Rapid and ultrasensitive detection of Candida tropicalis using multiple cross-displacement amplification combined with CRISPR/Cas12a.

BACKGROUND: Candida tropicalis (C. tropicalis), classified as a World Health Organization "critical priority" pathogen, causes life-threatening invasive infections with high mortality due to diagnostic delays. Therefore, the development of rapid fungal detection platforms is an urgent scientific and clinical priority.

METHODS: We designed multiple cross-displacement amplification (MCDA) primers to target the internal transcribed spacer 2 (ITS II) gene of C. tropicalis for specific amplification. Subsequently, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-crRNA complex bound to the amplified products, activating its trans-cleavage activity to generate a detectable fluorescent signal. Finally, clinical samples were used to validate the detection results, which were further compared with those obtained using fungal culture and multiplex polymerase chain reaction (Multiplex PCR).

RESULTS: Under optimized conditions, the C. tropicalis-MCDA-CRISPR/Cas12a assay was completed within approximately 53 min, with a limit of detection of 30 fg of genomic DNA per reaction. The assay showed no cross-reactivity with non-C. tropicalis pathogens. Clinical validation using 128 specimens demonstrated a sensitivity of 100.0% (95% CI: 93.4-100.0%) and a specificity of 97.3% (95% CI: 90.6-99.7%) against a composite reference standard, with near-perfect agreement (κ= 0.968).

DISCUSSION: The C. tropicalis-MCDA-CRISPR/Cas12a assay is an efficient, accurate, and practical diagnostic tool suitable for use in resource-limited laboratories.