Dual one-step recombinase-aided PCR for rapid detection of Candida in blood

This study presents a novel dual one-step recombinase-aided PCR (DO-RAP) method, combined with recombinant human mannan-binding lectin protein (rhMBL; i.e., M1 protein)-conjugated magnetic bead (M1 bead) enrichment, for the early detection of Candida krusei and Candida parapsilosis bloodstream infections. Unlike previous studies that utilized the characteristic of docosane being solid at room temperature and melting above 44 °C as an impermeable barrier to separate two reaction steps, DO-RAP simplifies the process by eliminating this step. Specificity tests with the genomic DNAs from 11 bacterial strains and 3 fungi related to bloodstream infections (BSIs) confirmed no cross-reactivity, while sensitivity analysis demonstrated detection limits of 1 copy/μL for recombinant plasmids containing 26S ribosomal RNA gene fragment from C. krusei and NADH5 mitochondrial gene fragment from C. parapsilosis and 10⁻⁷ ng/μL for DNAs from standard strains of C. krusei and C. parapsilosis. In simulated infection samples enriched with M1 beads, DO-RAP achieved detection thresholds of 1 CFU/mL (colony-forming unit per milliliter) in simulated samples within 3.5 h, surpassing quantitative PCR (qPCR) performance, which has detection limits of 3-5 CFU/mL. Clinical validation showed strong agreement between DO-RAP and qPCR, with Kappa values of 0.936 for C. krusei and 0.904 for C. parapsilosis (P < 0.05). This integrated approach improves detection speed and sensitivity, eliminates the need for culturing, and offers a more efficient alternative to qPCR for diagnosing invasive Candida infections. KEY POINTS: • The DO-RAP method achieves a detection sensitivity of 1 CFU/mL, surpassing conventional qPCR. • This approach eliminates the need for docosane, streamlining operations, and accelerating detection. • M1 magnetic bead enrichment enhances pathogen capture, facilitating rapid Candida diagnosis.