Abstract: This study was conducted to establish a fluorescence detection method of recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a for rapid, sensitive and visual detection of dermatophytes in dogs and cats. Taking Microsporum canis, Nannizia gypsea and Trichophyton mentagrophytes as research objects, specific RPA primers and CRISPR RNA (crRNA) were designed and synthesized in the internal transcribed spacer region. A fluorescence detection method for simultaneous and separate detection of the dermatophytes was established, and the limit of detection was evaluated. The sensitivity and specificity of the method were evaluated by detecting clinical samples. Under the condition of 37 ℃, the limit of detection (LOD) for three kinds of dermatophytes can be as low as a single copy in 30 min. Twenty-four clinical samples were tested, and the results of fungal culture and colony sequencing were taken as standard results. The sensitivity and specificity of this assay were 100% when using the dermatophyte crRNA (crRNA-DM) that can simultaneously detect M. canis, N. gypsum and T. mentagrophytes, and the Microsporum canis crRNA (crRNA-Mc) that can specifically detect M. canis in the RPA-CRISPR/Cas12a reaction. The RPA-CRISPR/Cas12a established in this study can detect M. canis, N. gypsum and T. mentagrophytes simultaneously and separately. This technique required short time, visualization, high sensitivity and specificity, did not need expensive instruments, and was suitable for rapid clinical diagnosis.

Key words: recombinase polymerase amplification, CRISPR/Cas12a, dermatophytes in dogs and cats, rapid detection