A new method for rapid detection of Listeria monocytogenes in food

Abstract

Listeria monocytogenes represents serious danger for human health. Thus detection of this pathogen in food, which represents its main means of entry into the organism, is a topic of special importance. The original classic methods for the determination of Listeria monocytogenes are in general laborious and time-consuming procedures. In order to address this issue we developed a new rapid method for specific detection of Listeria monocytogenes in food samples. The method consists of three steps: i) enrichment of food microflora (16 h), ii) selective isolation of Listeria sp. exploiting immunomagnetic separation (2–3 h) followed by iii) precise identification of Listeria sp. and Listeria monocytogenes using duplex PCR. PCR primers specific to part of 16S rRNA were used in order to identify the members of Listeria genus. The specific identification of Listeria monocytogenes was accomplished exploiting a pair of primers specific to gene encoding invasion-associated protein – iap (4–5 h). Amplification products, 1003 bp and 593 bp respectively, were separated by electrophoresis and visualized by UV detection. The optimized IMS-PCR method was used to test the presence of Listeria sp. and Listeria monocytogenes in food samples (ground meat, low-fat milk and cheese [olomoucké tvarůžky]). A comparison of the efficiency of the bacteria enrichment step by IMS and centrifugation was also performed. The analysis time including enrichment is less than 24 h. The detection limit for Listeria monocytogenes was found between 101–102 cfu per 25 g of food sample.