USE OF A MOLECULAR PROBE ASSAY FOR MONITORING SALMONELLA SPP. IN BIOSOLID SAMPLES

Current federal regulations (40 CFR 503) require enumeration of fecal coliform or salmonellae prior to land application of biosolids. This regulation specifies use of enumeration methods included in “Standard Methods for the Examination of Water and Wastewater 18th Edition,” (SM), or when analyzing for salmonellae, a cultural method developed by Kenner and Clark. For “Class A” biosolids the regulation requires use of multiple-tube fermentation techniques for fecal coliform or multiple tube enrichment techniques for Salmonella spp. followed by isolation and biochemical and serological confirmation. The difficulties in completing the procedure for enumeration of salmonellae in biosolids and sludges has limited the use of this assay for meeting regulatory requirements. Moreover, the time required to confirm the presence of salmonellae in a given sample is considerably longer than the time required to estimate the density of fecal coliforms. Analytical methods for Salmonella spp. which provide results expeditiously may provide the means for more frequent and routine monitoring of biosolid samples for these organisms.Molecular probes have been used for assaying clinical samples and food samples for several years. These probes are short strands of genetic material (oligonucleotides) which have a chromogenic or other type of marker attached to the probe. The probe is mated to the genetic material lysed from cells contained within a given sample. The genetic material will only anneal to probes which complement the host's DNA. Consequently, it is important for the probe's oligonucleotide to be specific for the organism(s) of interest.This study was conducted to determine if a commercially available molecular probe system could be used to determine and enumerate Salmonella spp. in biosolids or sludges. Samples of untreated, and treated sludges were assayed using the GENE-TRAK® Salmonella assay. Split samples were assayed using conventional multiple fermentation tube techniques and the DNA probe based diagnostic test. The results indicate that the molecular probe and the conventional fermentation tube technique yielded equivalent results. Interestingly, the probe technique yielded results within 52 hours following sample collection compared to the conventional fermentation tube technique with confirmation which required approximately 120 hours. These results suggest that the molecular probe system used for this work may be used to determine the presence or absence of Salmonella spp. in biosolids within a relatively short time frame.