Establishment of a Method for Describing stx Genes Expression of E. coli O157:H7 in Ground Beef Matrix during Refrigerated Storage

Abstract

AbstractIngredients and complexity of food matrices are inhibitory factors for gene expression studies of bacterial pathogens directly from food matrix. The aim of this study was to develop a simple and fast method for RNA extraction from ground beef matrix as well as stx genes expression of Escherichia coli O157:H7 by relative quantitative real‐time reverse transcription‐polymerase chain reaction (RT‐PCR). Inoculated ground beef samples were kept 7 days in refrigerated storage (4 ± 1C) and bacterial RNA was extracted according to the described method at 0, 2, 5 and 7 days after storage. Results indicate that refrigerated storage temperature decreased gene expression of stx1A (−3.35 relative changes on day 7) and stx2A (−2.9 relative changes on day 7) in ground beef matrix. In the current study, a method requiring minimal equipment and limited interference for RNA extraction from bacteria associated with a meat matrix was evaluated (developed, invested, etc.). Results of the study suggest that the quantitative real‐time RT‐PCR method could be used as a reliable and sensitive method for gene expression studies.Practical ApplicationsPolymerase chain reaction (PCR), known as a sensitive and specific method, was extensively used for the detection of genes involved in stress and virulence of bacteria. In this way, real‐time PCR shows a good sensitivity and diversity for detection and gene expression studies of pathogens. This technique frequently has been used for the study of bacterial behavior in broth or food‐like broth models. Complexity of food matrix and presence of proteins, and also other PCR‐inhibiting factors, restricted gene expression studies in food matrix. There has been great interest in the survival and pathogenicity of Escherichia coli O157:H7 because it is a great concern of ground meat industry. Shiga toxins (Stx1 and Stx2) as manifest characteristics of E. coli O157:H7 are known as significant virulence factors in the pathogenicity of bacterium so its behavior and gene expression profile in meat matrix and during cold chain were evaluated.