Isothermal Amplification and Lateral Flow Nucleic Acid Test for the Detection of Shiga Toxin-Producing Bacteria for Food Monitoring

Abstract

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 E. coli strains (such as E. coli O157:H7, E. coli O121, and E. coli O26) from contaminated food have been a major concern. They carry genes stx1 and/or stx2 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 E. coli 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.

Rapid detection methods for foodborne pathogens based on nucleic acid amplification: Recent advances, remaining challenges, and possible opportunities

“…In recent studies, researchers have used RPA and combined it with LFA systems, fluorescent dyes, fluorescent probes, or CRISPR/Cas-based technology to detect pathogens in food samples ( Cai et al, 2021 , Chen et al, 2021 , Jin et al, 2022 , Liu et al, 2022a , Ma et al, 2020 , Mao et al, 2022 , Petrucci et al, 2022 , Tian et al, 2021 , Wang et al, 2021 , Wang et al, 2022b ) ( Table S4 ). For example, Ma et al (2020) utilized RPA in combination with a lateral flow dipstick to identify various pathogens in different types of food samples.…”

“… Wang et al (2021) used a similar approach to detect V. cholerae and V. vulnificus in shrimps and found that these pathogens could be detected at a concentration of 1 cfu/10 g after 4 h of enrichment. This approach has also been used by Petrucci et al (2022) to detect E. coli O157:H7 in chicken meat and they found that this pathogen could be detected at a concentration of 10 cfu/mL after enriching their samples for 10 min.…”