FOOD BIOLOGICAL CONTAMINANTS

Assurance Polyclonal Enzyme Immunoassay for Detection of

Listeria monocytogenes and Related Listeria Species in Selected
Foods: Collaborative Study
FELDSINE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 80, NO. 4, 1997
PHILIP T. FELDSINE, ANDREW H. LIENAU, ROBIN L. FORGEY, and ROGER D. CALHOON
BioControl Systems, Inc., 19805 N. Creek Parkway, Bothell, WA 98011
Collaborators: S. Al-Hasani; V. Arling; T. Bandiera; M. Barnes; S. Beatty; A. Beaudoin; D. Beyer; J. Bryant; M. Burzynski; B.
Carey; F. Copeland; D. Culver; C. Destro; B. Diaz; W. Franke; D. Gallagher; J. Gary; M. Harper; C. Hermann; T. Isakson; P.
Jenkins; S. Johnson; J. Ke; C. Krause; K. Lange; Y.-L. Trottier; G. Maki; S. McDonagh; J. McLenaghan; L. Miller; R. Phebus;
E. Raghubeer; R. Redding; D. Retzlaff; D. Richter; C. Ritger; J. Robinson; L. Saunders; D. Schwants; E. Tuncan; K. Vanderbilt;
D. Ward; D. West; L. Woo; A. Zebchek

Six foods representing a variety of food products

were analyzed by the Assurance Listeria polyclonal
enzyme immunoassay (EIA) and by either the Bacteriological Analytical Manual or the U.S. Department of Agriculture culture method for detecting
Listeria monocytogenes and related Listeria species. Samples of each food type, at each inoculation level, were analyzed simultaneously by both
methods. A total of 19 laboratories representing
federal government agencies and private industry
in the United States and Canada participated. Food
types were inoculated with Listeria species including L. monocytogenes, with the exception of 3 lots
of green beans, which were naturally contaminated. During this study, 1764 samples and controls were analyzed and confirmed, of which 492
were positive and 947 were negative by both methods. There were 159 samples that were positive by
culture method but negative by the EIA and 188
that were negative by culture method but positive
by EIA. Twenty-two samples were negative by EIA
and by culture method but confirmed positive
when Assurance selective enrichment broths were
subcultured to selective agar. The Assurance polyclonal EIA for detecting L. monocytogenes and related Listeria species in foods has been adopted
first action by AOAC INTERNATIONAL.

Submitted for publication July 2, 1996.

The recommendation was approved by the Methods Committee on
Microbiology and Extraneous Materials, and was adopted by the Official
Methods Board of the Association. See Official Methods Board Actions
(1996) J. AOAC Int. 79, 111A, and Official Methods Board Actions
(1996) The Referee, October issue.

collaborative validation study was conducted to demonstrate the equivalence of the Assurance Listeria polyclonal enzyme immunoassay (EIA) to either the Bacteriological Analytical Manual (BAM) or the U.S. Department
of Agriculture (USDA) culture methods (13) for detecting
Listeria monocytogenes and related Listeria spp. in selected
food types.
An assay has been developed that uses highly purified antibodies directed against antigens produced by Listeria. This assay is configured in a universally accepted microwell format
with a 96-well test plate. Samples are enriched in modified
Fraser Broth with lithium chloride (mFB + LiCl), transferred to
buffered Listeria enrichment broth (BLEB), and reincubated
prior to assay.
In a previously reported comparative validation study (publication pending), 20 foods representing a wide variety of products were analyzed. A total of 790 samples and controls were
analyzed and confirmed. In this study, 683 sample results were
equivalent, with 288 confirmed positives and 395 negatives reported by both methods. There were 40 samples that were
negative by Assurance but positive by culture method and
58 samples that were negative by culture method but confirmed
positive by Assurance.

Collaborative Study

Study Design
Six food types were prepared and analyzed. Foods were selected either because of published reports of Listeria isolation
from a certain food type or to ensure coverage of various food
matrixes. Foods were prescreened for the presence of Listeria
and analyzed as natural samples where possible. When the food
was not naturally contaminated, it was inoculated with Listeria
at 2 levels: a high level where predominantly positive results
were expected and a low level where fractional recovery was
anticipated. If fractional recovery was not achieved on initial
analysis, additional samples were prepared and reexamined af-

ter inoculation levels were adjusted to achieve partial recovery.

Five samples representing the high contamination level and
5 samples representing the low contamination level were tested
for each food type. Five uninoculated samples were included as
controls on each day that a particular food group was analyzed.

Sample Preparation
Listeria strains were grown in brain heart infusion (BHI)
broth for 1824 h at 3537C. Each food type, except nonfat
dry milk, received inocula of stationary-phase cells sufficient
to achieve 2 seed levels: 15 colony-forming units (cfu)/25 g
(low) and 1050 cfu/25 g (high) when measured by the most
probable number (MPN) technique at the time of sample analysis. Inoculation levels were adjusted so that fractional recovery
of Listeria cultures from seeded samples occurred at the low
level.
For nonfat dry milk, cultures were centrifuged, suspended
in 10% nonfat dry milk, lyophilized, and stored at 0C until
used. Milk samples were prepared by adding the lyophilized
culture to approximately 25 g food. This concentrated sample
was vigorously mixed in a nonbreakable sealed container and
completely transferred to about 200 g food. The 200 g quantity
was vigorously shaken in a nonbreakable bottle for several
minutes, added to about 1000 g food, and remixed. MPN levels
were then estimated. The entire quantity was added to the appropriate quantity of food to achieve the target inoculation level
and vigorously remixed.
MPN procedures were conducted on the day of initiation of
analyses. Three replicates of 100, 10, 1, and 0.1 g samples were
evaluated after enrichment in either Listeria enrichment broth
(LEB) or Fraser broth (FB), depending on the food analyzed to
calculate Listeria population/g of food.

Sample Distribution
Each collaborating laboratory received 15 duplicate test
samples of each of the food types they had agreed to analyze,
containing 25 g/sample. Five sample pairs were uninoculated
controls, 5 pairs were inoculated with a low level of Listeria,
and 5 pairs were inoculated with a high level of Listeria. Test
samples were distributed by overnight delivery service and sent
to participating collaborators to arrive no later than the week
preceding the day that analyses were to be initiated. Raw poultry, cooked roast beef, ice cream, raw shrimp, and fresh green
bean products were frozen before shipment, transported with
dry ice, and maintained in a frozen state after receipt by collaborators prior to initiating testing. Nonfat dry milk was inoculated, shipped, and held under ambient conditions prior to
analysis. Collaborators were instructed to analyze each paired
sample by both the BAM or USDA culture procedure and the
Assurance method. Results were submitted on summary forms
with the raw data.

Sample Analysis
MPN quantitation samples and test samples were incubated
in either LEB or FB at 30C for 24 h. MPN levels were determined by performing the referenced culture method on 3 replicates at each MPN level. The Assurance method was conducted

according to manufacturers directions for use, provided below.

Results were confirmed by the BAM culture method. Raw
poultry and cooked roast beef were enriched and MPN quantitation was performed by the USDA enrichment protocol. Fresh
greens beans, raw shrimp, ice cream, and nonfat dry milk were
enriched and quantitated according to the BAM method.

Culture Methods
Reference culture procedures used in this study were either
the USDA method for raw poultry and cooked roast beef or the
BAM method for fresh green beans, raw shrimp, ice cream, and
nonfat dry milk. Regardless of the enrichment method, the
BAM method was performed to confirm presumptive results.

Statistical Analysis
A pair-wise statistical analysis of the methods was performed for each food group according to the method of McNemar (4). A 2 value greater than 3.84 indicated a significant
difference at the 5% probability level. The methods were compared on a per-level and per-food-type basis. The data presented in Table 996.14 include analyses of sensitivity, specificity, and incidence of false negatives and false positives for each
level of each food type according to the method of McClure (5).
996.14 Listeria monocytogenes and Related
Listeria Species Detection in Selected Foods,
Assurance Polyclonal Enzyme Immunoassay
First Action 1996
(Applicable to detection of Listeria monocytogenes and related Listeria species in dairy foods, red meats, pork, poultry
products, fruits, nutmeats, seafood, pasta, vegetables, cheese,
animal meal, chocolate, and eggs.)
Caution: See Appendix B, safety notes on handling microorganisms. Listeria monocytogenes infection can cause fetal
death. It is recommended that pregnant women avoid handling
this organism. Attention should be given to sterilization of contaminated equipment and media before disposal or reuse.
Method Performance:
See Table 996.14 for method performance data.

A. Principle
In Assurance polyclonal enzyme immunoassay (EIA), proprietary antibodies with high specificity to Listeria monocytogenes and related Listeria species antigens are bound to microwell plates. Enriched test samples and positive controls are
added to plates. If any Listeria antigens are present, they bind
to microwells, forming antibodyantigen complexes. Nonreactive sample material is washed away.
Listeria-specific antibody is added, and the incubation and
wash procedures are repeated. Alkaline phosphatase conjugate
is then added to bind enzyme to Listeria antigens. After incubation, unbound conjugate is washed away. The substrate, p-nitrophenylphosphate, is added, and the absorbance of resulting
colored product is read spectrophotometrically at 405410 nm.

Reading above calculated cutoff value indicates presumptive

positive and should be confirmed by culture method.

B. Media and Reagents

(a) Wash solution concentrate.2% Polyoxyethylene 20
sorbitan monolaurate (Tween 20) in H2O.
(b) Substrate tablets.5 mg p-nitrophenylphosphate tablets.
(c) Substrate diluent.1M Diethanolamine in H2O.
(d) Positive control.Stabilized, inactivated Listeria antigen.
(e) Antibody solution.Highly purified antibodies reactive with Listeria antigen.
(f) Conjugate solution.Alkaline phosphataseantibody
conjugate.
(g) Stop solution.20% Ethylenediaminetetraacetic acid
(EDTA) in H2O.
(h) Antibody-coated microwells.Microwell strips, each
well coated with Listeria antibody, 96-well holder, and cover.
(i) Modified Fraser broth with lithium chloride (mFB +
LiCl).Suspend 55 g commercial Fraser broth base in 1 L purified H2O. Stir until completely dissolved. Do not heat mixture
to dissolve powder. Only after powder has completely dissolved, add 4 g LiCl and stir until completely dissolved. Autoclave broth 15 min at 121C. Alternatively, prepare 45% (w/v)
LiCl solution as follows: transfer 45 g LiCl into 100 mL volumetric flask and dissolve in purified H2O. Dilute solution to
100 mL with purified H2O. Filter-sterilize LiCl solution
through 0.2 m filter. Add 2 mL sterile LiCl solution to 225 mL
presterilized mFB. Do not add ferric ammonium citrate to mFB
+ LiCl broth.
(j) Buffered Listeria enrichment broth (BLEB).Suspend
36.1 g commercial Listeria enrichment broth, 8.5 g 3-[N-morpholino] propanesulfonic acid (MOPS), and 13.7 g MOPS sodium salt in 1 L purified H2O. Heat mixture while stirring until
solids dissolve. Autoclave broth 15 min at 121C.
(k) Diagnostic reagents.Necessary for culture confirmation of presumptive positive EIA tests. See chapter on Listeria
monocytogenes in BAM, current edition, AOAC INTERNATIONAL, Gaithersburg, MD. For red meats and poultry, see
Laboratory Communication No. 57, revised May 24, 1989, and
February 8, 1994, Microbiology Div., U.S. Department of Agriculture, Food Safety Inspection Service, Beltsville, MD.
Items (a)(h) are available as Assurance Listeria enzyme
immunoassay (EIA) test kit from BioControl Systems, Inc.,
19805 N. Creek Parkway, Bothell, WA 98011.

C. Apparatus
(a) Incubators.Capable of maintaining 30 1C and
3537C.
(b) Water bath.Capable of maintaining 100 2C. Alternatively, flowing steam autoclave set at 100C can be used.
(c) Microplate washer.For washing microwell strips.
Plastic squeeze bottle can be used.
(d) Microplate reader.Photometer equipped with 405
410 nm filter, capable of reading microwell plates. May include
optional printer.
(e) Micropipets.Capable of accurately dispensing 100
and 250 L.

(f) Vortex mixer.

(g) Top-loading balance.For weighing test samples.

D. General Instructions
All reagents must be stored at 28C when not in use. Allow reagents to equilibrate to room temperature before use. Include 2 positive controls and 1 blank test well with each run of
test samples. Use separate pipet for each sample and reagent to
avoid cross-contamination. Kit reagents and components must
be used as an integrated unit and may not be mixed with components from other manufacturing batches or sources. Use
dedicated trough or glassware for each reagent to avoid crosscontamination. Do not use reagents after expiration date. Do
not reuse microwells.

E. Preparation of Test Samples

Enrichment.Aseptically weigh 25.0 g sample into
225 mL mFB + LiCl, B(i). If larger sample sizes are analyzed,
proportionately increase volume of mFB + LiCl to maintain 1:9
dilution ratio. Mix well. Incubate 28 h at 30C. Transfer 1.0 mL
incubated mFB + LiCl to 9.0 mL BLEB, B(j), and incubate
24 h at 30C.
After incubation, mix contents of bottle thoroughly. Allow
particulate matter to settle 1 min and then transfer 1.0 mL into
test tube. Refrigerate remaining enrichment broth and use for
confirmation of presumptive positive results.
Submerge test tubes in boiling H2O bath 15 min to inactivate microorganisms. Cool tubes to 2537C before testing.
Boiled samples can be stored at 28C up to 4 days prior to
testing. Note: Test tubes that have been stored must be thoroughly mixed on a vortex mixer before sample addition to microwell.

F. Enzyme Immunoassay
(1) Prepare following reagents and allow them to equilibrate to room temperature before starting assay: Prepare wash
solution by adding 1.0 mL wash solution concentrate, B(a), to
100 mL H2O (sufficient to wash 48 wells). Label container.
Wash solution is stable up to 30 days when stored at 28C.
Prepare substrate solution by adding 1 substrate tablet, B(b),
to 5.0 mL substrate diluent, B(c), (sufficient for 48 wells). Label container. Prepare only amount needed for immediate use.
Discard unused solution.
(2) Install 405410 nm filter in microwell plate reader.
(3) Fit required number of microwell strips into holder, allowing for 2 positive controls and 1 blank. Reseal unused microwells in foil pouch containing desiccant. Carefully record
positions of positive controls, blank, and test samples in holder.
(4) Mix test sample from E and, separately, positive control, B(d), on vortex mixer before pipetting. Pipet 100 L test
sample into sample well; pipet 100 L positive control into
each positive control well. Leave blank well empty.
(5) Cover microplate with plastic cover provided and incubate 30 min at 3537C. Do not stack anything on microwell
holder during incubation.
(6) Wash each well 3 by alternative (a) or (b) below. Note:
Effective washing is critical to obtaining accurate data.

(a) Completely remove contents of wells with microwell

washer. Immediately fill wells completely with wash solution
(ca 250 L). Repeat 2. Avoid overfilling or underfilling wells
(to prevent ineffective washing or cross-contamination).
(b) Remove contents of well by inverting and vigorously
tapping plate. Completely fill each well with wash solution using precleaned wash bottle. Repeat 2.
(7) Immediately after aspiration of last wash, mix antibody
solution, B(e), by gently inverting bottle several times. Add
100 L antibody solution to each well, including control and
blank wells. Cover plate with plastic cover and incubate 30 min
at 3537C.
(8) Wash each well 3 as in step (6).
(9) Immediately after aspiration of last wash, mix conjugate solution, B(f), by gently inverting bottle several times. Add
100 L conjugate solution to each well, including control and
blank wells. Cover plate with plastic cover and incubate 30 min
at 3537C.
(10) Wash each well 3 as in step (6).
(11) Immediately after aspiration of last wash, add 100 L
substrate solution to each well, including control and blank
wells. Cover plate with plastic cover and incubate 30 min at
3537C.
After incubation immediately proceed to G, Reading. If
reading of results is delayed, add 50 L stop solution, B(g), to
each well and then read results within 1 h.

G. Reading
Read control and sample absorbances, A, at 405410 nm.
Calibrate microwell plate reader against blank well before
reading controls and test samples. Standardize reader by adjusting optical density (OD) of blank well to zero. Read 2 positive
control wells before reading sample wells. Note: Certain samples may read <0; this is not uncommon and indicates a negative result.

H. Interpretation of Results
(a) Control value.Positive control reading should be
>0.8 OD units. Readings below this value may indicate problems with washing procedure. If readings are below 0.8 OD
units, repeat test starting from step F(3) or contact test kit
manufacturer. If readings are above the upper limit of microplate reader, use reading of 2.5 to calculate cutoff value, (b).
(b) Cutoff value.Calculate average value of 2 positive
control readings (in OD units) and multiply by 0.25 to determine cutoff value.
(c) Negative results.Samples with OD readings less than
cutoff value are negative.

I. Confirmation of Positive EIA Samples

Samples with readings cutoff value are presumptively
positive. Positive samples must be confirmed by culture methods as described in BAM, current edition, AOAC INTERNATIONAL, Gaithersburg, MD. For red meats and poultry, see
Laboratory Communication No. 57, revised May 24, 1989, and
February 8, 1994, Microbiology Div., U.S. Department of Agriculture, Food Safety Inspection Service, Beltsville, MD.

To isolate microorganism, use refrigerated enrichment broth

from E.
Ref.: J. AOAC Int. 80, 775790(1997)
Results
Nineteen laboratories participated in the study. Twelve
agreed to analyze all 6 food types, one analyzed 5 foods, 4 analyzed 4 foods, one analyzed 3 foods, and one analyzed 2 foods
(Table 1). Fifteen samples per food type as well as positive and
negative culture controls were analyzed by each participating
laboratory. At the end of the study, valid data were submitted
for 1764 samples that included 194 naturally contaminated,
1046 inoculated, and 524 control samples.
Of 1764 total reported samples, 492 were confirmed positive and 947 were negative by both Assurance and culture
methods. Of remaining 325 samples, 188 were confirmed positive by Assurance method but negative by culture method,
159 samples were negative by Assurance and positive by culture method, and 22 samples were negative by Assurance and
by culture method, but confirmed positive when Assurance selective enrichment broths were subcultured to selective agar.
Tables 210 present results of individual collaborators. Results are analyzed by food type in the following sections.
Table 996.14 summarizes test results, as well as sensitivity
and specificity data for each sample type and inoculation level.
Significant differences in number of positive samples were detected for one naturally contaminated lot of green beans and raw
shrimp. These results are discussed under individual food types.
For each inoculation level, the actual population of Listeria
was quantitated by MPN determination on the day of initiation
of analysis of each food type.

Nonfat Dry Milk

Eighteen laboratories analyzed nonfat dry milk. Laboratory 6 reported an invalid EIA run. The positive Listeria culture
control, which was included as a procedural control with the
enrichment broths, was analyzed as negative, indicating that
the EIA was performed improperly and the resulting data were
invalid. Laboratory 19 did not complete sample confirmations.
Data from these laboratories were eliminated. Laboratory 1 reported uninoculated controls as confirmed positive for Listeria.
This laboratorys data are reported but not included in statistical
analysis. Fifteen laboratories followed study instructions and
appeared to have valid data as indicated by data summary
worksheets. All remaining uninoculated control samples were
negative (Table 2).
For several food types, including nonfat dry milk, laboratory 12 reported difficulty in preparing the primary enrichment
media for the EIA protocol. This necessitated a significant pH
adjustment to solubilize ingredients, which appeared to have
had a negative impact on recovery of Listeria from these samples. Later in the study, prepared enrichment media were provided to this collaborator. Results of this laboratorys analyses
using the prepared media correlated well between the methods.
This laboratorys data are reported but not included in the statistical analysis.

Samples inoculated at the low level contained 0.07 cfu/g.

Ten samples were confirmed positive and 40 samples were
negative by both methods. Twelve samples were negative by
culture method but confirmed positive by EIA. Eleven samples
were confirmed positive by culture method but negative by
EIA. There were 21 unconfirmed positive samples reported by
culture method and 5 unconfirmed positive samples reported
by EIA.
The high-inoculation-level samples contained 11 cfu/g.
Fifty-four samples were confirmed positive and 10 samples
were negative by both methods. Three samples were negatives
by culture method and confirmed positive by EIA, and 3 samples were confirmed positive by culture method but negative by
EIA. There were 12 unconfirmed positive samples reported by
culture method and 10 unconfirmed positive samples reported
by EIA. All uninoculated controls were negative; however,
there were 19 unconfirmed positive samples reported by culture method and 6 unconfirmed positive samples by EIA. 2
analysis for nonfat dry milk gave 0.0 at the low level and 0.17
at the high level, indicating the methods were comparable.

Ice Cream
Sixteen laboratories participated in analysis of ice cream
samples. Laboratory 6 did not complete sample confirmation.
The remaining 15 laboratories followed study instructions and
appeared to have valid data as indicated by data summary
worksheets (Table 3). Laboratory 10 reported that sample 2
ruptured in transit and was not analyzed.
Samples inoculated at the low level contained 0.043 cfu/g.
Twelve samples were confirmed positive and 31 samples were
negative by both methods. Thirteen samples were negative by
BAM but confirmed positive by EIA. Twenty samples were
positive by BAM but negative EIA. Thirteen unconfirmed positive samples were reported by culture method and none by EIA.
The high-inoculation-level samples contained 1.1 cfu/g.
Sixty-two samples were confirmed positive and 8 samples
were confirmed negative by both methods. One sample was
negative by culture but confirmed positive by EIA and 5 samples were confirmed positive by culture method, but negative
by EIA. One unconfirmed positive sample was reported by culture method and 2 unconfirmed positive samples were reported
by EIA. All uninoculated samples were negative by BAM and
EIA, but 3 unconfirmed positive EIA samples were reported. 2
analysis for ice cream gave 1.09 at the low level and 1.5 for the
high level, indicating comparable results.

Raw Poultry
Raw poultry was prepared twice. Across both runs, a total of
17 laboratories participated and 24 valid data sets were reported per level. In the first run, laboratories 6 and 7 did not
complete confirmation of presumptive results. Data from laboratory 12 are reported but not included in statistical analysis
because of media preparation problem noted under nonfat dry
milk.
For run 1, samples inoculated at low level contained
0.009 cfu/g. Eight samples were confirmed positive and
27 samples were negative by both methods. Nine samples were

negative by culture method but confirmed positive by EIA.

Fourteen samples were confirmed positive by culture method
but negative by EIA. There were 5 unconfirmed positive samples reported by EIA. The high inoculation level contained
0.043 cfu/g. Thirty-nine samples were confirmed positive and
6 samples were negative by both methods. Two samples were
negative by the culture method and confirmed positive by EIA.
Eight samples were confirmed positive by culture method but
negative by EIA. There were no unconfirmed presumptive
positive samples for either culture method or EIA. For uninoculated controls, laboratory 9 reported one sample from the culture method as confirmed positive but identified it as a species
different from the inoculating microorganism. Data from this
laboratory are included in the analysis. 2 analysis for raw poultry, run 1, gave 0.70 for the low level and 2.50 for the high level.
For both runs, all uninoculated control samples were negative
(Tables 4 and 5).
For run 2, laboratory 8 inadvertently combined samples 11
and 12 in a common broth, therefore, these samples were not
analyzed. Samples inoculated at the low level contained
0.43 cfu/g. Thirty-nine samples were confirmed positive and
6 samples were negative by both methods. Thirteen samples
were negative by culture method but confirmed positive by
EIA, and 7 samples were confirmed positive by culture method
but negative by EIA. Four unconfirmed positive samples were
reported by culture, and 3 unconfirmed positive samples were
reported by EIA. The high inoculation level contained
0.93 cfu/g. Fifty-one samples were confirmed positive and
4 samples were negative by both methods. Five samples were
negative by culture but confirmed positive by EIA. Three samples were confirmed positive by culture but negative by EIA.
Four unconfirmed positive samples were reported by culture,
and 7 unconfirmed positive samples were reported by EIA. 2
analysis for raw poultry, run 2, gave 1.25 at the low level and
0.13 at the high level, indicating comparable results.

Raw Shrimp
Raw shrimp was prepared twice. Across both runs, a total of
17 laboratories participated and 25 valid data sets were reported. For run 1, laboratory 6 did not begin analysis on the
correct day and was eliminated. Laboratory 9 did not follow
study instructions and used the USDA rather than BAM-specified enrichment protocol for this food type. Laboratory 19 did
not complete sample confirmations. Data from laboratory 12
are reported but not included in statistical analysis for reasons
mentioned above. Laboratory 7 did not receive sample 11. For
run 2, all participating laboratories followed study instructions
and appeared to have valid data as indicated by data summary
worksheets. All uninoculated control samples were reported as
negative (Tables 6 and 7).
For run 1, samples inoculated at the low level contained
0.003 cfu/g. Eight samples were confirmed positive and
21 samples were negative by both methods. Fourteen samples
were negative by culture method but confirmed positive by
EIA. Fourteen samples were confirmed positive by culture but
negative by EIA. Twenty-one unconfirmed positive samples

were reported by culture and 2 unconfirmed positive results

were reported by EIA.
The high inoculation level contained 0.15 cfu/g. Thirty-four
samples were confirmed positive and 8 samples were negative
by both methods. Two samples were negative by culture but
confirmed positive by EIA, and 11 samples were confirmed
positive by culture but negative by EIA. Eight unconfirmed
positive samples were reported by culture, and 5 unconfirmed
positive samples were reported by EIA. 2 analysis for raw
shrimp, run 1, gave 0.04 at the low level.
At the high level, 2 was 4.92, indicating a statistically significant difference between methods, with the culture method
producing more confirmed positive results.
Run 2 was conducted to test the validity of this difference
and to determine if the result was foodlot specific or represented a difference between method recovery for this food type.
This was particularly interesting because the methods produced
equivalent results for the low seed level but did not for the high
seed level.
For run 2, samples inoculated at the low level contained
0.003 cfu/g. Six samples were confirmed positive and 24 samples were negative by both methods. Thirty-three samples were
negative by culture method but confirmed positive by EIA.
Twelve samples were confirmed positive by culture method,
but negative by EIA. There were 15 unconfirmed positive samples reported by culture method and 9 unconfirmed positive
samples reported by EIA. The high-inoculation-level samples
contained 0.043 cfu/g. Twenty-six samples were confirmed
positive and 6 samples were negative by both methods. Thirtythree samples were negative by culture method but confirmed
positive by EIA. Six samples were confirmed positive by culture method, but negative by EIA. Ten unconfirmed positive
samples were reported by culture method and 4 unconfirmed
positive samples were reported by EIA. 2 analysis for raw
shrimp, run 2, gave 8.9 at the low level and 17.3 at the high
level. This indicates a statistically significant difference at both
levels, with EIA detecting significantly more confirmed positives than the culture method for both levels. This may be partly
explained by the very low inoculation levels used in this analysis. The paired nature of the sampling necessitated by the use
of different primary enrichment broths may also contribute to
these results. The overall data suggest, however, that enrichment conditions used in EIA may be more permissive to the
growth of Listeria in raw shrimp products.

Cooked Roast Beef

Cooked roast beef was prepared twice. Across both runs, a
total of 18 laboratories participated and 27 valid data sets were
reported per level. In the first run, laboratory 11 lost selective
agar plates and was not able to perform confirmations. Laboratory 19 did not complete sample confirmations. Data from
laboratory 12 are reported but not included in statistical analysis for the reasons mentioned above. All uninoculated control
samples were negative (Tables 8 and 9). For run 1, samples inoculated at the low level contained 0.003 cfu/g. Two samples
were confirmed positive and 61 samples were negative by both
methods. Five samples were negative by culture method but

confirmed positive by EIA, and 4 samples were confirmed

positive by culture method but negative by EIA. Two unconfirmed positive samples were reported by EIA.
The high inoculation level contained 2.4 cfu/g. Forty-six
samples were confirmed positive and 4 samples were negative
by both methods. Seven samples were negative by culture
method but confirmed positive by EIA. Thirteen samples were
confirmed positive by culture method but negative by EIA.
Four unconfirmed positive samples were reported for culture
method. Four unconfirmed positive samples were reported for
EIA. 2 analysis for cooked roast beef, run 1, gave 0.00 at the
low level and 1.25 at the high level, indicating comparable results.
There were very few positive samples detected by either
method, however, for the low inoculation level. Therefore, it
was decided to rerun this food type at a slightly higher inoculation level. For run 2, all uninoculated control samples were
negative. Samples inoculated at the low level contained
0.07 cfu/g. Three samples were confirmed positive and
46 samples were negative by both methods. Nine samples were
negative by culture method but confirmed positive by EIA.
Eight samples were confirmed positive by culture method but
negative by EIA. Five unconfirmed positive samples were reported by EIA. The high inoculation level contained 0.93 cfu/g.
Sixty-two samples were confirmed positive and no samples
were negative by both methods. No additional samples were
confirmed negative by culture method but found positive by
EIA. Three samples were confirmed positive by culture method
but negative by EIA. Two unconfirmed positive samples were
reported by EIA. 2 analysis for cooked roast beef, run 2, gave
0.00 at the low level and 1.33 at the high level, indicating comparable results.

Green Beans
Thirteen laboratories analyzed green beans. Laboratory 3
did not receive sample 14. This food was naturally contaminated with L. monocytogenes and L. innocua. Three separate
lots of naturally contaminated green beans were analyzed. The
Listeria contamination level for lot 1 was 0.009 cfu/g. Four
samples were confirmed positive and 50 samples were negative by both methods (Table 10). Four samples were negative
by culture method but confirmed positive by EIA. Seven samples were confirmed positive by culture method but negative by
EIA. Fourteen unconfirmed positive samples were reported by
culture method, and one unconfirmed positive sample was reported by EIA. Listeria contamination for lot 2 was
0.075 cfu/g. Eighteen samples were confirmed positive and
33 samples were negative by both methods. Eight samples
were negative by culture method but confirmed positive by
EIA, and 6 samples were confirmed positive by culture method
but negative by EIA. Sixteen unconfirmed positive samples
were reported by culture method. Four unconfirmed positive
samples were reported by EIA. Listeria contamination for lot 3
was 0.01 cfu/g. Eight samples were confirmed positive and
38 samples were negative by both methods. Fifteen samples
were negative by culture method but confirmed positive by
EIA, and 4 samples were confirmed positive by culture method
but negative by EIA. Fourteen unconfirmed positive samples

were reported by culture method. Four unconfirmed positive

samples were reported by EIA. 2 for fresh green beans were
0.36 for lot 1, 0.07 for lot 2, and 5.26 for lot 3. For lot 3, the EIA
detected a statistically significant greater number of confirmed
positive samples than the culture method. This may be attributed to the improved selective properties of the primary enrichment medium used in the EIA procedure. This medium appears
to suppress more effectively the growth of non-Listeria microflora contained in this individual lot of fresh green beans.
Discussion
Of 1764 samples, 492 were confirmed positive and 947
were negative by both methods; 188 samples were negative by
culture method but confirmed positive by EIA; and 159 samples were confirmed positive by culture method but negative by
EIA. An analysis of the data for each food type at each inoculation level, as well as the 3 lots of naturally contaminated
green beans, reveals that the methods were comparable except
for one of the 3 lots of green beans and raw shrimp. For the
green beans, lot 3, 2 was 5.26, indicating that the EIA method
detected a significantly greater number of confirmed positive
samples than the culture method. This may be attributed to the
improved selective properties of the enrichment protocol used
in the EIA for isolating Listeria from the natural microflora
resident in the green beans.
For raw shrimp, 4 levels were analyzed. In run 1, the low
level showed no significant difference, but the higher inoculation level revealed a statistically significant difference, with the
culture method detecting more positives than the EIA. This appeared somewhat curious in that, at the lower inoculation level,
the data were equivalent. Upon reanalysis of 2 additional levels
in run 2, strongly significant differences were detected for both
the low and high levels with 2 of 8.9 and 17.3, respectively.
The EIA method detected significantly more confirmed positives than the culture method for both levels. It is, again, likely
that the EIA enrichment protocol may be more productive for
highly contaminated raw food types by more effectively suppressing the growth of competitive microflora. This will allow
sufficient time for the slower growing Listeria to resuscitate
and propagate. Some of the differences observed between the
methods may be explained by the fact that duplicate paired
samples are used in this study. Other collaborative studies that
have required paired sampling because of different primary enrichment media have reported similar results (68). This is necessitated by the use of a different primary broth for both culture and EIA methods. Listeria contamination levels in this
study, by design, are quite low and Listeria are relatively slow
growing microorganisms. Any variability associated with organism content uniformity because of the paired sample requirement will be magnified. This could result in greater 2
determinations. Overall, the data tend to indicate that the enrichment and detection procedures used in the EIA may be preferred for raw products.
A large number of unconfirmed positive results were reported for both the EIA and each of the 2 culture procedures.
Upon further analyzing these results, it was determined that of

the 19 laboratories that participated in this study, 6 laboratories

accounted for 84% of the unconfirmed positive determinations
for the EIA method. Similarly, of the 19 participants, 6 laboratories also accounted for 82% of the unconfirmed positive determinations for the culture methods. Upon reexamining the
reported results from all laboratories, but particularly those referenced above, the data summaries were reported completely
and accurately; however, biochemical confirmations did not
correctly identify the inoculating Listeria microorganisms.
Certain laboratories appeared to have great difficulty with
confirmations, particularly CAMP testing, HOLT determinations, nitrate, and sugar biochemical reactions. Because there
was no error evident in their data, results of these laboratories
were reported and included in statistical evaluation. It is likely,
however, that a certain level of difficulty in cultural confirmations may exist with laboratories that do not perform Listeria
identifications routinely. This challenge is also likely to occur
in routine samples analysis situations in a food industry production environment. It underscores the need for better diagnostic
and confirmation methodologies.
The data indicate that the Assurance Listeria enzyme immunoassay method and the BAM/USDA culture methods are
comparable for detection of L. monocytogenes and related Listeria spp. in selected food types, with the exception of green
beans and raw shrimp, where the EIA method produced significantly more confirmed positives. This difference may be attributed to a more permissive enrichment environment for raw
food types. Accurate confirmation of presumptive results generally requires experienced analysts trained in biochemical
confirmation of Listeria.
Recommendation
On the basis of this study, it is recommended that the Assurance Listeria EIA assay for detection of L. monocytogenes and
related Listeria spp. in selected foods be adopted first action.
Acknowledgments
The participation of the following collaborators is acknowledged with appreciation:
Victoria Arling and Shelagh McDonagh, Agriculture
Canada, Calgary, AB, Canada
Mary Burzynski, Joan McLenaghan, and Anne Zebchek,
Agriculture Canada, Ottawa, ON, Canada
Yvon-Louis Trottier, Agriculture Canada, St. Hyacinth,
PQ, Canada
Donna Gallagher and Michael Harper, BioControl
Systems, Inc., Bothell, WA
Tony Bandiera, Chris Destro, and Linda Saunders,
Birchwood Meats, Columbus, OH
Sami Al-Hasani, Leslie Miller, and Erdal Tuncan,
ConAgra Analytical Laboratory, Columbia, MO
Diane West, ConAgra Frozen Foods, Omaha, NE
Jim Bryant, U.S. Food and Drug Administration Regional
Laboratory, Bothell, WA

Frederica Copeland, Goldkist Poultry Research Center,

Sumter, SC
Sandy Johnson, Hibbs Analytical Labs, Boise, ID
Becky Carey and Kristina Lange, Industrial Laboratories,
Denver, CO
Michael Barnes, Don Culver, Randall Phebus, and
Deanna Retzlaff, Kansas State University, Manhattan, KS
Sandra Beatty, Teresa Isakson, and Galen Maki, Mak-Bea
Laboratory, Inc., Blue Earth, MN
Bob Diaz, James Gary, Phylis Jenkins, Rhoda Redding,
John Robinson, and Karen Vanderbilt, Nabisco Brands, East
Hanover, NJ
Jim Ke and Errol Raghubeer, Nalleys Fine Foods,
Tacoma, WA
Daryck Beyer, Chris Hermann, Connie Krause, and Dana
Ward, Northland Food Laboratories, Fort Atkinson, WI
Dawn Richter, Ore-Ida Foods, Ontario, OR
Aaron Beaudoin and Lydia Woo, Professional Service
Industry, San Antonio, TX

Wendy Franke, Claudia Ritger, and Doug Schwants, Red

Star Bioproducts, Juneau, WI
References
(1) Bacteriological Analytical Manual (1992) 7th Ed., AOAC
INTERNATIONAL, Arlington, VA
(2) McClain, D., & Lee, W.H. (1989) Laboratory Communication, No. 57, revised May, 24, 1989, Microbiology Division,
U.S. Department of Agriculture, Food Safety Inspection Service, Beltsville, MD
(3) Johnson, J.L. (1994) Modification to Laboratory Communication, No. 57, revised February 8, 1994, U.S. Department of
Agriculture, Food Safety Inspection Service, Beltsville, MD
(4) Siegel, S. (1956) Nonparametric Statistics for the Behavioral
Sciences, McGraw-Hill Book Co., New York, NY
(5) McClure, F. (1990) J. Assoc. Off. Anal. Chem. 73, 953960
(6) Gibson, M. (1992) J. AOAC Int. 75, 293302
(7) Curiale, M.S., & Lepper, W. (1994) J. AOAC Int. 77, 14721489
(8) Curiale, M.S., Sons, T., Fanning, L., Lepper, W., & McIver,
D. (1994) J. AOAC Int. 77, 602617

Table 996.14. Method performance for detection of Listeria monocytogenes and related Listeria spp. in selected foods by Assurance enzyme immunoassay
Incidence of false
negatives among
total positive
samples, % d

Samples positive
Culture
method

EIA

Sample

Microorganism

Nonfat dry milk

L. monocytogenes 4a

Ice cream

L. welshimeri

Raw poultry 1

L. monocytogenes 4b

Raw poultry 2

L. monocytogenes 4b

Raw shrimp 1

L. innocua

Raw shrimp 2

L. innocua

Cooked roast beef 1 L. monocytogenes 1/2

Cooked roast beef 2 L. monocytogenes 1/2

Green beans
(naturally
contaminated)
a
b
c
d
e
f
g

Type

MPN,
cfu/g

Total

Pres

Conf

Pres

Conf

High
Low
Control
High
Low
Control
High
Low
Control
High
Low
Control
High
Low
Control
High
Low
Control
High
Low
Control
High
Low
Control
Lot 1
Lot 2
Lot 3

11
0.07
<0.003
1.1
0.043
<0.003
0.043
0.009
<0.003
0.93
0.43
<0.003
0.15
0.003
<0.003
0.043
0.003
<0.003
2.4
0.003
<0.003
0.93
0.07
<0.003
0.009
0.075
0.011

60
33
0
68
45
0
49
31
0
59
59
0
47
36
0
65
51
0
66
11
0
65
20
0
15
32
27

67
22
6
64
23
3
41
19
2
65
55
1
41
21
3
62
44
2
59
8
9
64
16
2
9
30
27

57
17
0
62
23
0
41
14
0
56
52
0
36
19
0
58
35
0
53
5
0
62
11
0
8
26
22

69
37
19
68
43
0
47
19
0
58
50
0
54
40
27
41
29
15
63
4
0
65
10
1
25
40
25

57
16
0
66
30
0
47
19
0
54
46
0
45
19
0
31
14
0
59
4
0
65
10
0
11
24
11

2b

0.17
0.00
f
1.50
1.09
f
2.50
0.70
f
0.13
1.25
f
4.92
0.04
f
17.33
8.89
f
1.25
0.00
f
1.33
0.00
f
0.36
0.07
5.26

Method
agreement,
%c

EIA

Culture

191.4
171.4
100
193.3
158.1
100
181.8
163.6
100
187.3
169.2
100
176.4
153.7
100
145.7
142.9
100
171.4
190.0
100
195.4
175.4
100
183.1
178.5
171.9

5.0
33.3
g
7.4
44.4

16.3
45.2

5.1
11.9

23.4
38.9

9.2
23.5

19.7
36.4

4.6
40.0

46.7
18.8
14.8

5.0
36.4

1.5
28.9

4.1
29.0

8.5
22.0

4.3
38.9

50.8
64.7

10.6
45.5

0.0
45.0

26.7
25.0
55.6

Sensitivity
rated

EIA Culture
95.0
66.7

92.6
55.6

83.7
54.8

94.9
88.1

76.6
61.1

90.8
76.5

80.3
63.6

95.4
60.0

53.3
81.3
85.2

95.0
63.6

98.5
71.1

95.9
71.0

91.5
78.0

95.7
61.1

49.2
35.3

89.4
54.5

100.0
55.0

73.3
75.0
44.4

Incidence of false
positives among total
negative samples, % e

Specificity ratee

EIA

Culture

EIA

Culture

8.6

4.0

3.7

1.5

0.0

2.9

12.91

3.1

27.1

0.0

49.1

21.4

0.0

1.5

91.4

96.0

96.3

98.5

100.0

97.1

87.1

96.9

72.9

100

100

100

50.9

78.6

100

98.5

Pres = presumptive data; conf = presumptively positive and confirmed data.

Chi square ( 2), as defined by McNemar = ( a b 1)2/(a + b), where a = samples positive by EIA and negative by culture method, and b = samples negative by EIA and positive by culture method.
Rate reflects number of confirmed determinations that were equivalent between EIA and culture method.
Sensitivity rate = total number of analyzed positive test portions among known positive test portions/laboratory divided by t otal number of known positive test portions/laboratory, where known positive is defined
as samples confirmed positive by the reference method. Incidence of false negative = (100 sensitivity rate). Low number of total number of confirmed positives will result in high false-negative data.
Specificity rate = total number of analyzed negative test portions among known negative test portions/laboratory divided by total number of known negative test portions/laboratory, where known negative is
defined as samples confirmed negative by the reference method and negative controls.
Statistical analysis not applicable; methods gave equivalent results.
Uninoculated control samples are by definition known negatives in study. Performance rates not calculated.

Table 1. Participation of laboratories in collaborative study

Laboratory
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Sample sets
a
b
c
d
e
f

Nonfat dry
milk
a a

y
y
y
y
y
b b
y
y
y
y
y
y
y
y
y
y

y
y
c c
y
18

Ice cream

Raw
poultry 1

Raw
poultry 2

Raw
shrimp 1

Raw
shrimp 2

Roast
beef 1

Roast
beef 2

Green
beans

y
y
y
y
y
c c
y
y
y
y
y
y
y
y
y

y
y

16

y
y
y
c c
y
c c
y
y
y

y
y
y
y

y
y

14

y
y
y
y
y
y
y
y
y

y
13

y
y
y
y

d d
y
y
y
e e
y

y
y
y
y
y

y
y
c c
y
16

y
y
y
y

y
y
y
y

y
y

y
y
y

y
14

y
y
y
y
y
y
y
y
y
y
f f
y
y
y
y

y
y
c c
y
17

y
y
y
y
y
y
y
y
y

y
y

13

y
y
y
y

y
y
y
y
y

y
y
y

13

Key: y = collaborator analyzed this food type; = collaborator did not analyze this food type.
Invalid EIA run; positive control reported as negative value; results not used.
Incomplete/incorrect sample confirmation; results not statistically analyzed for this food type.
Laboratory did not start on correct day and did not complete analyses; no data available.
Laboratory did not follow study directions. USDA instead of specified BAM protocol used for enrichment; results not used.
Laboratory lost plates and not able to perform confirmation; results not used.

Table 2. Results of nonfat dry milk analyses for presence of Listeriaa

Sample
Laboratory

10

11

12

+
+

e e

+
+

h h

h h

+
h h

+
+

h h

h h

13

14

15

EIA method
b

1
2
3
4
5
7
8
9
10
11
g
12
13
14
15
17
18

c c

+
d d

+
+
e e

+
+
+
+
+
d d

+
+
+
+

+
d d

+
+
d d

+
+
+
+
+
d d

+
+
+
+

+
+
+
+
+
+
+
+
+
+
d d

+
+
+
+

+
d d

+
+
d d

+
+
+
+
+
d d

+
+
+
+

+
+
d d

+
+

+
+
+
+
+
+
+
+

+
d d

+
+

d d

f f

d d

f f

+
d d

+
e e

d d

f f

d d

d d

d d

d d

d d

d d

BAM method
1
2
3
4
5
7
8
9
10
11
12
13
14
15
17
18
a

b
c
d
e
f
g
h

+
+
h h

+
+
+
+
+
+
+
+
h h

+
+
+
+

+
+
+
+
+
h h

+
+
+
+
+
h h

+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
h h

+
+
+
+

+
+
h h

+
+
+
h h

+
+
h h

+
h h

+
+
h h

+
+
h h

+
+
h h

+
+
+
+
+
+
+
+

+
h h

h h

+
h h

h h

h h

f f

h h

h h

+
+
h h

h h

f f

h h

h h

h h

h h

h h

f f

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

h h

b h

h h

Most probable number for the following: 15, high inoculum samples, 11 cfu/g; 610, low inoculum samples, 0.07 cfu/g; 1115, uninoculated
samples, <0.003 cfu/g. Samples inoculated with L. monocytogenes 4a.
Uninoculated controls confirmed as positive. Data not included in analyses.
+, Listeria detected in sample; , Listeria not detected in sample.
The EIA was positive and the assay confirmation was negative.
The EIA was negative, but Listeria was recovered from assay broth.
BAM method was negative, EIA was negative, but Listeria was recovered from assay broth.
Collaborator had difficulty preparing EIA enrichment media. Data not included in analyses.
Selective plates had colonies typical of Listeria but were not Listeria on confirmation.

Table 3. Results of ice cream analyses for presence of Listeriaa

Sample
Laboratory

10

11

12

13

14

c c

+
+
+
+
+
+
+
+
+
+
+
+

+
+
e e

+
+
+
+
+
+
+
+
+

+
+
d d

+
+
+
+
+
+
+
+
+

+
+
+
+
c c

+
+
+
+
+
+
d d

+
+
+
+
+
+
+
+
+
+
+
+

+
+
e,g e,g

+
+
+
+
+
+
+
+
+

+
+

+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+

15

EIA method
1
2
3
4
5
7
8
9
10
11
12
13
14
17
18

b b

+
+

e e

+
+
+

+
f f
X

+
+
+
+

e e

+
+

+
+
+
d d

d d

+
+

d d

c c

c c

d d

+
+
+
+
+
+
+
+
+
+
+
+
+

c c

BAM method
1
2
3
4
5
7
8
9
10
11
12
13
14
17
18
a

b
c
d
e
f
g

+
+
g g

g g

e,g e,g

+
g g

+
g g

+
+

+
+
f f
X
g g

+
+
+
g g

e e

g g

+
+
g g

+
+

+
g g

+
+

g g

g g

+
+
+
+
+
g g

+
+
+
+
+
+
+
+
+
+
+
+
+

g g

Most probable number for the following: 15, low inoculum samples, 0.043 cfu/g; 610, uninoculated samples, <0.003 cfu/g; 11-15, high
inoculum samples, 1.1 cfu/g. Samples inoculated with L. welshimeri.
+, Listeria detected in sample; , Listeria not detected in sample.
The EIA was positive and the assay confirmation was negative.
The EIA was negative, but Listeria was recovered from assay broth.
BAM method was negative, EIA was negative, but Listeria was recovered from assay broth.
Sample bag ruptured during shipment. Sample not analyzed.
Selective plates had colonies typical of Listeria but were not Listeria on confirmation.

Table 4. Results of raw poultry 1 analyses for presence of Listeriaa

Sample
Laboratory

10

11

12

13

14

15

c c

+
e e

d d

+
+
+

+
+
+
+
+
e e

+
+
+
e e

+
+

+
+
+
+
e e

+
+
+
+

+
+
+

+
+

+
+
+

e e

+
+
+
+

d d

+
+
+
+
+
+
+

+
+

+
+
+
+
+

+
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+

+
+

EIA method
1
3
4
5
8
9
11
g
12
13
14
17
18

b b

f f
X

c c

c c

c c

+
+
+

c c

c c

d d

c c

d d

USDA method
1
3
4
5
8
9
11
12
13
14
17
18
a

b
c
d
e
f
g

f f
X

+
+

+
+
+

+
d d

+
d d

Most probable number for the following: 15, uninoculated samples, <0.003 cfu/g; 610, low inoculum samples, 0.09 cfu/g; 1115, high
inoculum samples, 0.043 cfu/g. Samples inoculated with L. monocytogenes 4b.
+, Listeria detected in sample; , Listeria not detected in sample.
The EIA was positive and the assay confirmation was negative.
USDA method was negative, EIA was negative, but Listeria was recovered from assay broth.
The EIA was negative, but Listeria was recovered from assay broth.
Uninoculated control confirmed positive with different Listeria species from inoculating organism.
Collaborator had difficulty preparing EIA enrichment media. Data not included in analyses.

Table 5. Results of raw poultry 2 analyses for presence of Listeriaa

Sample
Laboratory

10

11

12

13

+
+
c c

+
+
+
+
+
+
+
+
+

+
+
c c

+
+
+
+
+
+
+
+

c c

+
+
+
+
+
e e

+
+
+

c c

+
+
+
+
d d
X
+
+
+
+
+

+
+
c c

+
+
+
+
d d
X
+
+
+
+
+

+
+
c c

+
+
+
+
+
+
+
+
+
+

f f

+
+

+
+
+
+
+

f f

+
+

+
+
+
+
+

+
+

+
+

+
d d
X
+
+
+
+
+

+
+
f f

+
+

+
d d
X
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

14

15

+
+
+
+
+
+
+
+
+
+

+
+
c c

+
+
+
+
+
+
+
+
+
+

+
+
f f

+
+
+
+
+
+
+
+
+
+

f f

+
+

+
+
+
+
+
+
+

EIA method
1
2
3
4
5
6
7
8
9
12
14
16
19

b b

c c

+
+
+

+
+
+
+
+
+
+

+
+
+
+
+
+
+
+

c c

cc
c c

USDA method
1
2
3
4
5
6
7
8
9
12
14
16
19
a

b
c
d
e
f

+
+
f f

+
+
+
+
+
+
+

+
+

f f

+
+
+

+
+
+

+
+

+
+
+
+
+

f f

Most probable number for the following: 15, uninoculated samples, <0.003 cfu/g; 610, low inoculum samples, 0.043 cfu/g; 1115, high
inoculum samples, 0.093 cfu/g. Samples inoculated with L. monocytogenes 4b.
+, Listeria detected in sample; , Listeria not detected in sample.
The EIA was positive and the assay confirmation was negative.
Collaborator combined samples. Not analyzed.
The EIA was negative, but Listeria was recovered from assay broth.
Selective plates had colonies typical of Listeria but were not Listeria on confirmation.

Table 6. Results of raw shrimp 1 analyses for presence of Listeriaa

Sample
Laboratory

10

11

d d

c c

+
g g
X
+

+
+

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

+
g g
X
i i

i i

+
+
+

12

13

14

15

c c

e e

c c

+
+
d d

e e

+
+

+
i i

+
e,i e,i

i i

i i

+
+

i i

+
i i

i i

i i

i i

+
+
+
e,i e,i

i i

+
+
+

EIA method
1
2
f
3
4
7
8
11
h
12
13
14
15
17
18

b,c b,c

+
+
+
+

c c

+
+
+
+

d d

c c

+
+
+
+
+
c c

+
+
+
+

c c

+
+
+
+

c c

c c

+
+
+

c c

d d

d d

+
+
+

+
+
d d

+
+
+
+

+
+
d d

d d

d d

+
+

d d

e e

BAM method
1
2
3
4
7
8
11
12
13
14
15
17
18
a

b
c
d
e
f
g
h
i

i i

+
+
+
+
+
+
+
+
+
i i

i i

+
+
+
+
+
+
+
+
+
i i

+
+
+
+
+
+
+
+
+
+

i i

+
+
+
+
+
i i

+
+
i i

+
+
+
+
+
+
i i

+
+
+
+

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

i i

+
+
i i

i i

i i

e,i e,i

Most probable number for the following: 15, high inoculum samples, 0.15 cfu/g; 610, uninoculated samples, <0.003 cfu/g; 1115, low
inoculum samples, 0.003 cfu/g. Samples inoculated with L. innocua.
+, Listeria detected in sample; , Listeria not detected in sample.
The EIA was negative, but Listeria was recovered from assay broth.
The EIA was positive and the assay confirmation was negative.
BAM method was negative, EIA was negative, but Listeria was recovered from assay broth.
Uninoculated control confirmed positive. Data not included in analyses.
Collaborator did not receive sample. No analysis.
Collaborator had difficulty preparing EIA enrichment media. Data not included in analyses.
Selective plates had colonies typical of Listeria but were not Listeria on confirmation.

Table 7. Results of raw shrimp 2 analyses for presence of Listeriaa

Sample
Laboratory

10

11

d d

+
+
+
d d

+
+

12

13

14

15

EIA method
1
2
3
4
6
7
8
9
11
12
14
15
16
19

b b

d d

+
+
+
+
+
+
+
+
+

+
d d

+
+
d d

+
+
+
+
+
+
+
+
+

+
+
d d

+
+
+
+
+
+
+

+
+
+

+
+
+
+
+
+
+
+

c c

+
+
+
+
+
+
+
+
+
+

d d

c c

c c

d d

d d

+
+

+
+

c c

+
+
d d

+
d d

+
+
+
+
+

d d

d d

+
+
+
+

e e

c c

d d

d d

+
+
+

+
+

+
+

c c

f f

f f

f f

+
c c

f f

f f

+
+
f f

c c

f f

f f

+
f f

+
c c

BAM method
1
2
3
4
6
7
8
9
11
12
14
15
16
19
a

b
c
d
e
f

f f

+
+
+

f f

+
+
+

+
+
+
+

f f

f f

+
+
+
+

+
+
f f

f f

+
f f

+
+
+
+

c,f c,f

+
f f

f f

+
+
+
+

f f

f f

f f

f f

f
f f

f f

f f

f f

f f

f f

f f

f f

f f

f f

c,f c,f

f f

f f

+
f f

+
c c

f f

f f

Most probable number for the following: 15, high inoculum samples, 0.043 cfu/g; 610, uninoculated samples, <0.003 cfu/g; 1115, low
inoculum samples, 0.003 cfu/g. Samples inoculated with L. innocua.
+, Listeria detected in sample; , Listeria not detected in sample.
BAM method was negative, EIA was negative, but Listeria was recovered from assay broth.
The EIA was positive and the assay confirmation was negative.
The EIA was negative, but Listeria was recovered from assay broth.
Selective plates had colonies typical of Listeria but were not Listeria on confirmation.

Table 8. Results of cooked roast beef 1 analyses for presence of Listeriaa

Sample
Laboratory

10

11

12

13

14

15

c c

+
+
+
c c

+
+
d d

d d

+
d d

+
+
+
+
+
+

+
+
+

d d

+
+

+
+
+
+
+
+
c c

+
+
+

+
+
+

e e

c c

c c

e e

+
+

+
+
g g

+
+
+
+
+
+
+
+
+
+

+
+
g g

+
+
+
+
+
+
+
+

+
+
+

e e

e e

EIA method
1
2
3
4
5
6
7
8
9
10
f
12
13
14
17
18

b b

c c

c c

c c

c c

c c

c c

c c

c c

c c

+
+
+
+
+
+
d d

+
c c

d d

+
+
+
+

+
+
+

+
+
+
+
+
+
+
+

c c

c c

c c

USDA method
1
2
3
4
5
6
7
8
9
10
12
13
14
17
18
a

b
c
d
e
f
g

+
g g

+
+
+
+
+

+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

g g

Most probable number for the following: 15, uninoculated samples, <0.003 cfu/g; 610, high inoculum samples, 2.4 cfu/g; 1115, low
inoculum samples, 0.003 cfu/g. Samples inoculated with L. monocytogenes 1/2b.
+, Listeria detected in sample; , Listeria not detected in sample.
The EIA was positive and the assay confirmation was negative.
The EIA was negative, but Listeria was recovered from assay broth.
USDA was negative, EIA was negative, but Listeria was recovered from assay broth.
Collaborator had difficulty preparing EIA enrichment broth. Data not included in analyses.
Selective plates had colonies typical of Listeria but were not Listeria on confirmation.

Table 9. Results of cooked roast beef 2 analyses for presence of Listeriaa

Sample
Laboratory

10

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

11

12

13

14

15

c c

c c

c c

e e

c c

+
+

e e

EIA method
1
2
3
4
5
6
7
8
9
11
12
14
16

b b

c c

c c

+
+
+
+
+
+
c c

+
+
+
+
+
+

+
+
+
+
c c

+
+
+
+
+
+

d d

c c

+
+

USDA method
1
2
3
4
5
6
7
8
9
11
12
14
16
a

b
c
d
e
f

f f

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+
+
+

Most probable number for the following: 15, uninoculated samples, <0.003 cfu/g; 610, high inoculum samples, 0.93 cfu/g; 1115, low
inoculum samples, 0.07 cfu/g. Samples inoculated with L. monocytogenes 1/2b.
+, Listeria detected in sample; , Listeria not detected in sample.
The EIA was positive and the assay confirmation was negative.
The EIA was negative, but Listeria was recovered from assay broth.
USDA was negative, EIA was negative, but Listeria was recovered from assay broth.
Selective plates had colonies typical of Listeria but were not Listeria on confirmation.

Table 10. Results of green bean analyses for presence of Listeriaa

Sample
Laboratory

10

11

12

+
+
+

+
+
+
+
+
c c

+
c c

c c

d d

+
+

c c

+
+

+
+

+
c c

c c

+
f f

+
+
+
c c

+
+

+
+
+
g g

g g

g g

g g

g g

+
g g

+
g g

g g

g g

f f

+
g g

g g

13

14

15

+
+
+

e e
X

+
+

g g

+
+

g g

g g

e e
X

g g

g g

g g

g g

g g

EIA method
1
2
3
4
6
7
8
9
10
12
13
14
17

b b

d d

c c

+
+

+
+
c c

c c

BAM method
1
2
3
4
6
7
8
9
10
12
13
14
17
a

b
c
d
e
f
g

g g

g g

g g

g g

g g

g g

g g

g g

g g

g g

g g

g g

g g

g g

g g

g g

+
+
+

g g

g g

g g

g g

+
+
g g

+
+
+
g g

g g

g g

g g

All green bean samples were naturally contaminated with L. monocytogenes and L. innocua. Most probable number for the following samples
are: 15, lot 1, 0.009 cfu/g; 610, lot 2, 0.075 cfu/g; 1115, lot 3, 0.011 cfu/g.
+, Listeria detected in sample; , Listeria not detected in sample.
The EIA was positive and the assay confirmation was negative.
The EIA was negative, but Listeria was recovered from assay broth.
Collaborator did not receive sample. No analysis.
BAM method was negative, EIA was negative, but Listeria was recovered from assay broth.
Selective plates had colonies typical of Listeria but were not Listeria on confirmation.