Development of a novel serogrouping method for the rapid detection of 21 Escherichia coli O-serotypes using multiplex real-time PCR

Escherichia coli O-serotypes are associated with pathogenesis; thus, rapid and accurate serogrouping is required to prevent foodborne outbreaks. In this study, a novel multiplex real-time PCR method was developed for E. coli serogrouping to overcome some limitations of conventional and in silico serogrouping. Two O-serotype genes, wzy and wzx, were selected, and 21 primer-probe sets were designed for determining O-serotypes. To evaluate this, 11 O-serotype reference strains and 43 environmental isolates were tested: serogrouping took <40 min and the detection limit was 1-10 pg DNA, indicating that rapid identification was achieved with high sensitivity and without false-positive results. Subsequent food tests revealed that the method worked perfectly even at 10(1) CFU per ground beef sample; thus, O-serotypes were identifiable in food environments. The developed multiplex real-time PCR serogrouping method can rapidly, inexpensively, and accurately identify E. coli O-serotypes; therefore, it could help prevent foodborne outbreaks.