Aims:  To characterize the specificity and effect of pH and ionic strength on the kinetics of virus binding to histo‐blood group antigens (HBGA)‐conjugated magnetic beads. Methods and Results:  HBGAs from porcine gastric mucin (PGM) have been conjugated to magnetic beads (PGM‐MB) for concentration of NoV. A GII.4 virus was used for the detailed binding kinetics study and a panel of genogroup I (GI) NoVs, genogroup II (GII) NoVs and recombinant NoVs (rNoVs) were used for specificity and binding efficiency assays. We determined that NoV can be captured after 15 min of incubation with PGM‐MB, and virus recovery efficiency is decreased after extended incubation times. rNoV binding as measured by ELISA and NoV recovery as measured by quantitative reverse transcriptase‐polymerase chain reaction (RT‐PCR), were both enhanced significantly at acidic pH conditions. rNoV binding to PGM as measured by ELISA was increased up to 66%. While real‐time RT‐PCR analyses suggest that NoV could be concentrated as much as 1000‐fold at neutral pH, up to 3·4‐fold further increase of NoV recovery was achieved by adjusting the pH of the sample to 3·0–4·2. Variation between GI and GII viral binding to the PGM‐MB at basic pH was observed. All five GI rNoVs tested and 6 of 9 GII rNoVs were captured by PGM. All eight GI strains tested were concentrated by PGM‐MB, ranging from 28‐fold (GI.4) to 1502‐fold (GI.1). Eleven of 13 GII strains were concentrated from 30‐fold (GII.5) to 1014‐fold (GII.4, lab strain) by PGM‐MB. GI and GII rNoVs viral capsid proteins were recovered with high salt conditions, but results were inconsistent for whole virus recovery. Conclusions:  All GI and 85% of GII NoVs tested could be captured and concentrated by PGM‐MB method. The binding occurred rapidly and was enhanced at low pH. Significance and Impact of the Study:  These results facilitated development of a prototype method for sensitive detection of NoV in samples requiring larger volumes.

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Shiga toxin-producing Escherichia coli (STEC) is a significant foodborne pathogen with great economic consequences. There has been an increased food safety concern with this organism since outbreaks of human illnesses caused by this pathogen were first reported in 1982. Therefore, developing a reliable, sensitive, and rapid assay capable of detecting E. coli O157 and the main toxins produced by STEC (i.e., Shiga toxins 1 [Stx(1)] and 2 [Stx(2)]) will directly benefit regulatory agencies by minimizing analysis time. Here, we use Luminex technology to detect multiple analytes in a single 50-ml sample. Using commercially available monoclonal antibodies coupled to carboxylated magnetic microbeads, we developed an immunoassay capable of simultaneously serotyping E. coli O157 and detecting Stx(1) and/or Stx(2). The specificity and sensitivity of this immunoassay was tested against a collection of 34 E. coli isolates belonging to various O serogroups phenotypically different for Stx. The results were compared with microplate sandwich enzyme-linked immunosorbent assay (ELISA), and no cross-reactivity was observed for any of the monoclonal antibodies used. An increased sensitivity up to 1,000 times was observed in the microbead-based immunoassay when compared with the microplate sandwich ELISA. The results indicate that Luminex technology has the potential to simultaneously detect multiple targets without loss of specificity and/or sensitivity. A blind experiment was conducted with 48 samples of ground beef, lettuce, and milk spiked with ≤2 CFU/g E. coli. All the samples were correctly identified, with no false positives or false negatives. This microbead-based immunoassay could be extended to simultaneously detect additional foodborne pathogens and their toxic markers.

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The DiversiLab™ rep-PCR system was used to amplify DNA regions of 28 well-characterized Escherichia coli O104 strains to generate a digital DNA fingerprint profile for strain differentiation. E. coli O104 strains from human stools and other sources were examined. The results indicate that this system can cluster similar O104 strains rapidly.