Identification of Escherichia coli O157:H7 by multiplex PCR with primers specific to the hylA, eaeA, stx1, stx2, fliC AND rfb genes

Escherichia coli O157:H7 can cause fatal diseases such as hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura. The early symptoms, of E. coli O157:H7 infection are similar to those of infection with other gastrointestinal bacteria, so the availability of a rapid and precise method to identify E. coli O157: H7 is important for early recognition. We report a multiplex polymerase chain reaction (PCR) with primers to detect the presence of the E. coli O157:H7 genes hlyA, which produces enterohemolysin, eaeA (intimin), stx1 (Shiga-like toxin 1), stx2 (Shiga-like toxin 11), fliC (flagella H-antigen), and rfb (surface O-antigen). The technique improves specificity compared to general PCR when used with greater than or equal to 10(3) CFU/assay. Specificity was examined using 32 E. coli O157:H7 strains from the USA, Japan, Canada and Taiwan, all of which were positive for hlyA, eaeA, fliC, rfb, and stx1 and/or stx2. Thirty-five non-O157 enterovirulent E. coli strains, two Shigella sp strains, and two Salmonella sp strains were also examined and none gave the positive gene expression results described above. This multiplex PCR was highly specific in identifying E. coli O157 and was more useful than general PCR in early clinical detection of this pathogen.