Browsing by Author "Uotila, Ida"

The aim of this thesis was to develop a new rapid and simple detection method for diastatic Saccharomyces cerevisiae (var. diastaticus). Diastatic S. cerevisiae is a typical contaminant in the brewing industry. It produces an extracellular glucoamylase enzyme, which is able to break down dextrin that typically remains unfermented. This leads to extended fermentation causing numerous quality problems in beer, such as excessive ethanol and carbon dioxide content. With current methods, diastatic S. cerevisiae can be detected either by traditional cultivation methods or by molecular methods. The disadvantage with cultivation is that it often takes a few days to get results. Molecular methods are more rapid, and the results are obtained in hours instead of days, however, the methods often require specialized laboratory equipment. Prior to the protocol development, a suitable DNA extraction method was chosen according to pre-experiments. The developed method identifies the STA1 gene specific to the diastatic S. cerevisiae by RNA-guided Cas12a endonuclease originating from CRISPR. The sensitivity of the method can be increased by STA1 pre-amplification. Therefore, primers were designed for the STA1 gene for three different amplification methods, which were polymerase chain reaction (PCR), recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP). The amplified gene was identified by the CRISPR-Cas12a reaction visualised using both fluorescence and lateral flow methods. For this, a suitable single-stranded DNA probe was added to the reaction. The developed method was confirmed using fermentation samples intentionally contaminated with diastatic yeast. Of the amplification methods tested, LAMP was chosen as part of the protocol, as it had the advantage of isothermal amplification and high sensitivity in the CRISPR-Cas12a reaction. The result read-out was successful with fluorescence and lateral flow method. The advantage of lateral flow was its simplicity, as it only need a commercial kit. Thus, no fluorescence reader or UV-light is needed. With the developed protocol it was possible to detect diastatic S. cerevisiae at a concentration of 10^-5 within 75 minutes.