| Summary: | Foodborne bacteria have persisted as a significant threat to public health and to the food and agriculture industry. Due to the widespread impact of these pathogens, there has been a push for the development of strategies that can rapidly detect foodborne bacteria on-site. Shiga toxin-producing <i>E. coli</i> strains (such as <i>E. coli</i> O157:H7, <i>E. coli</i> O121, and <i>E. coli</i> O26) from contaminated food have been a major concern. They carry genes <i>stx1</i> and/or <i>stx2</i> that produce two toxins, Shiga toxin 1 and Shiga toxin 2, which are virulent proteins. In this work, we demonstrate the development of a rapid test based on an isothermal recombinase polymerase amplification reaction for two Shiga toxin genes in a single reaction. Results of the amplification reaction are visualized simultaneously for both Shiga toxins on a single lateral flow paper strip. This strategy targets the DNA encoding Shiga toxin 1 and 2, allowing for broad detection of any Shiga toxin-producing bacterial species. From sample to answer, this method can achieve results in approximately 35 min with a detection limit of 10 CFU/mL. This strategy is sensitive and selective, detecting only Shiga toxin-producing bacteria. There was no interference observed from non-pathogenic or pathogenic non-Shiga toxin-producing bacteria. A detection limit of 10 CFU/mL for Shiga toxin-producing <i>E. coli</i> was also obtained in a food matrix. This strategy is advantageous as it allows for timely identification of Shiga toxin-related contamination for quick initial food contamination assessments.
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