Real-time reverse transcription pcr detection of viable shiga toxin-producing Escherichia coli O157:H7 in food

Shiga toxin (Stx)-producing Escherichia coli (STEC), particularly E. coli O157:H7, have been associated with food-related outbreaks and have become a global health concern. Detection of low numbers of viable STEC in food is undoubtedly challenging. The present study demonstrated that 7 × 10² to 7 × 10³ cfu E. coli O157:H7 cells in 50 mL of inoculated food samples including apple juice, lettuce and ground beef could be concentrated through a two-step filtration technique. Bacterial RNA was effectively isolated from the concentrated E. coli O157:H7 cells with the combination of Trizol Max Bacterial RNA Isolation Enhancement Reagent and TriReagent®. Real-time reverse transcription polymerase chain reaction with seven sets of primers targeting virulence and serotype genes of E. coli O157:H7 specifically detected the bacterial RNA representing 1 × 10² to 1 × 10³ cfu viable bactersial cells. The total detection time from sampling to measurement was approximately 4 h. This study provides a rapid and specific detection method for viable STEC in food. Practical Applications: Rapid detection of viable STEC including Escherichia coli O157:H7 cells in food has been difficult, especially when present in low numbers. This study has shown that 7 × 10² to 7 × 10³ cfu E. coli O157:H7 in 50 mL of inoculated food samples, i.e., 14-140 cfu/mL bacterial cells, could be readily concentrated and the bacterial RNA could be detected by real-time reverse transcription polymerase chain reaction (PCR) in a relatively short time. Use of seven primer sets makes this method very specific for real-time PCR detection of E. coli O157:H7.