A novel one-tube nested fluorescence melting curve analysis for rapid detection of drug-resistant Mycobacterium tuberculosis

BACKGROUND: Rapidly detecting drug-resistant Mycobacterium tuberculosis (MTB) is essential for effective tuberculosis (TB) treatment. We have developed a one-tube nested fluorescence melting curve assay (OTN-FMCA) that allows rapid and simultaneous detection of resistance to rifampicin, isoniazid, fluoroquinolones, and bedaquiline. RESULTS: The OTN-FMCA uses nested PCR and locked nucleic acid (LNA)-modified probes to identify 44 mutations in six key genes (rpoB, katG, inhA, gyrA, gyrB, and atpE), achieving a detection limit of 5 copies per microliter and identifying up to 10% heterogeneous resistance. When tested on 198 culture-positive isolates, OTN-FMCA demonstrated high concordance with microarray susceptibility testing, with 100% sensitivity and 96.89% specificity. Further validation with 518 sputum samples confirmed its ability to classify patients into different drug resistance categories, including DS-TB, RR-TB, MDR-TB, pre-XDR-TB, and XDR-TB. OTN-FMCA offers a faster, more affordable, and more accessible alternative to current comprehensive drug resistance profiling methods, especially in resource-limited settings. CONCLUSIONS: OTN-FMCA is a promising tool for the rapid and accurate detection of drug resistance in MTB, which could improve the management of drug-resistant TB.