AOAC RI ERP Final Action Recommendations eBook

FINAL (Version 4)

DuPont™ BAX ® System Real-Time PCR Assay for Salmonella : Collaborative Study

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i) PCR tubes with tablets

j) Flat optical caps for PCR tubes

k) Lysis buffer l) Protease

m) Incubators – For maintaining media at 35±1°C and 39-42°C 5 n) Stomacher – Seward model 400 or equivalent for mixing the sponge sample with enrichment 6 media 7 o) Appropriate confirmatory media for culture confirmation - Rappaport-Vassiliadis Soya Peptone 8 (RVS), Selenite Cystine (SC), Tetrathionate-Hajna (TT-Hajna) and Tetrathionate (TT) broths and 9 Xylose Lysine Desoxycholate (XLD), Xylose Lysine Tergitol 4 (XLT4), Hektoen Enteric (HE), Brilliant 10 Green Sulfa (BGS) and Bismuth Sulfite (BS) agars 11 *The DuPont™ Thermal Block (DuPont catalog D14614252) may also be used to maintain appropriate temperatures for 12 lysis tubes. 13 C. Media 14 a) BAX® System MP media – DuPont catalog D12404925 (bulk powder) or D12745725 (StatMedia) 15 b) Brain Heart Infusion (BHI) broth – Oxoid catalog CM 1032 or equivalent 16 c) Buffered Peptone Water (BPW) – Oxoid catalog CM 0509 or equivalent 17 D. Sample Enrichment 18 Ground beef, ground beef with soy, beef trim (25 g) – Weigh 25 g test portion into sterile container. 19 Use a stomacher, B(n), to homogenize sample for 2 minutes with 225 mL pre-warmed (35°C) BPW, 20 C(d). Incubate, B(m), at 35°C for 20-24 hours. 21 Ground beef (375 g) - Weigh 375 g test portion into sterile container. Use a stomacher, B(n), to 22 homogenize sample for 2 minutes with 1500 mL pre-warmed (35°C) mTSB+n, C(g). Incubate, B(m), at 23 35°C for 22-26 hours. 24 Ground beef with soy (325 g) - Weigh 325 g test portion into sterile container. Use a stomacher, 25 B(n), to homogenize sample for 2 minutes with 975 mL pre-warmed (35°C) mTSB+caa+n, C(h). 26 Incubate, B(m), at 35°C for 20-24 hours. 27 Beef trim (325 g) - Weigh 325 g test portion into sterile container. Hand massage to homogenize 28 sample for 2 minutes with 1500 mL pre-warmed (41°C) BAX® System MP media, C(a). Incubate, 29 B(m), at 39-42°C for 16-24 hours. 30 Frankfurters (325 g) - Weigh 325 g test portion into sterile container. Use a stomacher, B(n), to 31 homogenize sample for 2 minutes with 1400 mL pre-warmed (35°C) BPW, C(d). Add additional BPW 32 to reach a total media volume of 2925 mL. Incubate, B(m), at 35°C for 18-24 hours. 33 Shrimp, peanut butter (25 g) – Weigh 25 g test portion into sterile container. Use a stomacher, B(n), 34 to homogenize sample for 2 minutes with 225 mL pre-warmed (35°C) LB, C(e). Let stand at room 35 temperature for 55-65 minutes. Adjust pH to 6.8±0.2 using 1 N HCl or 1 N NaOH, C(k), if necessary. 36 Incubate, B(m), at 35°C for 22-26 hours. 37

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