Quantification of Escherichia coli O157:H7 in milk by most probable number –polymerase chain reaction (MPN-PCR) method

Abstract

Escherichia coli O157:H7 has been recognized as one of the most important causes of potentially life-threatening human diseases such as haemorrhagic colitis, haemolytic ureamic syndrome and thrombotic thrombocytopenic purpura. To combine the principles of most-probable-number (MPN) statistics and the conventional polymerase chain reaction (PCR) technique to enumerate E. coli O157:H7 in milk, from 101 to 105 ml-1 of bacterial cells were inoculated in sterilized milk. Different background microorganisms including Gram positive and Gram negative bacteria were also inoculated. Modified MPN dilutions from inoculated milk sample with three replicates per dilution were prepared and enumeration was performed by DNA extraction from tubes showing turbidity and performing multiplex-PCR (m-PCR) using primers specific for O157 and H7 antigens gene. This MPN-PCR proved to be a rapid and reliable method for enumerating E. coliO157:H7 in milk at the lowest level (101 cfu.ml-1), even in the presence of different Gram positive and Gram negative background microorganisms. It may facilitate the enumeration of E. coli O157:H7 for routine analyses in milk without excessive work and could be considered as an alternative to MPN- culture techniques. Key words: E. coli O157:H7, milk, most probable number-polymerase chain reaction (MPN-PCR), most probable number (MPN) - culture, multiplex- polymerase chain reaction (m-PCR).