A rapid molecular-based assay for direct quantification of viable bacteria in slaughterhouses

A rapid test for microbial quantification in carcass and environmental swabs that does not require enrichment and provides results in less than 4 h is described here. Steps in the assay include the rapid concentration of bacteria on sponge swabs by vacuum filtration followed by real-time PCR detection. The assay has been applied for the detection of coliforms, Escherichia coli O157:H7, Salmonella spp., and Listeria monocytogenes on carcass swabs and environmental samples in a slaughterhouse-processing line. Comparison of this rapid method with standard culture techniques for coliform counts on beef and pork carcass swabs revealed higher numbers of bacteria (2- to 50-fold) by the rapid test compared with the plate counts. This was due to the detection of all bacteria (live, dead, and non-culturable forms) in the rapid assay. To allow detection of only viable bacteria, concentrated samples were treated with ethidium monoazide (EMA) prior to DNA extraction and real-time PCR detection, thereby preventing the amplification of DNA from bacteria with damaged cell walls and allowing only the DNA from bacteria with intact membranes to be detected. EMA treatment resulted in a significant reduction (P < 0.001) in the number of coliforms detected compared to real-time PCR without EMA treatment. In beef swabs, the counts obtained in EMA real-time PCR were not significantly different (P < 0.08) from the culture counts and the correlation coefficient between the two assays was 0.7385. A lower correlation coefficient (0.402) was obtained with pork swabs. The assay described herein has the potential to be applied on a routine basis to slaughterhouse lines for the detection of indicator organisms or specific pathogens.