GeneQuence" Salmonella assay. Performance Tested Method 030201.

A study was conducted to validate the GeneQuence Salmonella DNA hybridization assay, Performance Tested Method 030201, for detection of Salmonella spp. in peanut butter. The study was organized by the AOAC Research Institute under its Emergency Response Validation program. Peanut butter samples inoculated with S. Typhimurium were prepared by an independent laboratory and shipped to study participants for testing. The set of blind-coded test samples consisted of five uninoculated controls, 20 portions inoculated with S. Typhimurium at a low level [determined by most probable number (MPN) analysis to contain 1.1 CFU/25 g portion], and 20 portions inoculated with S. Typhimurium at a higher level (11 CFU/25 g portion by MPN analysis). Samples were tested in parallel by the GeneQuence method and by the U.S. Food and Drug Administration's Bacteriological Analytical Manual reference culture procedure. All five control samples were negative by both methods. For the low-level samples, the same two samples were positive by both the GeneQuence and reference methods. For the high-level samples, the same 19 samples were positive by both methods. All positive GeneQuence assays were confirmed by plating from associated broth cultures. Agreement between the GeneQuence and reference methods was 100%. Sensitivity and specificity of the GeneQuence method were both 100%. Because neither the low- nor the high-level samples yielded the desired fractional positive results (5-15 positives out of 20 samples), a second trial was conducted. Samples in the second trial contained 0.1 and 0.5 CFU/25 g portion for the low and high levels, respectively. All five control samples were negative by both methods. For the low-level samples, the same two samples were positive by both the GeneQuence and reference methods. For the high-level samples, the same three samples were positive by both methods. All positive GeneQuence assays were confirmed by plating from associated broth cultures. Sensitivity and specificity of the GeneQuence method were both 100%. Although once again the desired level of fractional positive results was not obtained, there was 100% agreement between the GeneQuence and reference methods. Based on the results of both trials, it is recommended that the validated claims for Performance Tested Method 030201 be expanded to include peanut butter.