Real Time PCR versus Conventional Methods For Detection of Viable But NonCulturable E. Coli O157 Isolated From Food of Animal Origin

Abstract

Escherichia coliO157:H7 consider an important zoonotic food borne pathogen that cause bloody diarrhea, hemorrhagic colitis (HC), hemolytic-uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP) in human. E. coli O157:H7 use a viable but non culturable (VBNC) state as a survival strategy which regarded as a threat to food security and the health of society. In this dormant stage, pathogens can evade identification by standard techniques. So, PCR methods are used for identifying all viable (culturable and non-culturable). This study was performed on 500 samples (300 raw meat and 200 raw milk samples) collected from different supermarkets allover Menoufia, Egypt. Samples were subjected to conventional culture method then positive-culture samples were confirmed by biochemical and serological identification while negative-culture sample subjected to conventional PCR and PMA in combination with SYBR green real-time PCR for VBNC E. coli O157:H7 detection. The obtained data showed that the incidence of E. coli O157:H7 by the conventional method was 3% while the incidence of VBNC E. coli O157:H7 by PMA with SYBR green real-time PCR together was 10% for ten tested samples and 0.21% of 475 negative-culture samples. This study revealed that conventional and immunoassay methods were unable to identify VBNC case, also conventional PCR is unable to distinguish between dead and live bacterial cells. However, employing a real-time PCR technique alongside PMA has the necessary efficiency and sensitivity for identifying all viable (culturable and non-culturable) E. coli O157:H7