DNA extraction for sensitive detection of Shiga toxin-producing Escherichia coli in food by real-time PCR assays

Abstract

Alkali-heat DNA extraction, a rapid and economical method, was evaluated for use in the detection of Shiga toxin-producing Escherichia coli in food using real-time PCR assays. Alkali-heat DNA extracts led to highly sensitive detection (102-104 CFU/mL) of stx and O-antigen genes in beef liver, ground beef, sliced pork, cheese, lettuce, radish sprouts, tomato, and spinach, equivalent to the sensitivity obtained using a commercial DNA extraction kit that utilizes proteinase K lysis, and silica membrane purification. Although there were differences in DNA concentration and purity between DNA extraction methods, the sensitivity of real-time PCR assays was similar. These results indicate that alkali-heat DNA extraction is a viable method when testing food products with real-time PCR assays for the presence of stx and O-antigen genes.