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ORIGINAL RESEARCH article
Front. Microbiol.
Sec. Microbiotechnology
Volume 15 - 2024 |
doi: 10.3389/fmicb.2024.1331656
High-throughput nanopore targeted sequencing for efficient drug resistance assay of Mycobacterium tuberculosis
Provisionally accepted
Chen Tang
1,2
Lianpeng Wu
3
Machao Li
4
Jianyi Dai
5
Ye Shi
1,2
Qiongdan Wang
6
Feng Xu
1,2
Laibao Zheng
1,2
Xingxing Xiao
1,2
Junwen Cai
1,2
Yanjun Zhang
1,2
Yuting Yang
1,2
Xiaoqun Zheng
1,2*
Guangxin Xiang
1,2*- 1 School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang Province, China
- 2 Key Laboratory of Laboratory Medicine, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang Province, China
- 3 Department of Clinical Laboratory, Wenzhou Central Hospital, Wenzhou, China
- 4 National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
- 5 Department of Infectious Diseases, Wenzhou Central Hospital, Wenzhou, China
- 6 Department of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China
Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), is one of the urgent clinical problems and public health challenges. Culture-based phenotypic drug susceptibility testings (pDSTs) are time-consuming and PCR-based assays are limited to hotspot mutations. In this study we developed and validated a convenient and efficient approach based on the high-throughput nanopore sequencing technology combined with multiplex PCR, namely nanopore targeted sequencing (NTS), to simultaneously sequence 18 genes associated with antibiotic resistance in Mycobacterium tuberculosis (MTB). Analytical performance of NTS was evaluated, and 99 clinical samples were collected for evaluation of NTS clinical performance. The NTS results showed that MTB and its drug resistance was successfully identified in about 7.5 hours. Furthermore, while compared to pDST and Xpert MTB/RIF assay, NTS provided much more drug resistance information with 14 anti-TB drugs, and 20 clinical MTB drug-resistant cases were revealed. The mutations underlying these drug-resistant cases were all verified by Sanger sequencing. Our approach for this TB drug resistance assay possesses the advantages of culture-free, efficiency, high-throughput, and high accuracy, which would be very helpful for clinical patient management and TB infection control.
Keywords: Mycobacterium tuberculosis, Drug Resistance, Drug susceptibility testing, nanopore sequencing, targeted sequencing, multiplex PCR
Received: 01 Nov 2023; Accepted: 06 May 2024.
Copyright: © 2024 Tang, Wu, Li, Dai, Shi, Wang, Xu, Zheng, Xiao, Cai, Zhang, Yang, Zheng and Xiang. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Xiaoqun Zheng, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang Province, China
Guangxin Xiang, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou, Zhejiang Province, China
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