A CRISPR-Cas12a and Quantum-Dot Lateral-Flow Assay for Rapid Species-Level Detection of Trichophyton rubrum.

BACKGROUND

Accurate species-level diagnosis of dermatophytes, particularly Trichophyton rubrum, is important for guiding targeted antifungal therapy and improving the management of recurrent or atypical superficial mycoses. However, routine microscopy has only moderate sensitivity and cannot resolve fungal species; fungal culture is time-consuming, and real-time quantitative PCR (qPCR) requires costly instrumentation.

OBJECTIVES

To develop and clinically evaluate CRI-RUB, a CRISPR-Cas12a-based assay integrating recombinase-aided amplification (RAA) and a quantum-dot (QD) fluorescent lateral-flow strip for rapid species-level detection of T. rubrum.

PATIENTS/

METHODS

Analytical sensitivity and specificity were assessed using plasmid standards and a panel of common cutaneous fungi. A total of 140 clinical specimens were tested by both CRI-RUB and TaqMan qPCR. Discrepant or grey-zone qPCR results (Ct 33-34), together with a subset of concordant cases, were further arbitrated by internal transcribed spacer (ITS) sequencing to establish a composite reference standard.

RESULTS

CRI-RUB achieved a visual limit of detection of 2.3 × 101 copies/μL and showed no cross-reactivity with other common cutaneous fungi. Among the 140 clinical specimens, CRI-RUB showed a sensitivity of 96.1% (73/76), specificity of 100.0% (64/64), positive predictive value of 100.0% (73/73), negative predictive value of 95.5% (64/67) and Cohen's κ of 0.96 compared with the composite reference standard, indicating excellent concordance. The complete workflow required approximately 85 min.

CONCLUSIONS

CRI-RUB provides a rapid, sensitive, and specific approach for species-level detection of T. rubrum. By combining CRISPR-Cas12a detection with QD-based lateral-flow readout, this assay shows potential for near-patient or point-of-care dermatophyte diagnosis.