Point-of-Care Lateral Flow Detection of Viable O157:H7 Using an Improved Propidium Monoazide-Recombinase Polymerase Amplification Method.

The detection of both viable and viable but non-culturable (VBNC) O157:H7 is a crucial part of food safety. Traditional culture-dependent methods are lengthy, expensive, laborious, and unable to detect VBNC. Hence, there is a need to develop a rapid, simple, and cost-effective detection method to differentiate between viable/dead O157:H7 and detect VBNC cells. In this work, recombinase polymerase amplification (RPA) was developed for the detection of viable O157:H7 through integration with propidium monoazide (PMAxx). Initially, two primer sets, targeting two different genes ( and ) were selected, and DNA amplification by RPA combined with PMAxx treatment and the lateral flow assay (LFA) was carried out. Subsequently, the gene target was found to be more effective in inhibiting the amplification from dead cells and detecting only viable O157:H7. The assay's detection limit was found to be 10 CFU/mL for VBNC O157:H7 when applied to spiked commercial beverages including milk, apple juice, and drinking water. pH values from 3 to 11 showed no significant effect on the efficacy of the assay. The PMAxx-RPA-LFA was completed at 39 °C within 40 min. This study introduces a rapid, robust, reliable, and reproducible method for detecting viable bacterial counts. In conclusion, the optimised assay has the potential to be used by the food and beverage industry in quality assurance related to O157:H7.