Development and evaluation of a highly specific detection method for beer spoilage strain Pediococcus acidilactici

Abstract Beer spoilage by microorganisms can lead to significant economic losses and quality defects in the brewing industry. Current molecular detection methods often lack sufficient specificity or require complex instrumentation, limiting their practicality for on-site use. This study developed a rapid and specific polymerase spiral reaction (PSR) assay for detecting Pediococcus acidilactici , a significant spoilage bacterium in beer production. From 13 candidate target genes, target PaciT2 was successfully cloned into a plasmid vector and used for subsequent assay development. Following multiple sequence alignment of its conserved region, three primer sets were designed and screened. The first primer set was ultimately used to establish and optimize the PSR assay protocol. The optimized PSR assay demonstrated exceptional specificity, producing positive results exclusively for P. acidilactici while showing no cross-reactivity with over 20 non-target strains, including six closely related lactic acid bacteria (LAB) species and various other foodborne pathogens. The assay achieved a high sensitivity, with a detection limit of 277.8 fg/μL. This combination of specificity and sensitivity renders the PSR method particularly suitable for monitoring P. acidilactici within the complex microbial communities of brewery environments. Overall, the established assay provides a rapid, equipment-independent, and highly reliable alternative to conventional PCR, offering significant advantages for on-site quality control in beer production.