Detection of Escherichia coli O157: H7 vt and rfb(O157) by multiplex polymerase chain reaction.

ABSTRACT

A rapid method for detection of Escherichia coli O157 H7 using multiplex PCR was developed. Two oligonucleotide primer pairs were used for simultaneously detection of vt encoding verotoxin genes for virulence factor and rfb(O157) encoding the O-antigen specific for E. coli O157 H7. Multiplex PCR generated two products of 215 bp and 420 bp for vt and rfb(O157), respectively. Multiplex PCR detected reference strain O157 H7 (NF-7777) with a sensitivity of 10(5) CFU per ml with no amplification of other 15 pathogenic bacteria. After incubation of 10(2) CFU/25 gram raw meat in tryptic soy broth at 37 degrees C for 8 hours, multiplex PCR conducted with the addition of 100 mg bovine serum albumin produced the two specific PCR products for E. coli O157 H7. This modified multiplex PCR is a rapid, sensitive, and specific technique for detecting and differentiating E. coli O157 H7 and has the potential to be used as an alternative to conventional methods for the screening of O157 H7 strains isolated from raw meat.