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DuPont™ BAX
®
System Real-Time PCR Assay for
Salmonella
: Collaborative Study
DuPont Nutrition & Health
Page 6
i)
PCR tubes with tablets
1
j)
Flat optical caps for PCR tubes
2
k)
Lysis buffer
3
l)
Protease
4
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
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media
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o)
Appropriate confirmatory media for culture confirmation - Rappaport-Vassiliadis Soya Peptone
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(RVS), Selenite Cystine (SC), Tetrathionate-Hajna (TT-Hajna) and Tetrathionate (TT) broths and
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Xylose Lysine Desoxycholate (XLD), Xylose Lysine Tergitol 4 (XLT4), Hektoen Enteric (HE), Brilliant
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Green Sulfa (BGS) and Bismuth Sulfite (BS) agars
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*The DuPont™ Thermal Block (DuPont catalog D14614252) may also be used to maintain appropriate temperatures for
12
lysis tubes.
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C.
Media
14
a)
BAX® System MP media – DuPont catalog D12404925 (bulk powder) or D12745725 (StatMedia)
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b)
Brain Heart Infusion (BHI) broth – Oxoid catalog CM 1032 or equivalent
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c)
Buffered Peptone Water (BPW) – Oxoid catalog CM 0509 or equivalent
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D.
Sample Enrichment
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Ground beef, ground beef with soy, beef trim (25 g)
– Weigh 25 g test portion into sterile container.
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Use a stomacher, B(n), to homogenize sample for 2 minutes with 225 mL pre-warmed (35°C) BPW,
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C(d). Incubate, B(m), at 35°C for 20-24 hours.
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Ground beef (375 g)
- Weigh 375 g test portion into sterile container. Use a stomacher, B(n), to
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homogenize sample for 2 minutes with 1500 mL pre-warmed (35°C) mTSB+n, C(g). Incubate, B(m), at
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35°C for 22-26 hours.
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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).
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Incubate, B(m), at 35°C for 20-24 hours.
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Beef trim (325 g)
- Weigh 325 g test portion into sterile container. Hand massage to homogenize
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sample for 2 minutes with 1500 mL pre-warmed (41°C) BAX® System MP media, C(a). Incubate,
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B(m), at 39-42°C for 16-24 hours.
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Frankfurters (325 g)
- Weigh 325 g test portion into sterile container. Use a stomacher, B(n), to
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homogenize sample for 2 minutes with 1400 mL pre-warmed (35°C) BPW, C(d). Add additional BPW
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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
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temperature for 55-65 minutes. Adjust pH to 6.8±0.2 using 1 N HCl or 1 N NaOH, C(k), if necessary.
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Incubate, B(m), at 35°C for 22-26 hours.
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FINAL (Version 4)