GenSeizer: a Multiplex PCR-Based Targeted Gene Sequencing Platform for Rapid and Accurate Identification of Major Mycobacterium Species

Bing Li; Liyun Xu; Qi Guo; Jianhui Chen; Yanan Zhang; Wenpan Huang; Zhemin Zhang; Lizhong Han; Xiaogang Xu; Haiqing Chu

doi:10.1128/JCM.00584-20

GenSeizer: a Multiplex PCR-Based Targeted Gene Sequencing Platform for Rapid and Accurate Identification of Major Mycobacterium Species

J Clin Microbiol. 2021 Jan 21;59(2):e00584-20. doi: 10.1128/JCM.00584-20. Print 2021 Jan 21.

Authors

Bing Li^#¹, Liyun Xu^#¹, Qi Guo^{1

2}, Jianhui Chen^{1

2}, Yanan Zhang³, Wenpan Huang³, Zhemin Zhang¹, Lizhong Han⁴, Xiaogang Xu⁵, Haiqing Chu^{6

7}

Affiliations

¹ Department of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China.
² Tongji University School of Medicine, Shanghai, China.
³ Shanghai Morgene Biotechnology Co., Ltd., Shanghai, China.
⁴ Department of Clinical Microbiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
⁵ Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China xxg5022@126.com chu_haiqing@126.com.
⁶ Department of Respiratory Medicine, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China xxg5022@126.com chu_haiqing@126.com.
⁷ Shanghai Key Laboratory of Tuberculosis, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China.

^# Contributed equally.

Abstract

Mycobacterium tuberculosis and nontuberculous mycobacterium (NTM) infections often exhibit similar clinical symptoms. Timely and effective treatment relies on the rapid and accurate identification of species and resistance genotypes. In this study, a new platform (GenSeizer), which combines bioinformatics analysis of a large data set and multiplex PCR-based targeted gene sequencing, was developed to identify 10 major Mycobacterium species that cause pulmonary, as well as extrapulmonary, human diseases. The simultaneous detection of certain erm(41) and rrl resistance genotypes in M. abscessus was also feasible. This platform was specific and sensitive and exhibited no cross-reactivity among reference strains and a detection limit of 5 DNA copies or 50 CFU Mycobacterium/ml. In a blind comparison, GenSeizer and multigene sequencing showed 100% agreement in the ability to identify 88 clinical Mycobacterium isolates. The resistance genotypes, confirmed by whole-genome sequencing of 30 M. abscessus strains, were also correctly identified by GenSeizer 100% of the time. These results indicate that GenSeizer is an efficient, reliable platform for detecting major pathogenic Mycobacterium species.

Keywords: Mycobacterium tuberculosis; multiplex PCR; nontuberculous mycobacteria; targeted gene sequencing.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Genotype
Humans
Multiplex Polymerase Chain Reaction
Mycobacterium Infections, Nontuberculous* / diagnosis
Mycobacterium tuberculosis* / genetics
Nontuberculous Mycobacteria / genetics