A Novel Real-Time Loop-Mediated Isothermal Amplification Combined with Immunomagnetic Beads Separation and Ethidium Bromide Monoazide Treatment for Rapid and Ultrasensitive Detection of Viable Escherichia coli O157:H7 in Milk

Abstract

In this study, a real-time loop-mediated isothermal amplification (Rti-LAMP) combined with immunomagnetic beads separation (IMS) and ethidium bromide monoazide (EMA) treatment was developed for the rapid and ultrasensitive detection of viable Escherichia coli (E. coli) O157:H7 in milk without enrichment. Polyclonal antibodies against E. coli O157:H7 flagellum and specific immunomagnetic beads (IMBs) were prepared. The capture efficiency of target bacteria was more than 95% by adding 50 μL of prepared IMBs into 1 mL of E. coli sample for incubation at 37 °C for 20 min. Then, Gly-HCl buffer (0.2 M, pH 2.2) was a key to the effective eluting and separating of bacteria from IMB-bacterial complexes. The eluted bacteria were resuspended with 20 μL TZ lysing solution and boiled at 99.5 °C for 10 min, as a DNA template of Rti-LAMP. The development of IMS-Rti-LAMP assay could specifically detect viable E. coli O157:H7 as low as 2.7 CFU/mL in pure culture. Furthermore, the optimum volume for detection of E. coli O157:H7 in milk was 1 mL of the sample by IMS-Rti-LAMP assay. Before Gly-HCl elution, 4 μg/mL of EMA was used to treat bacterial cells which significantly inhibited DNA amplification derived from dead cells in Rti-LAMP. The limit of detection (LOD) of viable E. coli O157:H7 in milk was 6 CFU/mL by the IMS-EMA-Rti-LAMP, and the whole assay could be completed in 4 h. The development of IMS-EMA-Rti-LAMP assay was a rapid, ultrasensitive, and specific detection of viable E. coli O157:H7 in milk without enrichment.