129 / 1195
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.
37
38
Dupont BAX Salmonella Pre- Collaborative Study
For Expert Review Panel Use Only
Do Not Distribute
AOAC INTERNATIONAL