A simple and rapid CRISPR-Cas12a-based detection test for diastaticSaccharomyces cerevisiae

Abstract

AbstractDiastaticSaccharomyces cerevisiaeis a common contaminant in the brewing industry. Currently available detection methods are either time-coansuming or require specialized equipment. The aim of this study was to develop a new rapid and simple assay for the detection of diastatic yeast from beer and yeast samples. More specifically, we aimed to develop a simple and rapid assay that requires minimal laboratory equipment or training, and ideally yields results as accurate as PCR-based methods. The developed assay consisted of three main steps: DNA extraction, pre-amplification of DNA, and CRISPR-Cas12a-based detection and visualisation. We compared different preamplification and visualisation techniques, and the final assay involved a one-pot reaction where LAMP and Cas12a were consecutively used to pre-amplify and detect a fragment from theSTA1gene in a single tube. These reactions only required a heat block, a pipette, and a centrifuge. The assay result was then visualised on a lateral flow strip. We used the developed assay to monitor an intentionally contaminated beer fermentation, and it was shown to yield results as accurate as PCR using previously published primers. Furthermore, the assay yielded results in approx. 75 minutes starting from a beer sample. The developed assay therefore offers reliable and rapid quality control for breweries of all sizes and can be performed without any expensive laboratory equipment. We believe the assay will be particularly useful for smaller breweries that don’t already have well-equipped laboratories and are looking to implement better quality control.