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

Abstract

Diastatic Saccharomyces cerevisiae is a common contaminant in the brewing industry. Currently available detection methods are either time consuming or require specialised equipment. The aim of this study was to develop a new rapid and simple assay for the detection of diastatic yeast from samples of beer and yeast. More specifically, the aim was to develop a simple and rapid assay that requires minimal laboratory equipment or training, and yields results as accurate as PCR-based methods. The assay consists of three main steps: DNA extraction, pre-amplification of DNA, and CRISPR-Cas12a based detection and visualisation. Different pre-amplification and visualisation techniques were compared, and the final assay involved a one-pot reaction where LAMP and Cas12a were consecutively used to pre-amplify and detect a fragment from the STA1 gene in a single tube. These reactions required a heat block, a pipette, and a centrifuge with the assay result visualised on a lateral flow strip. The assay was used to monitor an intentionally contaminated brewing fermentation and was shown to yield results as accurate as PCR with previously published primers. Furthermore, the assay yielded results in approximately 75 minutes. The developed assay offers reliable and rapid quality control for breweries of all sizes and can be performed without expensive laboratory equipment. It is suggested that the assay will be particularly useful for smaller breweries without well-equipped laboratories who are looking to implement better quality control.