Single-Sample Melt-Based Screening for Rifampicin Susceptibility in the Emerging Mutation Hotspot at rpoB Codon 491

Based on sequencing data, mutations at rpoB codon 491 ofMycobacterium tuberculosisare associated with rifampicin resistance, but current commercial and WHO-endorsed genotypic tests fail to detect them. As a result, resistant infections go untreated, driving transmission and multidrug resistance. A real-time PCR assay by André et al. specifically screens for I491F but omits other codon 491 mutations. To address this gap, a single-sample screening method using asymmetric PCR followed by melt analysis was developed for the three sequence-identified variants, I491F/N/M. Each sample contained a melt probe matching the susceptible sequence, which, after asymmetric PCR spanning codon 491, hybridized with the excess strand to form a duplex. The duplex's melt temperature (T_m) was then measured. To enable single-sample classification, each reaction also included double-stranded L-DNA identical to the probe and wild-type PCR product duplex. Susceptibility was determined by the within-sample T_m difference between the probe-product and L-DNA duplexes. The approach was evaluated and compared to the André assay across two calibrated PCR instruments using synthetic rpoB wild-type and variant sequences. As expected, the André assay distinguished wild-type from I491F samples but misclassified I491N and I491M samples based on multisample T_m comparison. In contrast, our single-sample classification strategy used within-sample T_m differences, classifying samples as rifampicin-susceptible when the within-sample T_m difference was less than 0.83 °C. With this approach, the method achieved 100% sensitivity and 100% specificity across both PCR instruments. Although demonstrated for rpoB codon 491, this assay design is readily adaptable to any other sequence-identified, clinically significant mutation hotspot.