Undergraduate Internship Project #4 of 9 for Fy06

Biosolids are produced as byproducts of waste water treatment. Salmonella spp. are major pathogens of concern in this material. Recently, the EPA proposed a new procedure for the detection of Salmonella in biosolids. In the newly proposed Method 1682, after an initial enrichment in a Trypticase Soy Broth (TSB) 15 tube Most Probable Number (MPN) assay, selection of Salmonella spp. occurs on modified semi-solid Rappaport Vassiliadis (MSRV). This medium contains an antibiotic (novobiocin) and a dye (malachite green) to inhibit nonSalmonella species. Method 1682 requires several days due to the multiple cultural steps involved. The standard polymerase chain reaction (PCR) assay is a molecular assay that can be used for the detection of Salmonella spp. This method takes less time, but DNA from dead as well as live organisms is amplified, and false positive results may be obtained. Other concerns are that DNA amplification may be inhibited by the presence of coliforms or by inhibitors present in biosolids. Our studies first tested biosolids samples using the cultural EPA method 1682 and compared them to the PCR assays run on the same samples. The results indicated that we could not use the TSB MPN samples in PCR assays because of inhibitory substances present in biosolids. Inhibition was again observed when cells isolated from MSRV plates were used as a source of DNA in the PCR assay. We determined that this inhibition was due to the MSRV media components. We then tested several different ways of separating cells or DNA from the inhibitors present in MSRV. It was found that heating the sample taken from MSRV in a hot water bath for 1-2 minutes to melt the agar, followed by centrifugation and removal of the supernatant prior to the addition of Instagene, gave results in the PCR assay consistent with those obtained from the cultural method.