Gene panel for MTBC drug resistance detection and genotyping by using third generation sequencing technologies

Tuberculosis represents one of the top ten causes of death being the first associated with an infectious disease. It is caused by Mycobacterium tuberculosis complex (MTBC) bacteria and it has high incidence and prevalence worldwide. To control the disease it is very important to perform an early and accurate diagnostic to prescribe a personal treatment for each patient since the first moment of the detection of the disease. Nowadays, the gold standard diagnosis technique requires bacterial culture, with diagnostic turnarounds of several weeks due to the slow growth of MTBC. Although effective first-line treatments for tuberculosis exist, the arise and spread of drug-resistance supposes a major public health concern. In this work, we developed a gene panel to detect drug-resistant tuberculosis strains by DNA Nanopore sequencing in a few hours. Our panel includes genes associated to MTBC resistance to first and second-line drugs, along with two phylogenetic determining regions. We designed this panel to be amplified in a single-tube multiplex PCR, and to be compatible with both short-read and long-read sequencing platforms. Importantly, we demonstrate that we are able to determine both the resistance profile and the lineage of MTBC strains directly from clinical samples, avoiding the long and cumbersome process of culturing the bacteria, providing an accurate diagnostic in few hours.