17.10.10 C.

Media and Reagents

AOAC Official Method 2003.12 Items (a)–(d) are available in the BAX system L. monocytogenes
Listeria monocytogenes in Foods test kit from DuPont Qualicon.
BAX® Automated System (a) Lysis buffer.
First Action 2003
(b) Protease.
Final Action 2006
(c) PCR tubes with tablets.
(Applicable to the detection of L. monocytogenes in dairy
products, fruits and vegetables [except radishes], seafoods, raw and (d) Optical caps for PCR tubes.
processed meats, and poultry.) Three primary enrichments are used for the BAX system
See Table 2003.12 for the results of the interlaboratory study L. monocytogenes assay. Each primary enrichment media is specific
supporting acceptance of the method. to the food type tested. A sec ond ary en rich ment, MOPS
[3-(N-morpholine)propanesulfonic acid (CAS
A. Principle 71119-22-7)]-Buffered Listeria Enrichment broth (MOPS-BLEB),
The BAX system L. monocytogenes test is an automated method is used for all foods.
that uses polymerase chain reaction (PCR) technology for the (e) Enrichment broth.—Add 30 g tryptone soya broth powder,
detection of L. monocytogenes in foods. The automated BAX 6 g yeast extract, 1.35 g monopotassium phosphate, and 9.6 g
sys tem fo cuses on a spe cific DNA frag ment, unique to anhydrous disodium phosphate to 1 L distilled water. Dispense into
L. monocytogenes. Only minute levels of organism are needed as the 225 mL containers and sterilize at 121°C for 15 min. Then add
DNA fragments will be amplified by the PCR technology of the 2.5 mL 10% (w/v) filter-sterilized sodium pyruvate. Prepare
BAX system. DNA from the test system is combined with DNA acriflavin and nalidixic acid supplements as 0.5% (w/v) stock
polymerase, nucleotides, and L. monocytogenes sequence-specific solutions in distilled water. Prepare cycloheximide supplement as
primers. The mixture then undergoes a series of timed heating and 1.0% (w/v) stock solution in 40% (v/v) solution of ethanol in water.
cooling cycles. Heating denatures the DNA, separating it into single After 4 h incubation, add stock solutions: 0.45 mL acriflavin, 1.8 mL
strands. As the mixture cools, the primers recognize and bind to the nalidixic acid, and 1.15 mL cycloheximide to 225 mL enrichment
targeted DNA sequences. The DNA polymerase then uses the broth. Final pH should be 7.3 ± 0.1. Base and supplements are
nucleotides to extend the primers, thus creating 2 copies of the commercially available through Oxoid, Inc. (Ogendsburg, NY
targeted DNA fragment. Repeating the cy cle of denaturing, 13669, USA).
annealing, and extending produces an exponential increase in the (f) Complete selective enrichment broth.—Add 30 g typtone
number of target DNA fragments. soya broth powder and 6 g yeast extract to 1 L distilled water. Adjust
Once this amplification process occurs, a fluorescent dye in each pH to 7.3. Dispense 225 mL and sterilize by autoclaving. Prepare
BAX system PCR tablet binds with double strand DNA and emits a acriflavin and nalidixic acid supplements as 0.5% (w/v) stock
fluorescent signal in response to light. After amplification, the BAX solutions in distilled water. Prepare cycloheximide supplement as
system begins a detection phase where the fluorescent signal is 1.0% (w/v) stock solution in 40% (v/v) solution of ethanol in water.
measured. During detection, the temperature of the system is raised Add stock solutions: 0.45 mL acriflavin, 1.8 mL nalidixic acid, and
to the point where the DNA strands separate, releasing the dye and 1.15 mL cycloheximide to 225 mL enrichment broth.
lowering the signal. The change in fluorescence is plotted against (g) UVM broth.—Add 5 g proteose peptone, 5 g tryptone, 5 g Lab
the temperature to generate a melting curve, which is interpreted by Lemco Powder (Oxoid), 5 g yeast extract, 20 g sodium chloride,
the BAX system software. 1.35 g potassium phosphate, 12 g sodium phosphate, 1 g esculin,
B. Apparatus 1 mL naladixic acid (2% in 0.1M NaOH), 12 mg acriflavin×HCl, and
1 L distilled water. Dispense into 225 mL containers and sterilize at
Items (a)–(i) are part of the BAX system Start-Up Package
available from DuPont Qualicon (Wilmington, DE 19810, USA; 121°C for 15 min.
www.qualicon.com/). (h) Demi-Fraser broth.—Add 55 g commercially available
(a) BAX system cycler/detector. Demi-Fraser broth (BD Biosciences) to 1 L distilled water. Adjust
pH to 7.2 ± 0.2. Dispense into 225 mL containers and sterilize at
(b) BAX system software.
121°C for 15 min.
(c) BAX system computer workstation.
(i) MOPS-BLEB.—Dissolve 30 g trypticase soy broth, 6.7 g
(d) Heating blocks.—Maintaining lysis tubes at 55° ± 1°C and MOPS free acid, 10.5 g MOPS sodium salt, and 6 g yeast extract in 1 L
95° ± 1°C. distilled water. Autoclave for 15 min at 121°C. Prepare 0.5% (w/v)
(e) Cooling block assembly. acriflavin and naladixic acid stock solutions in distilled water. Prepare
(f) PCR tube holders. cycloheximide as a 1% stock in 40% (v/v) ethanol/distilled water.
(g) Capping/decapping tools. Filter-sterilize all stock solutions. Add 3 mL acriflavin, 8 mL
naladixic acid, and 5 mL cycloheximide stock solutions to 1 L Listeria
(h) Lysis tubes with caps and racks.
enrichment broth.
(i) Calibrated pipets.—Mechanical, covering ranges of 1–20 and
(j) Universal preenrichment broth.—Add 5 g tryptone, 5 g
20–200 mL. One 8-channel pipet covering 5–50 mL for test solution
proteose peptone, 15 g potassium phosphate, 7 g sodium phosphate,
transfers.
5 g sodium chloride, 0.5 g dextrose, 0.25 g magnesium sulfate, 0.1 g
(j) Incubators.—Maintaining enrichment media at 30° ± 1°C and ferric ammonium citrate, and 0.2 g sodium pyruvate to 1 L distilled
35° ± 1°C. water. Heat ingredients with gentle agitation to dissolve; dispense
(k) Stomacher. and autoclave at 121°C for 15 min. The final pH should be 6.3 ± 0.2.

ã 2006 AOAC INTERNATIONAL

 Table 2003.12. Interlaboratory study results for detection of Listeria monocytogenes in foods by the BAX system
False
BAX Sensitivityd, % False negativee, % Specificityf, % positiveg, %
Total Culture
Food type Level MPN/ga samples Presumptive Confirmed method c2b,c Assay Culture Assay Culture Assay Assay
Frankfurters High 0.15 78 72 72 75 1.33 96 100 4 0 — —
Low <0.03 77 18 18 20 0.00 95 100 5 0 — —
Control <0.03 78 0 0 0 — — — — — 100 0
Soft cheese High 2.4 84 82 81 79 0.17 98 95 2 5 — —
Low 0.93 84 69 64 70 2.50 89 97 11 3 — —

ã 2006 AOAC INTERNATIONAL

Control <0.03 84 1 0 0 — — — — — 100 0
Smoked salmon 1 High >11.0 72 72 72 72 — 100 100 0 0 — —
Low 2.4 72 70 70 72 0.50 97 100 3 0 — —
Control <0.03 72 6 0 0 — — — — — 100 0
Smoked salmon 2 High 0.43 84 79 79 83 2.25 95 100 5 0 — —
Low 0.04 84 59 59 69 8.10 86 100 14 0 — —
Control <0.03 84 0 0 0 — — — — — 100 0
Smoked salmon 3 High 0.15 90 65 65 58 1.09 81 76 19 24 — —
Low <0.03 90 50 50 34 5.36 73 60 27 40 — —
Control <0.03 90 0 0 0 — — — — — 100 0
Ground beef 1h High 0.43 54 48 47 38 3.54 94 76 6 24 — —
Low 0.11 54 14 11 10 0.00 61 56 39 44 — —
Control <0.03 54 0 0 0 — — — — — 100 0
Ground beef 2 High 0.09 89 78 78 66 4.40 92 78 8 22 — —
Low 0.03 89 38 37 38 0.00 65 68 35 32 — —
Control <0.03 89 2 0 0 — — — — — 100 0
Ground beef 3 High <0.03 72 38 38 39 0.00 69 69 31 31 — —
Low <0.03 72 15 15 21 0.93 45 64 55 36 — —
Control <0.03 72 0 0 0 — — — — — 100 0
Radishes High 4.6 70 65 65 70 3.20 93 100 7 0 — —
Low 0.036 72 29 29 44 13.07 66 100 34 0 — —
Control <0.03 72 0 0 0 — — — — — 100 0
Peas High 4.6 84 84 84 84 — 100 100 0 0 — —
Low 0.23 84 74 74 73 0.00 98 97 2 3 — —
Control <0.03 84 0 0 0 — — — — — 100 0
a
MPN/g = 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. A
Chi square value $3.84 indicates significance at p # 0.05.
c 2 2
For ground beef and salmon 3, c is defined by Siegal 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 = test
suspensions negative by BAX, d = test suspensions negative by culture method.
d
Sensitivity rate was defined as 100 times the total number of assay positive test suspensions divided by the total number of positive test suspensions by both methods.
e
False negative rate is 100 – sensitivity rate.
f
Specificity rate was defined as 100 times the total number of assay negative test suspensions divided by the total number of negative test suspensions by both methods.
g
False positive rate is 100 – specificity rate.
h
There were only 9 valid data points for this food type, thus the food type was repeated.
D. Enrichment in cooling block, B(e), for 5 min. Arrange PCR tubes, C(c), in cooling
(a) Dairy products.—Weigh 25 g test portion into sterile block. Transfer 50 mL lysate from lysis tubes to PCR tubes. Cap PCR
container. Homogenize with 225 mL complete enrichment broth, tubes with optical caps, C(d), using capping/decapping tools, B(g).
C(f), and incubate 22–24 h at 30°C. Place PCR tubes in cycler/detector and run program.
(b) Processed meat and poultry.—Weigh 25 g test portion into G. Assay Results
sterile container. Homogenize with 225 mL UVM broth, C(g), and
After clicking on the FINISH button, a new window will display a
incubate 22–24 h at 30°C. modified rack view with each well appearing in a different color and
(c) Raw meat and poultry.—Weigh 25 g test portion into sterile a symbol in the center of the well.
container. Homogenize with 225 mL Demi-Fraser broth, C(h), and A minus sign indicates the test sample is negative for the target
incubate 22–24 h at 30°C. organism. A plus sign indicates the test sample is positive for the
(d) Fruits, veg e ta bles, and sea food (ex cept smoked target organism. A question mark indicates an indeterminate result.
fish).—Weigh 25 g test portion into sterile container. Homogenize An indeterminate result is a test result where the internal positive
with 225 mL enrichment broth, C(e), and incubate 4 h at 30°C. After control does not reach a sufficient level to be considered valid and
4 h, add se lec tive agents acriflavin, nalidixic acid, and the amount of target-specific product is at an undetectable level. An
cycloheximide. Continue incubating another 20 h at 30°C. indeterminate reading could be due to reagent or system failure or
(e) Smoked fish.—Weigh 25 g test portion into sterile container. operator error. Although common practice would be to immediately
Homogenize with 225 mL universal preenrichment broth, C(j), and repeat the analysis, this was not done with these tests. If, after a
incubate 24 ± 2 h at 35°C. repeat test, the result is still indeterminate, contact DuPont Qualicon
technical service. A question mark with a slash through it indicates a
E. Secondary Enrichment signal error; contact DuPont Qualicon technical service. When the
Af ter 22–24 h trans fer 0.1 mL en rich ment to 9.9 mL assay is finished, handle and dispose of all waste as a biohazard.
MOPS-BLEB, C(i), for all foods except smoked fish. For smoked H. Confirmation
fish, trans fer 1.0 mL into 9.0 mL MOPS-BLEB. In cu bate
Presumptive positive tests must be confirmed using culture
MOPS-BLEB for 18–24 h at 35°C for all foods.
methods as described in the current edition of Bacteriological
F. Assay Analytical Manual, AOAC INTERNATIONAL, Gaithersburg, MD
After 18–24 h, create rack file, B(b), and warm up cycler/detector, 20877, USA, or Microbiology Laboratory Guidebook, U.S.
B(a). Add 150 mL protease, C(b), to 12 mL lysis buffer, C(a). Add Department of Agriculture, Food Safety Inspection Service, Athens,
GA 30604, USA. Isolate from previously enriched MOPS-BLEB
200 mL lysis reagent to one lysis tube, B(h), for each test suspension to
tubes.
be tested. Add 5 mL enrichment in MOPS-BLEB to lysis tube. Heat
lysis tubes for 60 min at 55°C on heating block, B(d). After 60 min, Reference: J. AOAC Int. 87, 395(2004).
heat lysis tubes for 10 min at 95°C on heating block. Cool lysis tubes Posted: April 2006

ã 2006 AOAC INTERNATIONAL