Development of High-Precision Quantitative Analysis Detection for three Yeasts Based on CRISPR/Cas12a

Abstract Objectives Yeast plays a crucial role in fermented foods, yet the application of the CRISPR/Cas system for yeast detection has been rarely reported. The objective of this study is to develop a rapid and quantitative detection method for brewing yeast based on the CRISPR-Cas12a system, aiming to achieve higher sensitivity than the quantitative real-time polymerase chain reaction (qPCR) assay and fill the gap in the application of CRISPR for yeast detection in fermented foods. Materials and Methods: This work established one-step RPA-CRISPR/Cas12a (ORCC) detection for quantifying yeasts in fermented foods by integrating target amplification and the CRISPR system into a one-pot reaction. Results A numerical standardized definition was established for the quantifiable state in ORCC detection of yeasts. Using ORCC detection, which occurs at 37 °C, absolute quantification of Saccharomyces cerevisiae, Wickerhamomyces anomalus, and Zygosaccharomyces bailii can be completed in less than 45 min. Under optimal conditions, the limits of quantification and the limits of detection for three yeasts were 100 copies/μL and 1 copy/μL, with no cross-reaction observed among non-target yeasts. In pure yeast samples, ORCC detection of the target yeasts achieved a detection rate of 91.60% - 116.57%. In simulated fermentation samples, the recovery rate for spiked target yeasts using ORCC detection ranged from 92.78% to 106.90%. In original naturally fermented samples, target yeasts can be quantified by ORCC detection but not by qPCR assay. In spiked naturally fermented samples, the recovery rates of the qPCR assay were generally lower than those of ORCC detection. Conclusion ORCC detection is simple, rapid, accurate, and highly sensitive for detecting three yeasts in fermented foods. It has significant potential for precisely monitoring yeast growth and fermentation process in various fermentation systems.