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. 3, 2014
2925 mL BPW was added, and samples were incubated at 35°C
for 18–24
h. For the test method, samples were tested directly
from the BPW enrichment using the BAX System method.
For the USDA-FSIS MLG reference method, 0.5 mL aliquots
of each portion were transferred to 10 mL tetrathionate (TT)
Hajna broth, and 0.1 mL sample was added to 10 mL modified
Rappaport-Vassiliadis (mRV) broth. All secondary enrichments
were incubated at 42 ± 0.5°C for 22–24
h (or in a water bath
for 18–24
h). Secondary enrichments were streaked to brilliant
green sulfa and either double modified lysine iron agar (LIA) or
xylose lysine Tergitol
TM
4 agar plates and incubated 35 ± 2°C for
18–24
h. Isolated colonies were transferred to triple sugar iron
(TSI) agar and LIA slants and incubated 35 ± 2°C for 22–26
h.
Salmonella
colonies were confirmed using serological (Somatic
O and poly H agglutination) and biochemical procedures
according to USDA-FSIS MLG.
For testing orange juice, each collaborator received
12 low-spike, 12 high-spike, and 12 uncontaminated 25 mL
test portions blind-coded so that the contamination level was
unknown to the collaborator. For the test method, samples were
swirled with 225 mL BAX System MP media and incubated at
39–42°C for 22–26 h, then secondary enrichment was performed
by transferring 10 µL primary enrichment to 500 µL prewarmed
(37°C) BHI broth. Secondary enrichments were incubated at
37°C for 3 h, then tested with the BAX System method. For
the FDA-BAM reference method, portions were swirled with
225 mL Universal Preenrichment Broth (UPB) and incubated
at 35°C for 22–26
h. After primary enrichment, 1 mL of each
enriched portion was transferred to 10 mL TT broth and 0.1 mL
was transferred to 10 mL RV broth. RV tubes were incubated
at 42 ± 0.2°C for 22–26 h using a circulating, thermostatically
controlled water bath. TT tubes were incubated at 35 ± 2°C
for 22–26 h. Secondary enrichments were streaked to bismuth
sulfite, XLD, and Hektoen enteric agar plates and incubated
at 35°C for 22–26 h. Isolated colonies were transferred to TSI
and LIA slants and incubated 35 ± 2°C for 22–26 h.
Salmonella
colonies were confirmed using serological and biochemical
procedures according to FDA-BAM.
Statistical Analysis
Data analysis was performed using each of the metrics
below according to the format described in the
AOAC
INTERNATIONAL Methods Committee Guidelines for
Validation of Microbiological Methods for Food and
Environmental Surfaces
and using the Least Cost Formulations,
Ltd, AOAC Binary Data Interlaboratory Study Workbook (6).
For the purposes of this evaluation, POD was defined as the
number of positive outcomes divided by the total number of
trials. POD was estimated with a 95% confidence interval for
each of the following levels: candidate presumptive results
(POD
CP
); candidate confirmatory results (POD
CC
); candidate
method results based on the presumptive and confirmatory
results (POD
C
); and reference method results (POD
R
).
Candidate presumptive and confirmatory results were
compared by determining the difference between the POD
(dLOPD) values for each matrix and concentration (dLPOD
CP
= POD
CP
– POD
CC
). If the confidence interval of a dLPOD does
not contain zero, then the difference is statistically significant
at the 5% level.
Candidate and reference method results were compared by
determining the difference in POD values between the candidate
and reference methods for each matrix and concentration
(dLPOD
C
= POD
C
– POD
R
). If the confidence interval of a
dLPOD does not contain zero, then the difference is statistically
significant at the 5% level.
AOAC Official Method 2013.02
Salmonella
species in a Variety of Foods
and Environmental Surfaces
BAX
®
System Real-Time PCR Assay for
Salmonella
First Action 2013
[Applicable to the detection of
Salmonella
in a variety of
foods, including raw ground beef (25 and 375 g), ground beef
with soy (25 and 325 g), beef trim (25 and 325 g), frankfurters
(325 g), shrimp (25 g), ground turkey (25 g), chicken wings
(25 g), poultry rinse (30 mL), whole powdered (dried) eggs
(25 g), shell eggs (1000 mL), fresh bagged lettuce (25 g), frozen
peas (25 g), orange juice (pasteurized; 25 mL), cream cheese
(25 g), nonfat dry milk (25 g), ice cream (25 g), peanut butter
(25 g), cocoa (25 g), white pepper (25 g), milk-based infant
formula (25 mL), and dry pet food (375 g), and on stainless
steel, ceramic tile, and plastic surfaces.]
See
Table
2013.02
for a summary of results of the collaborative
study.
See
Appendix 4, Tables 1–6 for detailed results of the
collaborative study.
Caution: Kits.
—The reagents used in the BAX System
should pose no hazards when used as directed.
Dispose of lysate, PCR mixture, and other
waste according to your site practices.
Cycler/detector.
—Only
qualified
laboratory
personnel should operate the cycler/detector. Do
not attempt to repair the instrument. Live power
may still be available inside the unit even when
a fuse has blown or been removed. Refer to
the BAX System User Guide for maintenance
procedures when cleaning the unit or changing a
fuse. The heating block can become hot enough
during normal operation to cause burns or cause
liquids to boil. Wear safety glasses or other
eye protection at all times during operation.
Enrichment broths.
—All enrichment broths
may contain varying pathogens whether they
contain
Salmonella
or not and thus should be
sterilized and disposed of using proper procedures
following any culture-based confirmatory steps.
Reference cultures
.—When handling reference
Salmonella
cultures, always follow appropriate
biosafety containment procedures as provided by
your standard laboratory site practices, Centers
for Disease Control and Prevention (CDC), or
Canadian Pathogen Safety Data Sheets and Risk
Assessment.
A. Principle
The DuPont™ BAX System uses the polymerase chain
reaction (PCR) to amplify a specific fragment of bacterial