GenSeizer: A multiplex PCR-based targeted gene sequencing platform for rapid and accurate identification of major mycobacterium species

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.