Detection of Yersinia enterocolitica in raw pork by conventional culture methods and PCR based methods

Abstract

Yersinia enterocolitica is an emerging pathogenic microorganism associated with food. Its ingestion, through contaminated food, may cause different kinds of intestinal disorders. Since there is not much information about the presence of Y. enterocolitica concerning the consumption of food in Portugal and the conventional methodology is not very effective, this study proceeded, by implementing the PCR methodology, in order to detect the pathogenic microorganism in pork meat. One hundred samples of raw minced meat were acquired in supermarkets and butcher’s shops in the Greater Oporto and Braga area, with the purpose of determining the occurrence of Y. enterocolitica. The detection limit of the conventional method (ISO 10273: 2003) was determined to be 105 CFU/g using CIN culture medium and 104 CFU/g using a pre‐treatment step with KOH, which highlights the difficulties in detecting Yersinia using this methodology. A molecular PCR‐based method was implemented, using BDC followed by cellular alkaline lysis to extract the samples’ DNA (BDC‐PCR‐based method). The primers used in this study were the 16S rRNA gene, which allowed the detection of the genera Yersinia, and the yst gene to detect the pathogenic strains of the microorganism. The detection limit was studied in both sets of primers. The values obtained were 102 CFU/g for the 16S rRNA gene and 103 CFU/g for the yst gene for a pre‐enrichment time of 24 h. Nevertheless, we have implemented a combined culture and PCR method for detection of Yersinia that besides ensuring the viability of the cells detected, showed to be more sensitive than the BDC‐PCR‐based method. All the samples were analysed by the BDC‐PCR‐based method and 25 by the three methodologies. The different methodologies will be discussed and the incidence of Y. enterocolitica in raw minced meat pork evaluated. The results of this study show that the molecular methodology and the combined methodology that were here adopted are more sensitive than the common methodology. Therefore, it can become an important tool in food sample control, since it allows quicker results in a smallest time span. It is also more reliable and easier to work with when compared to other conventional methods.