Project Number: 3625-32000-080-01
Project Type: Specific Cooperative Agreement

Start Date: Sep 12, 2006
End Date: Aug 31, 2008

Objective:
A. Optimize detection and quantification of Salmonella using (1) a microtiter and (2) real-time PCR format. B. Compare the sensitivity of the microtiter and real-time PCR methods with conventional detection and Most Probable Number (MPN) methodologies.

Approach:
For microtiter format: A. Optimize media components, incubation time, microtiter well volume, etc. Screen both microtiter and real-time assay with artificial samples artificially spiked with 10-fold serial dilutions of Salmonella. Complete validation with field samples. B. Determine sensitivity and specificity of detection and enumeration when compared to conventional methods, including MPN. For real-time format: A. Optimize DNA extraction protocols from hog feces or fecal contents of turkeys using either the robotic Magna Pure LC instrument or manual extraction using commercially available kits. B. Select optimal DNA primers and probes specific for Salmonella. C. Alternatively, if the levels of Salmonella are below detection limits of the real-time format, a modified quantification protocol involving brief sample enrichment (~ 12hrs) may be needed with results extrapolated to CFU/gram in the original sample.