海市科学技术委员会第二批应急科技攻关专项(20411950200);上海市2020年度“科技创新行动计划”医学创新研究专项(20Z11900900);国家自然科学基金(81801991);中国肝炎防治基金会天晴肝病研究基金(TQGB20200164)
通讯作者: 卢洪洲     E-mail: luhongzhou@fudan.edu.cn
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引用本文:

贾小芳,张小楠,凌云,张欣宇,田棣,廖怡馨,易志刚,卢洪洲. 纳米孔测序技术在2019冠状病毒病重型患者继发感染诊断中的应用[J]. 浙江大学学报(医学版), 2021, 50(6): 748-754.

JIA Xiaofang,ZHANG Xiaonan,LING Yun,ZHANG Xinyu,TIAN Di,LIAO Yixin,YI Zhigang,LU Hongzhou. Application of nanopore sequencing in diagnosis of secondary infections in patients with severe COVID-19. J Zhejiang Univ (Med Sci), 2021, 50(6): 748-754.

链接本文:

https://www.zjujournals.com/med/CN/10.3724/zdxbyxb-2021-0158        https://www.zjujournals.com/med/CN/Y2021/V50/I6/748

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.

标本编号

患者编号

采集日期

标本类型

测序检出的

病原体名称

匹配总序列数

log2FC

序列覆盖率(%)

平均

覆盖深度

最大

覆盖深度

1

P1

2021年1月31日

痰液

黏滑罗斯菌

720

7.87

67.56

2.73

49

殊异韦荣菌

1401

4.94

36.09

0.10

71

2

P1

2021年2月16日

血浆

表皮葡萄球菌

3251

6.58

1.74

5.01

419

3

P2

2021年1月8日

肺泡灌洗液

嗜麦芽窄食单胞菌

113

3.50

3.32

0.06

8

4

P2

2021年1月8日

血浆

洛菲不动杆菌

1137

8.60

14.47

159.01

1110

5

P2

2021年1月12日

血浆

黄体微球菌

1968

6.66

0.28

0.65

390

6

P2

2021年1月17日

血浆

狡诈菌

4216

5.25

1.29

10.20

1762

7

P2

2021年1月27日

肺泡灌洗液

肺炎克雷伯菌

20?019

3.54

3.01

13.20

1561

8

P2

2021年1月31日

血浆

奥斯陆莫拉菌

2241

6.63

4.60

2.34

221

小韦荣球菌

570

无穷大

0.70

0.38

122

9

P3

2021年2月1日

痰液

屎肠球菌

36?863

5.02

6.20

24.83

3422

10

P3

2021年2月4日

痰液

纤细弯曲杆菌

550

9.97

0.30

1.61

543

11

P3

2021年2月12日

血浆

抗辐射不动杆菌

256

无穷大

11.17

7.01

144

12

P3

2021年2月16日

痰液

人γ疱疹病毒4

137

无穷大

47.70

2.93

30

表 1  来自2019冠状病毒病重型患者的77份临床标本经纳米孔测序鉴定的部分潜在病原体
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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