The development of an efficient and rapid enzyme linked fluorescent assay method for the detection of Listeria spp. from foods.

Conventional isolation methods, including the Health Products and Food Branch (HPFB), Health Canada method used for the isolation and identification of Listeria species and Listeria monocytogenes from foods and environmental samples, can take a week or more to complete and are usually labor-intensive. This has led to the development of various rapid methods which attempt to generate results comparable to standard methods but in a reduced time-frame with less hands-on operation. Our previous work with rapid detection systems indicated that the recommended enrichment protocols failed to grow the Listeria to detectable levels in a reliable and consistent manner. In the present study, a novel enrichment protocol is described and consists of samples being pre-enriched in Palcam broth (incubated at 35 degrees C for 26 h), enriched in UVM 2 (30 degrees C for 26 h) and then plated and analysed by a rapid detection kit, with results being generated after 52 h of incubation. In total, 200 naturally contaminated samples were analysed by both the HPFB standard method and the Palcam method. The results showed that the Palcam method is comparable to the HPFB method. Further analysis involved a rapid detection system, which applies ELISA techniques and automation in an enzyme-linked fluorescent assay (ELFA) system. This system, referred to as the Vitek Immuno Diagnostic Assay System or VIDAS, can identify Listeria to the genus or species (L. monocytogenes) level. In this comparison, an additional 324 naturally contaminated samples were analysed by both the Palcam and ELFA methods. The sensitivity and specificity of the ELFA method were 98.1% and 97.0%, respectively, while the efficiency was 97.5%. False-negative and false-positive rates were 1.9% and 3.0%, respectively. These results show that the ELFA method (when using the Palcam method for pre- and secondary enrichment) was efficient and gave reliable results after 52 h of incubation, and met Health Canada's criteria for approval as a rapid method.

The inhibitory properties of various sponges on Listeria spp.

Various retail and environmental sponges were tested for inhibitory properties against Listeria species and several other bacterial genera. Sterile sponges, unrinsed and rinsed in sterile distilled water or sterile neutralizing buffer, were placed on seeded plates of tryptic soy agar with 0.6% yeast extract. Plates were incubated at 30 degrees C for 24 h and zones of inhibition measured. The Systems Plus environmental sponge and the Technical Service Consultants Ltd sponge (sTc) proved to be the only sponges which consistently demonstrated no inhibitory properties. Results using scanning electron microscopy showed considerable bacterial attachment to the Systems Plus sponge, further corroborating these findings.

Fermentation of wort containing added ochratoxin A and fumonisins B1 and B2.

Ochratoxin A (OA), fumonisin B1 (FB1) and fumonisin B2 (FB2) were added to wort at levels of 0.19, 0.95 and 0.95 micrograms/ml, respectively, and fermented for up to 8 days by three strains of Saccharomyces cerevisiae. Decreases of OA in the beer over this period were estimated from straight line slopes to be 2-13%. Losses of FB1 and FB2 were estimated to be 3-28% and 9-17% respectively. Some OA was taken up by the yeast, up to 21% in a detailed study with one strain. In contrast, uptake of fumonisins by yeast was negligible (< 1% FB1 and < 2% FB2). In control experiments, OA, FB1 and FB2 were found to be stable when added to yeast-free wort and kept for up to 8 days at 25 degrees C. In addition, spiking experiments with blank day 0-8 fermenting wort samples showed method recoveries averaging 87-91%. None of the mycotoxins was detected in control fermentations where they were not added to the wort.

Fermentation of wort containing deoxynivalenol and zearalenone.

Wort containing deoxynivalenol and zearalenone, each added at a level of 1.9 µg/mL, was fermented by 3 strains ofSaccharomyces cerevisiae for 7 or 9 days to make beer. Analysis showed that deoxynivalenol was stable during this process. The major metabolite of zearalenone was ß - zearalenol, which formed in up to 69% of the initial zearalenone concentration, while up to 8.1% of the initial zearalenone was converted to α - zearalenol. The major part of the metabolism of zearalenone occurred by 1 - 2 days. Control experiments, where the yeasts were omitted and deoxynivalenol, zearalenone and α - and ß - zearalenol were added, showed good recovery and stability of the mycotoxins over the 7-9 day time period. No deoxynivalenol, zearalenone, α-zearalenol or ß-zearalenol was detected in control yeast fermentations where they were not added to the wort.