Multi-probe single melt profiling enables mutation screening of long genomic regions for RRDR rifampicin susceptibility

Abstract Tuberculosis drug resistance can arise from single-base mutations that often cluster within genomic hotspots. For example, mutations within the 81-base rifampicin resistance-determining region (RRDR) of Mycobacterium tuberculosis rpoB account for over 96% of rifampicin-resistant cases globally. Mutation screening by post-PCR melt analysis uses sequence-specific derivative melt temperatures (T m s) to identify single-base changes. These single-base changes are more difficult to identify in longer regions like the RRDR because the mutation-induced T m shift decreases as target length increases. In this report, we preserve single-nucleotide sensitivity by dividing the long region into smaller non-overlapping segments and using multiple unlabeled melt probes to screen for non-mutated PCR products. This approach, Multi-probe Single Melt Profiling (MSMP), was applied to the RRDR to generate one intercalated melt profile with two distinct, sequence-sensitive T m s. A Receiver Operating Characteristic (ROC)-defined cutoff for susceptibility was identified. 100% classification accuracy was achieved, with all clinically significant RRDR variant samples falling below at least one segment-specific cutoff and all wild-type samples satisfying both cutoffs. By enabling multi-probe, temperature-based multiplexing in a single intercalated melt profile, MSMP achieves high-resolution screening of long-span hotspots. As longer hotspots emerge, this cost-effective, accessible framework provides a practical approach for susceptibility screening, particularly in resource-limited settings.