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DuPont™ BAX
®
System Real-Time PCR Assay for
Salmonella
: Collaborative Study
DuPont Nutrition & Health
Page 3
reference culture methods. A total of 15 laboratories participated in this study, with 14 laboratories
1
reporting data for each matrix. Each collaborator received instructions for performing the study and
2
required materials prior to the start of the study. If necessary, training on the BAX® System was
3
provided to laboratory personnel by a DuPont representative.
4
This collaborative study was conducted in accordance with the AOAC INTERNATIONAL Methods
5
Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces
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[4]. Frankfurter samples were evaluated against the USDA-FSIS reference method [1] as a paired study,
7
as the test and reference method enrichment protocols are identical. Orange juice samples were
8
evaluated against the FDA-BAM reference method [2] as an unpaired study, as the BAX® System method
9
uses an enrichment in proprietary media. Estimates of repeatability, reproducibility, and probability of
10
detection (POD) were evaluated.
11
Test Sample Inoculation and Distribution
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Sample product was obtained from a local retail outlet and screened by the organizing laboratory to
13
identify any naturally-contaminating
Salmonella
and determine a total aerobic plate count. For each
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sample type five analytical size portions (25 g for orange juice and 325 g for hot dogs) were screened for
15
Salmonella
using the appropriate reference method. Although naturally contaminated samples would
16
have been preferred, all samples tested returned negative results for
Salmonella
. Therefore, each
17
sample matrix was artificially inoculated with a different serovar of
Salmonella
for use in this study.
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Portions of each sample type were inoculated at levels that on the day of initiation of analysis produced
19
a high spike level (POD ~1.0 or approximately 5 cfu/test portion) and a low spike level (POD 0.25–0.75 or
20
0.2–2.0 cfu/test portion). Additional matrix was left uninoculated to serve as negative controls.
21
To inoculate frankfurter samples, a pure colony of
Salmonella
Typhimurium was transferred from
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Trypticase Soy agar with 5% sheep’s blood (SBA) into Brain Heart Infusion broth (BHI) and incubated at
23
35°C for 18-24 hours. The inoculum was heat stressed in a 55°C water bath for 10 minutes to obtain a
24
percent injury of approximately 70% (as determined by plating onto selective Xylose Lysine
25
Desoxycholate agar and non-selective Tryptic Soy agar). The inoculation process started by inoculating
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four small portions of the bulk matrix (all equal sizes). After each of these portions had been inoculated
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dropwise with an 18-22 hr culture of the target organism, they were homogenized by hand. After
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homogenization, all four portions were combined one at a time into a single container, homogenizing
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the bulk material after each portion was added. The bulk lot was separated into two sampling
30
containers and analysts simultaneously prepared test portions from each container for both the
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candidate and reference method (four analysts total, with two analysts working from each bin, one of
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the analysts weighed out the candidate method samples, the other the reference method
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samples). After 40 samples had been removed (20 for each method, candidate and reference) at
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random locations throughout the sample bin, the matrix was re-homogenized by combining the material
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from both containers into one and mixing thoroughly for the purposes of maintaining an even
36
distribution of the organism.
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FINAL (Version 4)