17.9.
32 the temperature to generate a melting curve, which is interpreted by the
AOAC Official Method 2003.09
Salmonella in Selected Foods
BAX® Automated System
First Action 2003
Final Action 2006
Revised First Action 2009
Revised First Action 2011
(Applicable to the detection of Salmonella in frankfurters, raw
ground beef, raw ground chicken, mozzarella cheese, raw frozen
tilapia fish, and orange juice.)
See Tables 2003.09A (2003 study), B (2009 study), and C (2011
study) for the results of the interlaboratory and comparative studies
supporting acceptance of the method.
Caution: The BAX System User Guide details the electrical
hazards associated with the BAX instrument (and
indeed any piece of electrically energized piece of
laboratory equipment). Additionally the following
hazard analysis is offered: Kits.—Reagents used in the
BAX System should pose no hazards when used as
directed. Dispose of lysate, PCR mixture, and other
waste according to one's site practices.
Pathogens.—Biosafety Level-2 precautions should be
exercised when handling all enriched food products.
Salmonella and other potential pathogens may be
present in all enrichments. 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 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 liquids to boil. Wear safety glasses or
other eye protection at all times during operation.
A. Principle
The BAX System Salmonella test is an automated method that
uses polymerase chain reaction (PCR) technology for the detection
of Salmonella in foods. The automated BAX System focuses on a
specific DNA fragment, unique to Salmonella. Only minute levels
of organism are needed as the DNA fragments will be amplified by
the PCR technology of the BAX System. Sample DNA is combined
with DNA polymerase, nucleotides, and primers that are specific for
a given nucleotide sequence. The mixture then undergoes a series of
timed heating and cooling cycles. Heating denatures the DNA,
separating it into single strands. As the mixture cools, the primers
recognize and bind to the targeted DNA sequences. The DNA
polymerase then uses the nucleotides to extend the primers, thus
creating two copies of the targeted DNA fragment. Repeating the
cy cle of de na tur ing, an neal ing, and ex tend ing pro duces an
exponential increase in the number of target DNA fragments.
Once this amplification process occurs, a fluorescent dye in each
BAX System PCR tablet binds with double-strand DNA and emits a
fluorescent signal in response to light. After amplification, the BAX
System begins a detection phase in which the fluorescent signal is
measured. During detection, the temperature of the samples is raised to
the point where the DNA strands separate, releasing the dye and
lowering the signal. The change in fluorescence can be plotted against
BAX System software.
B. Apparatus
Items (a)–(i) are part of the BAX System Start-Up Package
avail able from DuPont Qualicon (Wilmington, DE, USA;
www.qualicon.com).
(a) BAX System Classic or Q7 cycler/detector.
(b) BAX System software.
(c) BAX System computer workstation.
(d) Heating blocks.—Maintaining lysis tubes at 37 ± 1°C and 95
± 1°C.
(e) Cooling block assembly.
(f) PCR tube holders.
(g) Capping/decapping tools.
(h) Lysis tubes with caps and racks.
(i) Calibrated pipets.—Mechanical; covering ranges of 1–20 and
20–200 mL. One 8-channel pipet covering 5–50 mL for sample
transfers.
(j) Incubators.—For maintaining enrichment media at 35° ± 1°C
and 37 ± 1°C.
(k) Blender.—High-speed (16 000–18 000 rpm) with sterile jar.
(l) Stomacher.—Seward model StomacherÒ 400 or equivalent.
C. Media and Reagents
Items (a)–(d) are available in the BAX System Salmonella test kit
from DuPont Qualicon.
(a) Lysis buffer.
(b) Protease.
(c) PCR tubes with tablets.
(d) Optical caps for PCR tubes.
Three primary enrichments are used for the BAX System
Salmonella assay. Each primary enrichment media is specific to the
food type tested. A secondary enrichment, brain heart infusion (BHI),
is used for all foods except meat, poultry, fish, and seafood.
(e) Buffered peptone water.—Add 10 g peptone, 5 g sodium
chloride, 3.5 g sodium phosphate (dibasic), and 1.5 g potassium
phosphate (monobasic) to 1 L distilled water. Dissolve dry ingredients
in distilled water, dispense, and autoclave at 121°C for 15 min. The
final pH should be 7.2 ± 0.2.
(f) Lactose broth.—Add 3 g beef extract, 5 g peptone, and 5 g
lactose to 1 L distilled water. Dissolve dry ingredients in distilled
water, dispense, and autoclave at 121°C for 15 min. The final pH
should be 6.9 ± 0.2.
(g) Universal pre-enrichment broth.—Add 5 g tryptone, 5 g
proteose peptone, 15 g potassium phosphate, 7 g sodium phosphate,
5 g sodium chloride, 0.5 g dextrose, 0.25 g magnesium sulfate, 0.1 g
ferric ammonium citrate, and 0.2 g sodium pyruvate to 1 L distilled
water. Heat ingredients with gentle agitation to dissolve, dispense,
and autoclave at 121°C for 15 min. The final pH should be 6.3 ± 0.2.
(h) BHI.—Add 200 g infusion from calf brains, 250 g infusion
from beef heart, 10 g proteose peptone, 2 g dextrose, 5 g sodium
chloride, and 2.5 g disodium phosphate to 1 L distilled water.
Dissolve dry ingredients in distilled water, dispense, and autoclave
at 121°C for 15 min. The final pH should be 7.4 ± 0.2.
© 2011 AOAC INTERNATIONAL
 Table 2003.09A. Interlaboratory results for the detection of Salmonella in foods by the BAX System (2003 study)
False False positive,
d ,e f g h
BAX Sensitivity, % n e g a ti v e , % Specificity, % %
Total
a 2 b ,c
Food Lev el MPN/g suspensions Presumptive Confirmed Ref. method c A s s ay Ref. A s s ay Ref. A s s ay A s s ay
j
Frankfurters High 0 .1 1 78 61 60 60 — 100 100 0 0 — —
S . poona Low 0 .0 3 78 23 21 21 — 100 100 0 0 — —
Control < 0 .0 3 78 2 0 0 — — — — — 100 0
Ground beef High 0 .4 3 72 70 69 69 — 100 100 0 0 — —
S. newport Low 0 .0 4 72 35 32 32 — 100 100 0 0 — —
Control < 0 .0 3 72 0 0 0 — — — — — 100 0
Ground chicken Lot 1 < 0 .0 3 90 6 6 6 — 100 100 0 0 — —
Lot 2 < 0 .0 3 90 12 9 12 1 .3 3 75 100 25 0 — —
Lot 3 < 0 .0 3 90 33 29 33 2 .2 5 88 100 12 0 — —
Cheese High < 0 .0 3 66 65 65 40 2 6 .8 2 98 61 2 39 — —
S. enteritidis Low < 0 .0 3 66 50 50 10 4 6 .4 8 76 15 24 85 — —
Control < 0 .0 3 66 2 0 0 — — — — — 100 0
Tilapia fish High 0 .9 3 78 74 73 73 0 .0 0 99 100 1 0 — —
S. typimurium Low 0 .0 4 78 53 50 51 0 .0 0 98 100 2 0 — —
Control < 0 .0 3 78 2 0 0 — — — — — 100 0
Orange juice High 0 .0 9 78 76 76 76 — 100 100 0 0 — —
S. muenster Low < 0 .0 3 78 41 39 43 0 .5 0 95 100 5 0 — —
Control < 0 .0 3 78 1 0 0 — — — — — 100 0
a
MPN = Most probable number of colony forming units per gram of food.
b 2 2
c is defined by McNemar as ([a – b] – 1) /(a + b) where a = test suspensions positive by the BAX and negative by culture method and b = test suspensions negative by the BAX and positive by culture method, except for
cheese.
c 2 2
For cheese, c is defined by Siegel as N*{[(a)(d) – (b)(c)] – N/2} /(a + b)(c + d)(b + d)], where a = test suspensions positive by BAX, b = test suspensions positive by culture method, c = suspensions negative by BAX, d =
suspensions negative by culture method.
d
Sensitivity rate was defined as 100 times the total number of positive test suspensions for the method divided by the total number of positive test portions for both methods, except for cheese.
e
Sensitivity rate was defined for cheese as the total number of positive test suspensions divided by the total number of test suspensions analyzed.
f
False negative rate is 100 – sensitivity rate.
g
Specificity rate is 100 times the total number of assay negative test suspensions divided by the total number of negative test suspensions by both methods.
h
False positive rate is 100 – specificity rate.
i
Method agreement is defined as the total number of assay negative suspensions divided by the total number of negative test suspensions by both methods.
j
— = There were no differences in the results between the BAX and the standard method, therefore the Chi square is divided by 0.

© 2011 AOAC INTERNATIONAL

 Table 2003.09B. Comparative results for detection of Salmonella in foods by the modified BAX and reference methods (2009 study)
BAX BAX
Reference No. test BAX BAX Reference BAX fa l s e n e g a ti v e , BAX fa l s e p o s i ti v e ,
a b c d e f g h i
Food m e th o d MPN/25 g portions presumptive confirmed m e th o d Chi-square sensitivity, % % specificity, % %
j
Frankfurters M LG 3 .8 20 18 18 18 0 .0 0 100 0 — —
5 0 0 0 — — — 100 0
Raw ground chicken M LG 0 .4 20 10 10 10 0 .0 0 100 0 — —
k
Orange juice BAM 1 .1 20 11 11 11 0 .0 0 100 0 — —
5 0 0 0 — — — 100 0
a
Most probable number of target microorganisms. MPN/25 g tested on day of assay using appropriate reference method.
b
Number of BAX assay positive samples, not considering subsequent confirmations.
c
Number of BAX assay positive samples subsequently confirmed.
d
Number of samples positive by reference method.
e 2
McNemar’s Chi-square for matched samples is defined as (|a – b| – 1) /(a + b), where a = results that were positive by BAX and negative by reference method, and b = results that were negative by BAX and
positive by reference method. A Chi-square value >3.84 indicates significance at P < 0.05.
f
BAX sensitivity rate is defined as the number of BAX confirmed positive test results divided by the number of reference positive test results, expressed as a percentage. Not calculated where the number of
confirmed BAX positive test results is higher than the number of reference positive test results.
g
BAX false-negative rate is 100 BAX – sensitivity rate.
h
BAX specificity rate is defined as the number of BAX assay negative samples divided by the number of negative samples, expressed as a percentage. Calculated only for control levels with no confirmed positive
results.
i
BAX false-positive rate is 100 – BAX specificity rate.
j
MLG = U.S. Department of Agriculture–Food Safety and Inspection Service Microbiology Laboratory Guidebook.
k
BAM = U.S. Food and Drug Administration Bacteriological Analytical Manual.

© 2011 AOAC INTERNATIONAL

 a
Table 2003.09C. Comparative results for the detection of Salmonella in foods by the modified BAX and reference methods (2011 study)

Reference No. test BAX BAX Reference Chi BAX BAX BAX BAX
b c d e f g h i j
Food m e th o d MPN/25 g portions presumptive confirmed m e th o d square s e n s i ti v i ty , % fa l s e n e g . , % s p e c i fi c i ty , % fa l s e p o s . , %
k l
Frankfurters M LG 0 .6 7 20 6 6 6 — 100 0 — —
NEG 5 0 0 0 — — — 100 0
Raw ground beef (85% lean) M LG 0 .7 9 20 12 12 12 — 100 0 — —
NEG 5 0 0 0 — — — 100 0
m n
Cream cheese BAM 0 .5 3 20 5 5 5 — 100 0 — —
NEG 5 0 0 0 — — — 100 0
Dry dog food BAM 0 .6 3 20 11 11 11 — 100 0 — —
NEG 5 0 0 0 — — — 100 0
a
All BAX and BAX Q7 results were identical, so test method results are presented as a single result for each value presented in the table.
b
Most probable number of target microorganisms. MPN/25 g tested on day of assay using the appropriate reference and calculated using the FDA-BAM MPN method. NEG = Negative control samples.
c
Number of BAX assay positive samples, not considering subsequent confirmations. Results were identical using both the BAX and BAX Q7 instruments.
d
Number of BAX assay positive samples subsequently confirmed.
e
Number of samples positive by reference method.
f 2
McNemar’s Chi square for paired samples is defined as (|a–b|–1) /(a+b), where a = results that were positive by BAX and negative by reference method, and b = results that were negative by BAX and positive by
reference method. A Chi-square value >3.84 indicates significance at P < 0.05.
g
BAX sensitivity rate is defined as the number of BAX confirmed positive test results divided by the number of reference positive test results, expressed as a percentage. Not calculated where the number of
confirmed BAX positive test results is higher than number of reference positive test results.
h
BAX false-negative rate is 100 – BAX sensitivity rate.
i
BAX specificity rate is defined as the number of BAX assay negative samples divided by the number of negative samples, expressed as a percentage. Calculated only for control levels with no confirmed positive
results.
j
BAX false-positive rate is 100 – BAX specificity rate.
k
Frankfurters were composed of pork, turkey, and beef.
l
MLG = U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook, http://www.fsis.usda.gov/Science/Microbiological_Lab_Guidebook/index.asp
m
Cream cheese was the regular (not low-fat or fat-free) formulation.
n
BAM = U.S. Food and Drug Administration's Bacteriological Analytical Manual, http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnalyticalManualBAM/UCM070149

© 2011 AOAC INTERNATIONAL

D. Sample Enrichment
(a) Frankfurters, ground beef, and ground chicken.—Weigh 25 ±
0.5 g test portion into sterile container. Use a stomacher, B(l), to
homogenize sample for 2 min with 225 mL buffered peptone water,
C(e), and incubate, B(j), 20–24 h at 35 ± 1°C.
(b) Orange juice.—Weigh 25 g test portion into 225 mL
universal pre-enrichment broth, C(g), swirl thoroughly, and let
stand for 60 ± 5 min at room temperature; then incubate, B(j), 24 ±
2 h at 35 ± 1°C.
(c) Tilapia fish and mozzarella cheese.—Weigh 25 g test portion
into sterile container. Blend, B(k), sample for 2 min with 225 mL
lactose broth, C(f), and let stand for 60 ± 5 min at room temperature.
If necessary, adjust pH to 6.8 ± 0.2 using 1 N HCl or 1 N NaOH; then
incubate, B(j), 24 ± 2 h at 35°C.
(d) Cream cheese and dry pet food.—Weigh 25 ± 0.5 g test
portion into sterile container. Use a high-speed blender, B(k), to
blend sample for 2 min with 225 mL lactose broth, C(f), and let stand
for 60 ± 5 min at room temperature. If necessary, adjust pH to 6.8 ±
0.2 using 1 N HCl or 1 N NaOH, then incubate, B(j), 24 ± 2 h at 35°C.
E. Secondary Enrichment
After 24 ± 2 h, transfer 10 mL enrichment to 0.5 mL BHI, C(h), for
all foods, except meat, poultry, fish, and seafood. Incubate BHI for
3 h at 37 ± 1°C.
F. Assay
(Protocol listed below is applicable to and was performed on both
BAX System Classic and Q7 instruments.)
After enriching the sample, create rack file, B(b), and warm up
cycler/detector, B(a). Add 150 mL protease, C(b), to 12 mL lysis
buffer, C(a). Add 200 mL lysis reagent to one lysis tube, B(h), for
each sample to be tested. Add 5 mL enriched sample to lysis tube.
Heat lysis tubes for 20 min at 37°C on heating block, B(d). After
20 min, heat lysis tubes for 10 min at 95°C on heating block. Cool
lysis tubes in cooling block, B(e), for 5 min. Arrange PCR tubes,
C(c), in PCR holder, B(f), then in cooling block. Pipet, B(i), 50 mL
lysate from lysis tubes to PCR tubes very carefully, avoiding
cross-contamination. Cap PCR tubes with optical caps, C(d), using
the cap ping/decapping tool, B(g). Place PCR tubes in
cycler/detector and run program.
G. Assay Results
After amplification, a window displays a modified rack view, with
each well appearing in a different color and a symbol in the center of
the well. A minus sign indicates that the sample is negative for the
target organism. A plus sign indicates that the sample is positive for
the target organism. A question mark indicates an indeterminate
result, and DuPont Qualicon technical service should be contacted. A
question mark with a slash through it indicates there was a signal
error, and DuPont Qualicon technical service should be contacted.
When the assay is finished, handle all waste as biohazard waste and
dispose accordingly.
H. Confirmation
Presumptive positive samples must be confirmed culturally as
described in 967.26 (see 17.9.02) and 2000.06 (see 17.9.27); the
FDA Bacteriological Analytical Manual (http://www.fda.gov/Food/
ScienceResearch/LaboratoryMethods/BacteriologicalAnalytical
ManualBAM/ucm070149); or the USDA-FSIS Microbiology
Laboratory Guidebook (http://www.fsis.usda.gov/PDF/MLG_
4_05.pdf), as appropriate.
References: J. AOAC Int. 86, 1149(2003); 92, 989(2009);
94, 1490(2011).
© 2011 AOAC INTERNATIONAL