Development of a real-time PCR assay with an internal amplification control for the detection of spoilage fungi in fruit preparations. (May 2022)

• Record Type: Journal Article
• Authors: Roumani, Foteini; Azinheiro, Sarah; Rodrigues, Cristina; Barros-Velázquez, Jorge; Garrido-Maestu, Alejandro; Prado, Marta
• Is Part Of: Food control. Volume 135(2022)
• Publication Date: 2022-05
• Subjects: Real-time PCR -- Spoilage fungi -- Fast molecular methods -- Yeast -- Moulds -- ITS; Food -- Quality -- Periodicals; Food -- Analysis -- Periodicals; Food handling -- Periodicals; Food industry and trade -- Quality control -- Periodicals; Aliments -- Industrie et commerce -- Qualité -- Contrôle -- Périodiques; Aliments -- Qualité -- Périodiques; Aliments -- Analyse -- Périodiques; Hygiène alimentaire -- Périodiques; Food -- Analysis; Food handling; Food -- Quality; Periodicals; Electronic journals; 664.07
• DOI: 10.1016/j.foodcont.2021.108783
• Languages: English
• ISSNs: 0956-7135

Abstract

Abstract: Fungal spoilage of food commodities is still of great concern for food industry due to increased costs regarding food waste and product recalls. Traditional culture-based methods are laborious and require huge amounts of reagents and media. In addition, results are available after seven days rendering these techniques unsuitable for the intense production that exists nowadays. As a result, there is a need for faster and more sensitive methods for fungal detection. The aim of this study was the development and evaluation of a method (enrichment, sample treatment, DNA extraction, and qPCR) for the fast detection of spoilage fungi in fruit preparations. In this sense, a set of universal primers was selected and a hydrolysis probe was designed in-house. In addition, a non-competitive internal amplification control was included in the assay to eliminate false negative results due to reaction inhibition. It was demonstrated that the method could reliably detect yeasts with a LOD95 of 1.0 CFU/50 g. Regarding moulds detection, two different enrichment times were examined, namely 24 h and 48 h in order to increase the sensitivity. The obtained LOD95 values were 123.5 spores/50 g and 37.1 spores/50 g for 24 h and 48 h respectively. During the method evaluation all the performance parameters resulted in values higher than 85.0% and the Cohen's k was determined to be above 0.86 for yeasts and moulds. Overall, a reliable and sensitive next-day detection method for fungi was achieved. Highlights: Next-day detection of yeast and moulds was successfully achieved by qPCR. High result confidence due to implementation of a non-competitive internal amplification control. LOD95 of 1.0 cfu/50 g for yeast detection in 24 h. LOD95 of 123.5 spores/50 g and 37.1 spores/50 g after 24 h and 48 h or enrichment respectively.