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

Abstract

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.