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

Genotypic methods for the determination of antimicrobial resistance in Mycobacterium tuberculosis (M.tb) require expensive instruments, which limits their availability in peripheral locations. We present a minimally instrumented method for the detection of the four most common mutations associated with rifampicin resistance in M.tb: S531L, H526Y, H526D, and D516V. The detection is based on the oligonucleotide ligation assay, coupled with lateral flow readout. The assay can detect wild-type and mutant DNA from as few as 10 and 100 gene copies per reaction, respectively. In heterozygous samples, the assay can detect <3% mutant DNA for all 4 mutations. Preliminary validation of the assay was carried out using genomic DNA extracted from 29 M.tb isolates being cultured at the ICMR-National Institute for Research in Tuberculosis in Chennai, India. The assay achieved a sensitivity and specificity of 100% for the detection of M.tb, and 90.90% and 100% respectively, for the detection of rifampicin resistance. The assay is simple to extend to other resistance-causing mutations and may aid in the reduction of instrumentation associated with current TB genotypic AMR detection.