A minimally instrumented method for the detection of rifampicin resistance-causing mutations in Mycobacterium tuberculosis utilizing lateral flow readout.

BACKGROUND: Genotypic methods for detecting antimicrobial resistance in Mycobacterium tuberculosis (M.tb) typically require expensive instrumentation, limiting their accessibility in peripheral and resource-limited settings. Rifampicin resistance is a key marker of multidrug-resistant tuberculosis (MDR-TB) and is primarily caused by mutations in the rpoB gene. Rapid, cost-effective, and minimally instrumented methods for detecting these mutations are essential for improving tuberculosis diagnostics, particularly in low-resource environments. This study addresses the need for a simple, sensitive, and specific assay to detect the four most common rifampicin resistance-associated mutations without relying on complex laboratory infrastructure.

RESULTS: We developed a minimally instrumented method to detect the four most prevalent mutations causing rifampicin resistance in M.tb-S531L, H526Y, H526D, and D516V. The method is based on the oligonucleotide ligation assay (OLA), coupled with lateral flow readout. The method successfully detected wild-type and mutant DNA at limits of 10 and 100 gene copies per reaction, respectively. It exhibited high sensitivity in heterozygous samples, detecting as low as 3 % mutant DNA for all four mutations. Validation with genomic DNA extracted from 29 M.tb isolates from the ICMR-National Institute for Research in Tuberculosis (NIRT), Chennai, India, demonstrated a sensitivity and specificity of 100 % for M.tb detection and 90.90 % and 100 %, respectively, for rifampicin resistance detection.

SIGNIFICANCE: This method provides a cost-effective and minimally instrumented alternative to conventional genotypic resistance detection methods. Its high sensitivity and specificity, combined with its ease of adaptation to other resistance-causing mutations, make it a promising tool for tuberculosis diagnostics in resource-limited settings. By reducing reliance on expensive laboratory infrastructure, this approach could facilitate more accessible and rapid detection of drug-resistant M.tb, improving global TB control efforts.