AN AUTOMATED FLUORESCENT PCR METHOD FOR DETECTION OF SHIGA TOXIN-PRODUCING ESCHERICHIA-COLI IN FOODS

An automated fluorescence-based PCR system (a model AG-9600 AmpliSenso r analyzer) was investigated to determine whether it could detect Shig a toxin-producing Escherichia coli (STEC). The AmpliSensor PCR assay i nvolves amplification-mediated disruption of a fluorogenic DNA signal duplex (AmpliSensor) that is homologous to conserved target sequences in a 323-bp amplified fragment of Shiga toxin genes stx(1), stx(2), an d stx(e). Using the Amplisensor assay, me detected 113 strains of STEC belonging to 50 different serotypes, while 18 strains of non-Shiga-to xin-producing E. coli and 68 strains of other bacteria were not detect ed. The detection limits of the assay were less than 1 to 5 CFU per PC R mixture when pure cultures of five reference strains were used and 3 CFU per 25 g of food when spiked ground beef samples that were preenr iched overnight were used. The performance of the assay was also evalu ated by using 53 naturally contaminated meat samples and 18 raw milk s amples. Thirty-two STEC-positive samples that were confirmed to be pos itive by the culture assay were found to be positive when the AmpliSen sor assay was used. Nine samples that were found to be positive when t he PCR assay was used were culture negative. The system described here is an automated PCR-based system that can be used for detection of al l serotypes of STEC in food or clinical samples.