Identification of <em>Mycobacterium tuberculosis</em> and its Drug Resistance by Targeted Nanopore Sequencing Technology

Tuberculosis (TB) remains the leading cause of human mortality in infectious diseases. Drug-resistant TB, particularly multidrug-resistant TB and extensively drug-resistant TB, poses a pressing clinical and public health challenge. The main causative agents of TB are known as <i>Mycobacterium tuberculosis</i> (MTB), which exhibits a highly complex drug resistance profile. Traditional culture-based phenotypic drug susceptibility testing is time-consuming, and PCR-based assays are restricted to detecting known mutational hotspots. In this study, we present a protocol leveraging high-throughput nanopore sequencing technology in conjunction with multiplex PCR, termed targeted nanopore sequencing, for the identification of MTB and analysis of its drug resistance. Our method for MTB drug resistance assessment offers the benefits of being culture-free, efficient, high-throughput, and highly accurate, which could significantly aid in clinical patient management and the control of TB infections. Key features • Targeted nanopore sequencing detects 18 genes simultaneously linked to antibiotic resistance in MTB. • The method provides broad drug resistance profiles for 14 first- and second-line anti-TB drugs without bacterial culture. • The expedited turnaround time of the process is approximately 7.5 h with a detection limit of 10² bacteria/mL.