Detection of viable Escherichia coli O157:H7 by ethidium monoazide real-time PCR

摘要: Aims:  The aim of this study was to develop and optimize a novel method that combines ethidium bromide monoazide (EMA) staining with real-time PCR for the detection viable Escherichia  coli O157:H7 in ground beef. EMA can penetrate dead cells bind intracellular DNA, preventing its amplification via PCR. Methods Results:  Samples were stained 5 min, iced 1 min exposed bright visible light 10 min prior DNA extraction, allow binding from cells. then extracted amplified by TaqMan® detect only E. coli primers probe used target uidA gene O157:H7. An internal control (IAC), consisting 0·25 pg plasmid pUC19, added each reaction prevent occurrence false-negative results. Results showed reproducible application technique both broth culture EMA, at final concentration 10 μg ml−1, demonstrated effectively 108 CFU ml−1 cells, optimized could as low 104 CFU g−1 beef without interference 108 CFU g−1 cells. Conclusions:  IAC separate cells. Significance Impact Study:  has potential be highly sensitive quantitative assess contamination other meat or food products.