|KONG, QIULIAN - Shanghai Academy Of Agricultural Sciences|
Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/12/2016
Publication Date: 1/29/2016
Publication URL: https://handle.nal.usda.gov/10113/63151
Citation: He, X., Kong, Q., Patfield, S.A., Skinner, C.B., Rasooly, R. 2016. A new immunoassay for detecting all subtypes of Shiga toxins produced by Shiga toxin-producing E. coli in ground beef. PLoS One. 11(1):e0148092.
Interpretive Summary: Shiga toxin –producing E. coli (STEC) are food-borne pathogens. As few as 100 cells can cause disease leading to serious illness or even death. Shiga toxin (Stx) is the common trait and major virulence determinant of all STEC strains. Therefore, reliable methods for detection of Stxs are crucial for identification of STEC. Many antigenically distinct Stxs, including 3 subtypes of Stx1 and 7 subtypes of Stx2 have been identified. Currently, very few commercial kits are available and they are normally overwhelmingly expansive. In addition, none of them detect all subtypes of Stxs. In this study, we developed a highly sensitive and universal immunoassay that is capable of detecting all subtypes of Stxs. When this assay is applied to ground beef samples, it was able to identify beef samples spiked with a single STEC cell after overnight enrichment. This assay will be useful for prompt detection of STEC contamination at early stage in the food chain, therefore, avoiding the need for possible recall.
Technical Abstract: Background Shiga toxin (Stx) is a common virulence factor of all Shiga toxin producing E. coli (STEC) that cause a wide spectrum of disease, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although several commercial kits are available for detection of Stx produced by STEC, none of them is capable of recognizing all subtypes of Stxs, which include three subtypes of Stx1 and seven subtypes of Stx2. Methods and Findings New monoclonal and polyclonal antibodies against Stx1 and Stx2 were developed. A universal sandwich ELISA capable of detecting all known subtypes of Stx1 and Stx2 was established using a pool of newly developed antibodies. To precisely monitor the sensitivity of the assay for each subtype of Stxs, recombinant toxoids were created and used as standards in ELISAs. Because of the high affinity of the antibodies incorporated, the ELISA assay is highly sensitive with a limit of detection for different subtypes of Stx1a and Stx2a between 10 and 50 pg/mL in phosphate buffered saline (PBS). The assay was also able to identify STEC based on the production of Stxs using the supernatants of culture fluids or even single colonies on agar plates without lengthy enrichment in liquid medium. When applied to ground beef samples, this newly developed ELISA was capable of distinguishing beef samples spiked with a single bacterial cell. Conclusions A highly sensitive and universal assay for all subtypes of Stx1 and Stx2 was developed. It significantly improved the current technologies by avoiding false negative results due to the narrow detection range of the assay. The assay developed in this study can be useful for prompt detection of new and emerging serotypes and screening ground beef samples for contamination of STEC at the early stage in the food chain, thus avoiding the need for possible recall.