Rapid simultaneous detection of katG S315T and rpoB S531L mutations for molecular screening of multidrug-resistant tuberculosis using a dual LAMP-lateral flow assay

ABSTRACT The global spread of multidrug-resistant tuberculosis (MDR-TB) remains a major challenge to tuberculosis control programs, particularly in resource-limited settings where access to rapid molecular drug-susceptibility testing is limited. Resistance to isoniazid and rifampicin is most commonly associated with mutations in the katG and rpoB genes, respectively, making these loci valuable molecular markers for early MDR-TB screening. In this study, we developed a dual loop-mediated isothermal amplification assay coupled with lateral flow detection for the simultaneous identification of the katG S315T and rpoB S531L mutations in Mycobacterium tuberculosis . Mutation-specific primer sets were designed using mismatch amplification principles to achieve single-nucleotide specificity. Amplification was performed at 61°C for 60 min, followed by visual detection on a three-line lateral flow strip within 5 min. The assay demonstrated a preliminary detection threshold of approximately 10 CFU/mL equivalents and exhibited no cross-reactivity with wild-type M. tuberculosis H37Rv, non-tuberculous mycobacteria, or non-target resistance-associated mutations, including the inhA −15 promoter mutation and rpoB codon 526 variants. Preliminary clinical feasibility was evaluated using genetically characterized clinical sputum specimens and showed complete concordance with GenoType MTBDRplus and Sanger DNA sequencing results. Because the assay targets only two resistance-associated mutations, a negative result does not exclude isoniazid or rifampicin resistance mediated by alternative mutations. IMPORTANCE These proof-of-concept findings demonstrate the initial feasibility of a rapid, low-complexity molecular platform for the simultaneous detection of clinically important resistance-associated mutations in Mycobacterium tuberculosis . The assay represents a promising step toward low-complexity molecular screening for selected multidrug-resistant tuberculosis-associated mutations, bringing us closer to simplified testing in settings where access to conventional molecular drug-resistance testing remains limited.