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
6
[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
12
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
14
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.
18
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
22
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
26
four small portions of the bulk matrix (all equal sizes). After each of these portions had been inoculated
27
dropwise with an 18-22 hr culture of the target organism, they were homogenized by hand. After
28
homogenization, all four portions were combined one at a time into a single container, homogenizing
29
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
31
candidate and reference method (four analysts total, with two analysts working from each bin, one of
32
the analysts weighed out the candidate method samples, the other the reference method
33
samples). After 40 samples had been removed (20 for each method, candidate and reference) at
34
random locations throughout the sample bin, the matrix was re-homogenized by combining the material
35
from both containers into one and mixing thoroughly for the purposes of maintaining an even
36
distribution of the organism.
37
FINAL (Version 4)