In recent years, the number of product
recalls and contamination
incidents involving pathogenic bacteria has significantly increased,
and the ensuing infections continue to be an ongoing problem for public
health and agriculture. Due to the widespread impact of these pathogens,
there is a critical need for rapid, on-site assays that can provide
rapid results. In this work, we demonstrate the development of a rapid
and simple test based on the combination of reverse transcription
with recombinase polymerase amplification followed by lateral flow
strip detection of viable Escherichia coli O157:H7 cells by detecting the RNA of the pathogen. The optimized
method can be performed for approximately 2 h with a detection limit
of 10 CFU/mL of E. coli O157:H7 in
buffer, spinach, and ground beef samples. Our assay is sensitive,
detecting only E. coli O157:H7 and
not nonpathogenic E. coli or other
similar pathogens. This strategy was able to distinguish viable from
nonviable bacteria and more significantly was able to detect viable
but nonculturable bacteria, which is a major issue when using culture-based
methods for monitoring pathogenic bacteria. An important advantage
of this test is that it can provide timely identification and removal
of contaminated consumables prior to distribution without an extensive
sample preparation.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.