Development of a Point-of-Care Dual One-Step Recombinase-Aided PCR Assay for Rapid Identification of Mycobacterium tuberculosis gyrA Mutations Conferring Fluoroquinolone Resistance

Abstract

Background: Fluoroquinolone (FQ) resistance in Mycobacterium tuberculosis (MTB) is a major cause of treatment failure in multidrug-resistant tuberculosis (MDR-TB). This resistance primarily results from mutations within the quinolone resistance-determining region (QRDR) of the gyrA gene encoding DNA gyrase. Conventional phenotypic drug susceptibility testing (DST) is labor-intensive and time-consuming, making it unsuitable for rapid clinical decision-making. Therefore, developing a rapid, sensitive, and point-of-care testing (POCT) assay is of great importance. Methods: A cartridge-based POCT dual one-step recombinase-aided PCR (POCT-DO-RAP) assay was established for rapid detection of FQ resistance-associated mutations in MTB. Locked nucleic acid (LNA) probes were designed to enhance single-nucleotide discrimination for gyrA A90V and D94G mutations. Magnetic bead-based extraction enabled fully automated nucleic acid purification, while recombinase-aided amplification (RAA) and quantitative PCR (qPCR) were sequentially performed within a real-time PCR-based POCT device. The analytical performance of the POCT-DO-RAP assay was evaluated using recombinant plasmids (1–10⁵ copies/μL), H37Rv-simulated sputum samples and 128 clinical isolates. The POCT-DO-RAP assay was further validated using 88clinical samples and the results were compared with the conventional qPCR and the nested PCR followed by Sanger sequencing. Results: The optimized POCT-DO-RAP assay achieved limits of detection of 1 copy/reaction for the wild-type (WT) tube and 10 CFU/mL for the mutant-type (MT) tube, representing a 10-fold increase in sensitivity compared with conventional qPCR. The assay reliably detected mutant alleles even when they represented only 1% of mixed templates. Among 128 clinical isolates, the assay accurately differentiated 50 wild-type and 78 resistant strains, showing complete concordance with Sanger sequencing and no cross-reactivity. In clinical validation,9 samples negative by qPCR were confirmed as positive by both DO-RAP assay and nested PCR followed by Sanger sequencing. Conclusion: The POCT-DO-RAP assay developed in this study achieves a fully integrated "sample-in, result-out" workflow on a single device, offering ultra-high sensitivity, precise mutation discrimination, and excellent clinical concordance. This approach provides a promising molecular diagnostic tool for rapid detection of drug-resistant tuberculosis, particularly suitable for primary healthcare and resource-limited settings.