Xylenol orange-based loop-mediated DNA isothermal amplification for sensitive naked-eye detection of Escherichia coli

Abstract

Loop-mediated isothermal amplification (LAMP) can amplify DNA specifically and sensitively. Under minimal buffering conditions, it produces hydrogen ions that lower the pH of the solution upon DNA amplification. This characteristic was applied to visually detect amplified DNA of Escherichia coli through the use of Xylenol Orange, a pH-dependent dye. Under the optimal conditions, 120 min at 63 °C, the Xylenol orange-dependent colorimetric LAMP revealed a detection limit as low as 1 CFU, namely 100,000 times more sensitive than typical multiplex PCR, and showed no cross-reactions with other foodborne pathogens. The colorimetric assay was successfully exploited to detect E. coli contaminations in milk samples, showing high reliability and the same high sensitivity with naked-eye readout. Together with robustness, simplicity, and visual detectability of amplification, this assay can serve as an alternative tool to PCR for detecting E. coli, which is suitable for both laboratory and on-field applications.

Keywords: Colorimetric assay; E. coli; LAMP; Milk; Xylenol orange.