Rapid and Visual RPA-Cas12a Fluorescence Assay for Accurate Detection of Dermatophytes in Cats and Dogs

Abstract

Dermatophytosis, an infectious disease caused by several fungi, can affect the hair, nails, and/or superficial layers of the skin and is of global significance. The most common dermatophytes in cats and dogs are Microsporum canis and Trichophyton mentagrophytes. Wood’s lamp examination, microscopic identification, and fungal culture are the conventional clinical diagnostic methods, while PCR (Polymerase Chain Reaction) and qPCR (Quantitative PCR) are playing an increasingly important role in the identification of dermatophytes. However, none of these methods could be applied to point-of-care testing (POCT). The recent development of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) based diagnostic platform promises a rapid, accurate, and portable diagnostic tool. In this paper, we present a Cas12a-fluorescence assay to detect and differentiate the main dermatophytes in clinical samples with high specificity and sensitivity. The Cas12a-based assay was performed with a combination of recombinase polymerase amplification (RPA). The results could be directly visualized by naked eyes under blue light, and all tested samples were consistent with fungal culture and sequencing results. Compared with traditional methods, the RPA-Cas12a-fluorescence assay requires less time (about 30 min) and less complicated equipment, and the visual changes can be clearly observed with naked eyes, which is suitable for on-site clinical diagnosis.

Harnessing multiplex crRNA enables an amplification-free/CRISPR-Cas12a-based diagnostic methodology for Nosema bombycis

“…The CRISPR/Cas system becomes a powerful tool for disease and pathogen detection and diagnosis especially when combined with on-site real-time monitoring equipment. It has been developed and successfully applied to nucleic acid detection of various pathogens, including virus ( 18 – 22 ), bacteria ( 23 – 27 ), fungi ( 28 – 30 ), and parasites ( 31 ). However, many pathogen detection technologies based on the CRISPR/Cas12a system rely on nucleic acid isothermal amplification technology.…”

Rapid and sensitive one-tube detection of mpox virus using RPA-coupled CRISPR-Cas12 assay

“…The RPA method combines isothermal recombinase-driven primer targeting of template with strand-displacement DNA synthesis, which can occur at room temperature with minimal sample pretreatment. 22 , 23 Thus far, RPA-based diagnostics have been developed for the detection of different pathogens, including viruses, 24 fungi, 25 bacteria, 26 and parasites. 27 In this study, we combined RPA and CRISPR-Cas12a and developed a rapid and ultrasensitive diagnosis system for mpox virus detection.…”