Development of SNP-LAMP Combined with Lateral Flow Dipstick to Detect the S531L rpoB Gene Mutation in...

Background: Tuberculosis (TB) remains a primary global health concern, despite the widespread availability of effective chemotherapeutic interventions. The emergence and dissemination of drug-resistant strains of Mycobacterium tuberculosis, particularly those exhibiting resistance to rifampicin, present significant obstacles to the success of TB control programs. Consequently, there is an urgent need for rapid, sensitive, and specific molecular diagnostic tools to inform timely clinical decision-making and reduce the transmission of disease. Loop-mediated isothermal amplification (LAMP) has gained attention as a promising alternative to conventional polymerase chain reaction (PCR) techniques. This method, which facilitates DNA amplification under constant temperature conditions, offers advantages including high specificity, rapid turnaround time, and operational simplicity-features that render it especially suitable for implementation in resource-limited settings. Methods: In this study, a LAMP assay targeting the rpoB gene was developed, with particular focus on detecting the codon 531 C→T mutation associated with rifampicin resistance. A set of four to six primers was designed to recognize six distinct regions of the target sequence. Allele-specific amplification was achieved by incorporating a deliberate single nucleotide mismatch at the 3' terminus of the B2 primer to enable precise discrimination between wild-type and mutant alleles. The assay was conducted at an optimized temperature of 61 °C for 60 min, followed by visual detection using a lateral flow dipstick (LFD) within five minutes. Results: The LAMP-LFD assay demonstrated 100% concordance with drug susceptibility testing (DST) and DNA sequencing. No cross-reactivity with wild-type strains was observed, underscoring the assay's high specificity. Conclusions: This platform offers a robust, field-deployable solution for detecting the codon 531 C→T mutation associated with rifampicin resistance in low-resource settings.