Response surface methodology to optimize peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) in Saccharomyces cerevisiae

Abstract

Saccharomyces cerevisiae (S. cerevisiae) is a crucial fermenting microorganism for the beer, wine andbread industry. As such, an accurate and rapid method for its identification and monitoring is required. Inhere, a peptide nucleic acid (PNA) probe was first designed to target S. cerevisiae by fluorescence in situhybridization (FISH). This PNA-FISH method was then systematically optimized, employing responsesurface methodology (RSM). The interaction between the critical hybridization temperature, time andformamide concentration and their effect on the FISH efficiency were modelled. The model predictedoptimal fluorescence intensity upon hybridization at 53.9 C, during 57.8 min and using 43.8% (v/v)formamide in the hybridization buffer, which was experimentally confirmed. RSM showed to be avaluable tool to optimize and better understand the dynamics of yeast FISH, which can impact theperformance evaluation of related fermentation processes.