Simultaneous detection of two verotoxin genes using dual-label time-resolved fluorescence immunoassay with duplex PCR

Verotoxin (VT) produced by several Escherichia coli serotypes causes haemorrhagic colitis and has been associated with haemolytic uraemic syndrome in humans. Two types of verotoxin are known. Conventional diagnosis of verotoxin-producing Escherichia coli (VTEC) is conducted after isolation of bacteria from clinical specimens, followed by serological determination and identification of VTs. This method is complicated and time-consuming. Recently, rapid, direct immunological methods for identification of VTEC, i.e. immunochromatography and latex agglutination, have been developed. However, these techniques continue to suffer from limited sensitivity and a lack of specificity. These difficulties arise from the fact that the antibody used in these procedures reacts exclusively with the O157 antigen; moreover, VTEC strains with non-O157 antigens, such as O26, O103 and O111 antigens, exist. These VTEC groups did not react with anti-O157 antibody. Consequently, it is necessary to diagnose the VT gene in these bacteria. Therefore, we have designed a sensitive and specific method for the detection of two VT genes simultaneously, utilizing duplex PCR with time-resolved fluorescence immunoassay (TRFIA).