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

## TL;DR

This study developed a fast and accurate test to distinguish two drug-resistant Candida species, enabling better treatment decisions in healthcare settings.

## Contribution

A novel duplex RPA-LFS system for rapid, visual discrimination of Candida glabrata and Candida krusei with high sensitivity and specificity.

## Key findings

- The system achieved detection limits comparable to qPCR (10-100 copies/mL).
- It showed 100% detection rate for 30 target strains with no cross-reactivity.
- High concordance with qPCR in 328 clinical samples (100% sensitivity, specificity, and agreement).

## 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.