{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T07:02:52Z","timestamp":1772434972347,"version":"3.50.1"},"reference-count":35,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T00:00:00Z","timestamp":1772409600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Microbiol."],"abstract":"<jats:sec>\n                    <jats:title>Background<\/jats:title>\n                    <jats:p>\n                      Fluoroquinolone (FQ) resistance in\n                      <jats:italic>Mycobacterium tuberculosis<\/jats:italic>\n                      (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\n                      <jats:italic>gyrA<\/jats:italic>\n                      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.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Methods<\/jats:title>\n                    <jats:p>\n                      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\n                      <jats:italic>gyrA<\/jats:italic>\n                      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\u2013105 copies\/\u03bcL), H37Rv-simulated sputum samples and 128 clinical isolates. The POCT-DO-RAP assay was further validated using 88 clinical samples and the results were compared with the conventional qPCR and the nested PCR followed by Sanger sequencing.\n                    <\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Results<\/jats:title>\n                    <jats:p>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.<\/jats:p>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Conclusion<\/jats:title>\n                    <jats:p>The POCT-DO-RAP assay developed in this study achieves a fully integrated \u201csample-in, result-out\u201d 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.<\/jats:p>\n                  <\/jats:sec>","DOI":"10.3389\/fmicb.2026.1772984","type":"journal-article","created":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T06:32:47Z","timestamp":1772433167000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Development of a point-of-care dual one-step recombinase-aided PCR assay for rapid identification of Mycobacterium tuberculosis gyrA mutations conferring fluoroquinolone resistance"],"prefix":"10.3389","volume":"17","author":[{"given":"Xingyu","family":"Liu","sequence":"first","affiliation":[{"name":"Hebei Medical University, Shijiazhuang","place":["Hebei, China"]},{"name":"Department of Clinical Laboratory, Hebei General Hospital, Shijiazhuang","place":["Hebei, 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