CRISPR technology combined with isothermal amplification methods for the diagnosis of Candida albicans infection.

Abstract

Since Candida albicans, a type of fungus, causes severe infections that pose a significant threat to human health, its rapid detection is critical in clinical antifungal therapy. Traditional fungal diagnostic approaches are largely based on the culture method. This method is time-consuming and laborious, taking about 48-72 h, and cannot identify emerging species, making it unsuitable for critically ill patients with bloodstream infections, sepsis, and so on. Other antigen or nucleic acid amplification-based methods were also found to be unsuitable for Point-of-Care Testing (POCT) diagnosis due to various limitations. Therefore, establishing a new approach for the rapid diagnosis of Candida spp is imperative. Herein, we proposed a novel diagnostic method for invasive fungi detection. Specifically, we created a new CRISPR diagnostic platform for Candida albicans-specific Internal Transcriptional Spacer 2 (ITS2) gene by combining the DNase cleavage activity of Cas12a with Recombinase Polymerase Amplification (RPA). Furthermore, to achieve rapid on-site detection under low-resource conditions, we used a transverse lateral flow strip with a single target to visualize the Cas12a single enzyme digestion product. We designated the platform as a rapid molecular detection tool that integrates RPA and the CRISPR-Cas12a technology. The entire platform can accurately identify Candida albicans within 50 minwhile remaining unaffected by other fungi or bacteria. Furthermore, the detection limit of the platform could reach 10² CFU/ml. Moreover, this approach offers additional benefits, including easy operation, low set-up cost, and broad applicability for Candida albicans detection across medical institutions at all levels, especially in township health centers in resource-poor regions.