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. coli O157: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.