Potential use of multiplex real-time PCR to develop a kit for identification of beer-spoilage microorganisms

• Ano de defesa: 2022
• Autor(a) principal: Machado, Túlio Iglésias
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de Viçosa; Microbiologia Agrícola
• Palavras-chave em Português: Cerveja - Microbiologia; Cerveja - Controle de qualidade; Reação em cadeia da polimerase multiflex; Microbiologia Agrícola
• Link de acesso: https://locus.ufv.br//handle/123456789/32131; https://doi.org/10.47328/ufvbbt.2023.733

Beer is the most consumed alcoholic beverage globally. Despite being considered a microbiologically stable beverage due to its intrinsic properties such as low pH, high CO2, presence of antimicrobial compounds from hop and other factors, microbiological contamination in beer does happen, leading to off-flavor production with changes in flavor and aroma, viscosity, acidification, among other unwanted effects. This study explores the use of multiplex real-time PCR (qPCR) coupled to High Resolution Melting (HRM) analysis for the simultaneous detection and discrimination of beer-spoilage microorganisms genera. Orthologous sequences were identified using the OrthoMCL pipeline for primer design. The designed primers exhibited high specificity, generating distinct melting peaks for the target genera. Sensitivity was confirmed, with successful amplification at low DNA concentrations. The perfect alignment of primers with target regions significantly influenced sensitivity. The multiplex qPCR-HRM approach demonstrated efficacy in detecting beer-spoilage microorganisms in multiplex reactions. Nonetheless, sensitivity variations among primers underscore the importance of thoughtful design for multiplex reactions with primers within the same sensitivity range. Our pipeline is highly adaptable and can be applied not only to the detection of various beer-spoilage microorganisms but also to other segments within the food industry, pharmaceutical, oil & gas industry, among others, effectively enhancing cost-efficient quality control measures. Keywords: Beer-spoilage. Quality control. Multiplex qPCR. HRM. Orthologous genes.