PCR detection of enterohemorrhagic Escherichia coli O145 in food by targeting genes in the E. coli O145 O-antigen gene cluster and the shiga toxin 1 and shiga toxin 2 genes.
Shiga toxin-producing Escherichia coli (STEC) strains belonging to serogroup O145 are an important cause of hemorrhagic colitis and hemolytic uremic syndrome worldwide. Cattle and other animals are potential reservoirs for this pathogen. To develop PCR assays for detection and identification of E. coli O145, the wzx (O-antigen flippase) and wzy (O-antigen polymerase) genes in the O145 O-antigen gene cluster that are specific for this serogroup were selected as targets. Oligonucleotide primers complementary to regions in the E. coli O145 wzx and wzy genes were designed to perform PCR assays with DNA from strains of E. coli O145, non-O145 E. coli serogroups, and other bacterial genera. The assays were highly specific for E. coli O145. A multiplex PCR assay targeting the E. coli O145 wzx and wzy genes and the Shiga toxin 1 (stx(1)) and Shiga toxin 2 (stx(2)) genes and a real-time multiplex PCR assay targeting the O145 wzy, stx(1), and stx(2) genes were developed for detection of STEC O145. The assays were used for detecting STEC O145 in seeded ground beef, lettuce, and raw milk initially inoculated with ca. 2, 20, or 200 CFU/25 g or 25 mL after 8 or 20 h of enrichment at 42 degrees C in modified EC broth containing 20 mg/L of novobiocin. STEC O145 was detected in all samples inoculated with 2 CFU/25 g or 25 mL. The detection limit of the multiplex PCR assays was <or=7.9 x 10(4) CFU/mL, which corresponded to <or=400 CFU/PCR reaction. The PCR assays can be employed to identify enterohemorrhagic E. coli serogroup O145 and to detect low levels of the pathogen in food.
Microbial Food Safety Research Unit, Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania 19038, USA. pina.fratamico@ars.usda.gov
Pina M
PM
Fratamico
Chitrita
C
DebRoy
Takahisa
T
Miyamoto
Yanhong
Y
Liu
D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry; D054324:Enterohemorrhagic Escherichia coli / Q000235:genetics* / Q000302:isolation & purification*; D029968:Escherichia coli Proteins / Q000235:genetics; D005516:Food Microbiology; D016695:Glycosyltransferases / Q000235:genetics; D026901:Membrane Transport Proteins / Q000235:genetics; D019081:O Antigens / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA; D022622:Shiga Toxin 1 / Q000235:genetics*; D022641:Shiga Toxin 2 / Q000235:genetics*
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D019081:O Antigens; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2; C543926:Wzx protein, E coli; D016695:Glycosyltransferases; C513753:Wzy polymerase, E coli