Establishment and application of a duplex RPA-LFS detection system for Candida glabrata and Candida krusei

Abstract

Abstract Based on the differences in drug resistance characteristics between Candida glabrata and Candida krusei and the clinical need for rapid discrimination, this study established a duplex recombinase polymerase amplification-lateral flow strip (RPA-LFS) detection system using dual-labeled probes. By optimizing ITS2-targeted primers and probes (5′-end labeled with FITC/DIG, 3′-end labeled with biotin) and integrating dual test lines on the strip (streptavidin-T line for directional capture), simultaneous visual detection of both targets was achieved. Performance validation demonstrated: a detection limit of 10 copies/mL and 100 copies/mL, matching qPCR sensitivity; 100% detection rate for 30 target strains (including 12 reference and clinical isolates) with strict discrimination from 8 closely related pathogens (0% cross-reactivity); high concordance with qPCR in 328 clinical samples (sensitivity, specificity, and total concordance all 100%). The system delivers “sample-to-result” output within 30 min without complex instrumentation, providing technical support for precise point-of-care discrimination of drug-resistant Candida infections and rational antifungal drug use, particularly in primary healthcare settings and outbreak scenarios.