Evaluation of a Real-Time PCR Multiplex Assay For Detecting Foodborne Pathogenic Bacteria

Year 2015, Volume: 1 Issue: 1, 7 - 16, 01.06.2015

İsmail Hakkı Tekiner Haydar Özpınar

Abstract

Asbtract Molecular biological methods are feasible, quick and reliable tools of detecting major foodborne pathogens in food quality and safety monitoring. Among them, real-time PCR application is one of the testing platforms that optimally meets the criteria of performance against high throughput screening of microorganisms. The objective of this study was to evaluate the performance of a real-time PCR fourplex assay in simultaneous detection of major foodborne pathogenic bacteria, including Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. To do this, the reference variants were inoculated to UHT milk sample individually as well as a cocktail containing them at all hands. All the spiked suspensions were initially exposed to pre-enrichment in buffered peptone water. Subsequently, 10-fold serial dilutions were prepared from them. The serially diluted suspensions were then transferred to Plate Count PC agar plates, followed by an aerobic incubation under the conditions according to the mannufacturer’s instructions. After that, the plates expressing a viable count of 1-10 CFU/ml, 10-100 CFU/ml and 100-1000 CFU/ ml were selected. The DNAs extracted by Eurofins GENESpin DNA isolation kit were subjected to a real-time PCR fourplex application using PowerChek™ Pathogen 4-plex detection kit Kogene Biotech, South Korea . The screening results revealed that the tested multiplex assay simultaneously exhibited its best performance as triplex for Sta, Lm and Sal in a concentration of 10-100 CFU/ml, excluding Ec. To conclude, detection and differentiation of multiplex bacteria are not yet optimized due to some technical limitations, and still evolving

Keywords

Food, Multiplex assay, real-time PCR, Sensitivity

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