This study aimed to establish a digital loop-mediated isothermal amplification (LAMP) method to detect Escherichia coli in milk. This experiment was performed by including two polyethylene glycol (PEG) hydrogel monomers into the LAMP assay for hydrogel cross-linking, followed by isothermal incubation at 65 ℃. Due to the restriction effect of the PEG hydrogel matrix, one bacterium could only produce one fluorescent amplicon dot for simple digital counting. Therefore, the sample bacterial concentrations can be determined based on the number of fluorescent amplicon dots using a fluorescence microscope for imaging. Optimal results were obtained by systematically optimized the experimental conditions, such as amplification time, primer concentration and bovine serum albumin concentration. The result showed that the detection could be completed with sensitivity down to single bacterium. Considering its rapidity, simplicity, efficiency and sensitivity, the developed digital LAMP has an enormous potential for rapid detection of E. coli in food samples.
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