A (+/-) indicates amplification/no amplification by real time PCR. Figure 4 Detection of silent genes in dual BoNT containing strains of C. botulinum. Shown are amplification plots of three strains of C. botulinum that contain silent genes: CDC1436 A2b (A), strain 657 Ba4 (B), and strain An436 Bf (C). Copy numbers and the indicated gene detected by color are listed for each. We then tested DNA-spiked food samples and crude culture

supernatants for the presence of serotype-specific BoNT genes using the above assays. In spiked food samples, we were able to detect type-specific BoNT DNA down check details to at least three genomic copies of BoNT DNA

in each sample (Figure 5A and 5B). To determine relative levels of detections, we tested the four major causes of foodborne botulism, BoNT A, B, E, and F within crude toxin supernatants. Positive PCR signals were seen with sample dilutions Tariquidar price containing toxin concentrations of 0.000018 LD50 BoNT/A per ml and 0.00385 LD50 BoNT/B toxin per ml. The level of detection is greater than 50,000 times more sensitive than the mouse bioassay for BoNT/A and greater than 250 times more sensitive than the mouse Idelalisib cost bioassay for BoNT/B in equivalent samples. Positive PCR signals were observed with sample dilutions equal to 1LD50 in BoNT/E toxin/mL and 0.007 LD50 BoNT/F toxin/mL. Thus the level of detection for BoNT/E and BoNT/F matched or was 1000 times more sensitive than the mouse protection bioassay, respectively (Table 5). Figure 5 qPCR detection of type-specific BoNT DNA in food samples spiked with purified

C. botulinum DNA. Canned green beans or corned beef was spiked with ten-fold dilutions of purified type-specific BoNT DNA. Samples were processed and DNA extracted from each sample. Results show copy number of each type-specific BoNT BIBW2992 mouse dilution in both food types. Table 5 Detection limits of BoNT DNA in crude toxin supernatants   Bot A Bot B Bot E Bot F Crude Toxin 2 ng LOD       Crude Toxin 200 pg   LOD   LOD Crude Toxin 20 pg     0.8 (LOD)   Crude Toxin 2 pg         Crude Toxin 200 fg   11.7   2.58 Crude Toxin 20 fg 29.2       LOD indicates the averaged limits of detection for that subtype in our mouse protection bioassay with identical serotypes used in toxin complex preparations.