Quantification and Discrimination of Viable and Dead Escherichia coli O157:H7 Cells from Chicken Without Enrichment by Ethidium Bromide Monoazide Real-time Loop-Mediated Isothermal Amplification

In this study, a rapid and sensitive method of real-time loop-mediated isothermal amplification (Rti-LAMP) assays was developed for quantification and discrimination of viable and heat-killed E. coli O157:H7 cells treated with low concentration of ethidium bromide monoazide (EMA). Four micrograms per milliliter of EMA was chosen as the optimal concentration which did not inhibit DNA amplification derived from viable cells, but significantly increased the Tt values of dead cells in Rti-LAMP assays. When the DNA from 2.0 × 10 3 viable CFU of E. coli O157:H7 was subjected to EMA-Rti-LAMP, the resulting Tt value was 17.73 min. In contrast, the DNA from 2.0 × 10 3 CFU completely heat destroyed CFU of E. coli O157:H7 did not yield a positive amplification which Tt value was regarded as 60 min. When the DNA from viable plus heat-killed CFU at a ratio of 5:2995 was subjected to EMA-Rti-LAMP, the resulting Tt value was 23.06 min, which was statistically identical ( P