AOAC RI ERP Final Action Recommendations eBook

Dupont BAX Salmonella Pre- Collaborative Study For Expert Review Panel Use Only Do Not Distribute

This automated BAX® System method uses fluorescent detection to analyze PCR product. One PCR 1 primer for each target (one Salmonella -specific target and an internal control) contains a fluorescent dye 2 (two different dyes, one for each target) as a constituent of the primer as well as a quencher (the uni- 3 molecular combination of a primer, fluorescent dye and quencher constitute a Scorpion™ Probe). When 4 incorporated into a PCR product, the dye and quencher are spatially separated, which causes an 5 increase in emission signal. The BAX® System measures the magnitude and characteristics of fluorescent 6 signal change. An analysis by the BAX® System software algorithm then evaluates that data to determine 7 a positive or negative result which is displayed as described below. 8 B. Apparatus and Reagents 9 Items (a)–(h) are part of the DuPont™ BAX® System Start-Up Package available from DuPont Nutrition & 10 Health (Wilmington, DE, USA; www.fooddiagnostics.dupont.com). 11 Items (i)–(l) are part of the DuPont™ BAX® System Real-Time PCR Assay for Salmonella available from 12 DuPont Nutrition & Health (catalog D14306040). 13 a) DuPont™ BAX® System Q7 cycler/detector with computer workstation 14 b) DuPont™ BAX® System application software 15 c) Cluster tubes with caps and racks for lysis 16 d) Capping/de-capping tools – for removing and sealing cluster tube caps and PCR tube caps 17 without jarring the contents 18 e) Heating and cooling blocks* with inserts – for maintaining lysis tubes at 37±2°C, 95±2°C, and 4°C 19 f) Pipettes – for transferring reagents; two adjustable mechanical pipettes covering 20-200 µL and 20 5-50 µL; one repeating pipette; and one multi-channel pipette covering 8 channels and 550 µL. 21 Pipettes should be calibrated to deliver required volumes within 10%. 22 g) Pipette tips with barriers: 0.5-250 µL, 0.5-100 µL extended barrier; 5 mL repeater pipette tips 23 h) PCR tube holders – for transferring a rack of tubes from the cooling block to the cycler/detector 24 i) PCR tubes with tablets 25 j) Flat optical caps for PCR tubes 26 k) Lysis buffer 27 l) Protease 28 m) Incubators – For maintaining media at 35±1°C and 39-42°C 29 n) Stomacher – Seward model 400 or equivalent for mixing the sponge sample with enrichment 30 media 31 o) Appropriate confirmatory media for culture confirmation - Rappaport-Vassiliadis Soya Peptone 32 (RVS), Selenite Cystine (SC), Tetrathionate-Hajna (TT-Hajna) and Tetrathionate (TT) broths and 33 Xylose Lysine Desoxycholate (XLD), Xylose Lysine Tergitol 4 (XLT4), Hektoen Enteric (HE), Brilliant 34 Green Sulfa (BGS) and Bismuth Sulfite (BS) agars 35 *The DuPont™ Thermal Block (DuPont catalog D14614252) may also be used to maintain appropriate temperatures for 36 lysis tubes.

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