A Novel Multiplex qPCR Platform for Detection of Mycobacterium tuberculosis Drug Resistance Using Synthetic Mutation Standards and Fluorescence Quenching Probes

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis strains has intensified the need for rapid, accurate, and affordable molecular diagnostics. In this study, we report the development and analytical validation of a multiplex real-time PCR assay employing contact-quenching fluorescence detection, allele-specific primers, and 3′ blocked wild-type allele blockers for the detection of five clinically relevant resistance-associated mutations in the rpoB, katG , and inhA genes. Synthetic double-stranded linear DNA constructs bearing individual point mutations ( rpoB codons 516, 526, 531; katG codon 315; and inhA (–)15 promoter) were engineered using the Splicing by Overlap Extension (SOEing) technique and confirmed by Sanger sequencing. These were used as positive controls in a background of wild-type genomic DNA from clinical isolates. The assay demonstrated high specificity and sensitivity, with detection limits ranging from 25 to 50 copies per reaction and effective mutant allele detection even in the presence of a 70-fold excess of wild-type DNA. The multiplex qPCR platform showed consistent amplification efficiency (92 to 104%), minimal cross-reactivity, and reliable performance even under PCR-inhibitory conditions. Inclusion of internal amplification controls and compatibility with multiple fluorescence channels support the robustness of the system. This study presents the first application of contact-quenching-based allele-specific PCR for MDR TB detection and offers a scalable, cost-effective alternative to commercial diagnostics. With its modular design and high analytical performance, the platform shows strong potential for deployment in decentralized settings for early drug resistance surveillance and patient management.