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Abstract
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.
Supplementary materials
Title
Additional methods and full data sets
Description
Section 1 Bacterial transformation of plasmids
Table S1 Sequences of oligomers
Section 2 Urea-TBE gel
Table S2 Cycle condition for OLA
Section 3 Denaturation of OLA products
Section 4 Optimising concentration of probes for LFA
Section 5 Clinical study data
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