A simple filtration technique to detect enterohemorrhagic Escherichia coli O157:H7 and its toxins in beef by multiplex PCR.
Primers, specific for a unique base substitution in uidA of Escherichia coli O157:H7, were coupled with oligonucleotides for the shiga-like toxin I (SLT-I) and SLT-II genes in a multiplex PCR assay. A minimum of 10(2) CFU per PCR (10 microliters) was necessary to amplify E. coli O157:H7-specific bands by multiplex PCR. Food particles as well as various unknown metabolic by-products of bacteria inhibited the PCR, but a simple two-step filtration procedure eliminated this inhibition. To reliably generate PCR products, an E. coli inoculum of 10(3) CFU g of food slurry-1 in a nonspecific medium was required with 6 h of enrichment at 37 degrees C. However, when the food homogenate was incubated overnight, E. coli O157:H7 at an initial inoculum of even 1 CFU g-1 was detected.
Venkateswaran
K
K
Central Research Laboratory, Nippon Suisan Kaisha, Ltd., Tokyo, Japan. kjvenkat@csd.uwm.edu
Kamijoh
Y
Y
Ohashi
E
E
Nakanishi
H
H
D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics*; D002417:Cattle; D004927:Escherichia coli Infections / Q000209:etiology; D019453:Escherichia coli O157 / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D005374:Filtration / Q000379:methods; D005516:Food Microbiology; D005517:Foodborne Diseases / Q000209:etiology; D006801:Humans; D008460:Meat / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2
D001427:Bacterial Toxins; D022622:Shiga Toxin 1; D022641:Shiga Toxin 2