FOOD BIOLOGICAL CONTAMINANTS

Enzyme-Linked Immunoassay for Detection of Listeria

monocytogenes in Dairy Products, Seafoods, and Meats:
Collaborative Study
CURIALE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 77, NO. 6, 1994
MICHAEL S. CURIALE and WENDY LEPPER
Silliker Laboratories Group, Inc., 1304 Halsted St, Chicago Heights, IL 60411
BARBARA ROBISON
Organon Teknika, 100 Akzo Ave, Durham, NC 27704
Collaborators: D.A. Blank; J.L. Bryant; A. Codd; A. Cohen; C. Coles; H.S. Commager; P. Cook; N. Corristan; T. Donlevy; R.
Durham; A.W. Edwards; J. Faison; L. Fanning; G. Fisher; E.W. Frampton; C.J. Hagen; D. Higgins; A. Hildenbrand; C.D.
Hoffman; E. Hu; J.L. Johnson; C. Kasper; K.J. Keating; M.R. Lankford; C. Lord; J. Madden; G. Miklovic; C.W. Noah; S.
McNally; F. Moller; C. OBryan; M.J. Palmieri; S. Pao; R. Parmar; S.V. Patel; R. Pfundheller; D. Potel; M. Pratt; L. Restaino;
S.L. Richardson; E. Richter; M. Thomas; M. Thompson; L. Tilton; K. Warner; J.L. Witt; Y. Visier

A collaborative study was conducted to evaluate

Listeria-Tek, an enzyme-linked immunosorbent
assay (ELISA) for detection of Listeria monocytogenes and other Listeria spp. in foods. The present
ELISA method was compared to the U.S. Food and
Drug Administration culture method for detection
of L. monocytogenes in dairy products and seafoods and to the U.S. Department of Agriculture
Food Safety and Inspection Service method for detection of L. monocytogenes in meats. Replicate
samples of 6 food types (frankfurters, roast beef,
Brie cheese, 2% milk, raw shrimp, and crab meat)
inoculated with L. monocytogenes and uninoculated control samples were analyzed by the collaborators. L. monocytogenes was identified in
593 samples by the ELISA method and in 574 samples using culture procedures. Identical results
were obtained for 506 positive samples and
419 negative samples using the ELISA and culture
methods for an overall agreement rate of 85.6%.
The enzyme-linked immunoassay for detection of
L. monocytogenes in dairy, seafood, and meat
products has been adopted first action by AOAC INTERNATIONAL.

Submitted for publication August 4, 1993.

The recommendation was approved by the Committee on Microbiology
and Extraneous Materials, and was adopted by the Official Methods Board
of the Association. See Official Methods Board Actions (1994) J. AOAC
Int. 77, 43A, and Official Methods Board Actions (1994) The Referee 17,
February issue.

he Listeria-Tek method is a colorimetric enzymelinked immunosorbent assay (ELISA) for detecting Listeria monocytogenes in dairy products, seafoods, and
meats. The method uses monoclonal antibodies specific for
Listeria spp. (1). Several versions of the method have been issued since the assay was first developed. Generally, changes
have been made in the procedure recommended for enrichment
and have followed changes made in the protocols of the U.S.
Food and Drug Administration (U.S. FDA) and the United
States Department of Agriculture (USDA). The version evaluated in the present study differs from protocols recommended
before 1992. In addition, claims of the method have been modified at present time to detection of L. monocytogenes, instead
of Listeria spp. Although the method detects other species of
Listeria, all strains are not detected with equal sensitivity. These
observations were made during trials of the method (Robison
and Cunningham, in-house data).
The Listeria-Tek method can detect as few as one recoverable L. monocytogenes cell per 25 g sample (Robison, pre-collaborative study). To detect L. monocytogenes, foods are first
cultured to increase the Listeria density. Culture enrichment is
accomplished in 2 steps and requires 4856 h incubation. The
immunoassay performed on the enrichment culture takes 2 h
and represents a negative screen for the presence of L. monocytogenes. Thus, samples that are nonreactive in the assay are
considered negative for L. monocytogenes and do not require
further analysis. Positive assays represent presumptive results
for L. monocytogenes and must, therefore, be confirmed using
culture methods before the sample is reported as positive for L.
monocytogenes.
A pre-collaborative comparison of the ELISA and culture
methods for naturally contaminated foods and foods inoculated
with multiple levels of L. monocytogenes showed that the

methods were comparable (Robison and Cunningham, inhouse data).

This report describes results from a collaborative study in
which the Listeria-Tek and reference culture methods were
compared for their ability to detect L. monocytogenes in seafood, meat, and dairy products.
Collaborative Study
Six food types (frankfurters, roast beef, Brie cheese, 2%
milk, raw frozen shrimp, and crab meat) commonly tested for
Listeria, frequently contaminated with Listeria, or associated
with listeriosis in humans were selected for the study. Each
food was inoculated with a different serotype of L. monocytogenes. The shrimp contained naturally occurring L. innocua
and L. monocytogenes in addition to L. monocytogenes added
by inoculation.
Collaborating laboratories were sent 15 samples of each
food type (6075 g/sample) to be analyzed. Samples were numerically labeled from 1 to 15. Additional information on the
label included food type, storage temperature, and date for
analysis. The inoculation or contamination status of all samples
was known by the reference laboratory only. Frankfurters, roast
beef, cheese, and milk were shipped the day before analysis in
Styrofoam containers packed with cooling packs at 47C.
Shrimp and crab samples were shipped frozen 5 or 6 days before analysis in Styrofoam containers. Recommended storage
temperatures conformed to the shipping temperatures.
Collaborators were instructed to analyze each of the 15 samples using the Listeria-Tek assay and either the U.S. FDA (2)
or USDA (3) culture method. The U.S. FDA culture method
was used for the milk, cheese, shrimp, and crab samples. Frankfurters and roast beef were enriched and plated according to the
USDA protocols and identified as Listeria according to U.S.
FDA procedures. All Fraser broth enrichments used in the
USDA procedure were analyzed for Listeria irrespective of
broth color. Suspect Listeria isolates derived from streaking
U.S. FDA, USDA, and ELISA broths on selective plates were
identified to species using the U.S. FDA protocol.

Preparation of Listeria Cultures for Sample

Inoculation
L. monocytogenes stock cultures were stored at 70C in
trypticase soy broth (TSB) containing 15% glycerol. Stock cultures were streaked to trypticase soy agar (TSA) and incubated
for 24 h at 35C to obtain single colonies. A 10 mL portion of
TSB was inoculated with a single colony and incubated for 24 h
at 35C. The cell density of the culture was determined by direct microscopic count. The broth culture was then appropriately diluted in Butterfields phosphate buffer to achieve inoculation rates of 0.040.2 cells/g (low inoculum level) and
0.42 cells/g (high inoculum level) for the frankfurters, roast
beef, milk, shrimp, and crab meat. Inoculation levels used for
Brie cheese were higher because of die-off during storage. Plate
counts of the Listeria inocula were within 4-fold of the microscopic count determinations.

Preparation of Test Samples

(a) Frankfurters.Forty-five 1 lb packages of commercially available frankfurters (15 packages with same code dates
for each of 3 brands) were used. Frankfurters of a similar brand
were combined and blended in a Hobart mixer at 4C and inoculated with L. monocytogenes 1/2c. Sixty gram portions
were packaged in plastic bags and stored at 2C for 7 days prior
to shipping. Uninoculated controls were prepared in a similar
manner.
(b) Roast beef.Whole vacuum-packaged roast beefs
were purchased from 3 different stores and treated as 3 independent samples. Each sample was cut into small pieces, finely
chopped in a food processor, and then blended in a Hobart
mixer at 4C. Two samples were inoculated with L. monocytogenes 3b during blending. The third sample served as an uninoculated control. Sixty gram portions were packaged in
Whirl-Pak bags and stored at 2C for 7 days prior to shipping.
(c) Milk, 2%.Six gallons of 2% pasteurized milk with the
same code date were obtained for the study. Two gallons were
combined per inoculation level and uninoculated control. Four
days before the scheduled test dates, samples were inoculated
with appropriate dilutions of L. monocytogenes 4b to achieve
the desired inoculation levels. The milk was packaged in WhirlPak bags and stored at 2C prior to shipment.
(d) Brie cheese.Domestic Brie cheese was cut into sixty
75 g portions and placed in Whirl-Pak bags. Individual portions
were inoculated with 1 mL of a diluted culture containing 106
(high level inoculum) or 104 (low level inoculum) cells/mL of
L. monocytogenes 3a. The samples were mixed by hand and
stored at 2C. During 7 days of storage the viable count for
Listeria was observed to decrease by 46 log cycles (4).
(e) Shrimp.Frozen shelled shrimps were obtained from a
single source. The shrimp was divided into 3 subsamples and
was mixed thoroughly in a Hobart mixer. During mixing 2 subsamples were inoculated with L. monocytogenes 1/2a. The third
subsample served as an uninoculated control. Sixty gram portions of the subsamples were packaged in Whirl-Pak bags and
stored frozen for 7 days prior to shipping.
(f) Crab meat.Imported frozen blocks of cooked King
crab were purchased from a single source. After partial thawing
overnight at 4C, 3 blocks were mixed in a Hobart mixer at 4C
for each inoculum level and the control. Subsamples were inoculated with L. monocytogenes 1/2b during mixing. Inoculated and control portions were divided into 60 g test portions
in Whirl-Pak bags and stored frozen for 7 days before shipping.

Shipment of Samples
Sample sets consisting of 5 high level inoculum, 5 low level
inoculum, and 5 uninoculated controls were prepared for each
food and sent to collaborators for analysis. Refrigerated foods
were shipped overnight the day before analysis; the frozen
foods were shipped overnight 5 to 6 days before analysis. Each
sample was packaged in a double Whirl-Pak bag on which was
written a sample number from 1 to 15, the product type, storage
temperature, and date for analysis. Samples were sealed in
1 gallon metal cans containing refrigerated or frozen ice packs.

Cans were placed in Styrofoam shipping containers with additional refrigerated or frozen ice packs. Containers were labeled
in compliance with the Dangerous Goods Regulations of the
International Air Transport Association (5) and requirements of
the specific carrier.

Analysis of Samples
Each test sample contained a sufficient amount to prepare
2 enrichments (Figure 1). One enrichment was prepared as
specified for the Listeria-Tek ELISA method and the other enrichment was prepared in accordance with the U.S. FDA guidelines (2) for dairy products and seafoods or the USDA recommendations (3) for meats. According to the ELISA
immunoassay protocol only positive broths are streaked for
confirmation of Listeria. However, in this study all broths were
streaked for isolation regardless of the immunoassay result to
determine whether the assay was detecting all positive enrichments. All primary U.S. FDA and secondary USDA enrichment
broths were streaked to appropriate agar media for Listeria isolation. Listeria-like colonies appearing on the isolation agar
media for all 3 methods were confirmed as Listeria using the
U.S. FDA procedures (2).
Collaborators were instructed to record results on supplied
data sheets. The completed result forms were returned to the
author (MSC) for data compilation and analysis.
The level of Listeria in retained samples was determined by
the reference laboratory using 3-tube most probable number
techniques (MPN) (Table 1). The determination was made by
the collaborators on the day scheduled for analyses.
994.03 Listeria monocytogenes in Dairy Products,
Seafoods, and MeatsColorimetric Monoclonal
Enzyme-Linked Immunosorbent Assay Method
(Listeria-Tek)
First Action 1994
Procedure for presence of L. monocytogenes in dairy products, seafoods, and meats. Test is not confirmatory because
monoclonal antibodies used in test may cross-react with other
members of the genus Listeria.
Enrichment broths from samples positive by this test must
be streaked on selective media as in 992.18C and 992.19C and
E. Typical or suspicious colonies must be identified biochemically as L. monocytogenes.
Positive result is valid only when negative and positive controls show acceptable optical density readings.
(Caution: L. monocytogenes can cause fetal death. Pregnant
women should avoid handling this bacterium.)
Method Performance:
See Table 994.03 for method performance data.

A. Principle
Listeria antigens are detected based on enzyme immunoassay (EIA) using specific monoclonal antibodies. Polystyrene
microtiter wells are coated with monoclonal antibodies to Listeria antigens. Samples and controls along with enzyme-la-

beled antibody are placed into wells. Listeria antigens present

in sample attach to specific antibodies adsorbed on well and
present in conjugate. Wells are washed to remove unbound
conjugate, and enzyme substrate is added. Blue color is produced which turns yellow when reaction is stopped with stop
solution. Color development indicates the presence of Listeria
antigen in the sample.

B. Apparatus
(a) Microtiter strip plate holder.Polystyrene frame to
hold 18 microtiter strips.
(b) Plate sealers.Adhesive sheets to cover microtiter
plates.
(c) Clamp and rod.Closure for foil pack.
(d) Manual or automated EIA wash system.System designed to wash coated wells in 12 8 microwell plates, consists
of vacuum pump which aspirates well contents and dispensing
system which fills wells with wash solution.
(e) EIA plate reader.Photometer with 450 nm filter, capable of reading microtiter plates.
(f) Incubators.Capable of maintaining 30 1.0C,
35 1.0C, and 37 1.0C.
(g) Micropipets.Multichannel, capable of accurately delivering 50300 L; single channel, 50200 L. Disposable
micropipet tips.
(h) Reservoir troughs.Reagent reservoir used with multichannel pipets to hold EIA reagents for dispensing to microtiter plate wells.
(i) Hot plate or water bath.Capable of maintaining
100 1.0C.
(j) Screw-cap tubes.Glass, 16 125 mm.

C. Reagents
(a) Antibody coated microtiter strip plates.Polystyrene
microtiter strip plates containing 8 strips of 12 wells coated
with monoclonal antibodies to Listeria, packaged in foil with
silica desiccant. Store opened packages, properly resealed, at
28C for 2 months. Monoclonal antibodies in test kit must
react with an epitope common to all species of Listeria, but not
present in other Gram positive organisms.
(b) Control antigens.(1) Positive control.Lyophilized, purified Listeria antigen reactive to Listeria spp. antibodies. (2) Negative control.Lyophilized 1% nonfat dry milk.
Both controls preserved with 0.1 mg/mL gentamicin sulfate.
Store reconstituted antigens at 28C for 60 days.
(c) Conjugate and conjugate diluent.Anti-Listeria antibodies (murine) conjugated to commercially available horseradish peroxidase in liquid diluent with protein stabilizers and
antimicrobial agent in Tris buffer pH 7.6. Monoclonal antibodies
in conjugate must react with epitope common to all Listeria species, but not react with epitope adsorbed to microtiter strip plates.
(d) TMB peroxidase substrate (Solution A).3,3,5,5Tetramethylbenzidine, 0.4 g/L, in a proprietary organic base.
(e) TMB peroxidase substrate (Solution B).Citric acid
buffer containing 0.02% H2O2.
(f) Wash concentrate, 50.50% glycerol and 2.5%
Tween 20 in H2O.

(g) Stop solution.2N sulfuric acid (H2SO4).

(h) Modified Fraser broth.5.0 g pancreatic digest of casein, 5.0 g peptic digest of animal tissue, 5.0 g beef extract,
5.0 g yeast extract, 20.0 g NaCl, 9.6 g Na2HPO4, 1.35 g
KH2PO4, 1.0 g esculin, 0.02 g nalidixic acid, 0.012 g acriflavine HCl, 3 g LiCl. Suspend ingredients in 1 L H2O and heat to
boiling to dissolve ingredients completely. Dispense 225 mL
portions into flasks. Autoclave 15 min at 121C. Just before use,
add 2.25 mL filter-sterilized stock solution of ferric ammonium
citrate (5% in distilled H2O). Store stock solution at 28C.
(i) Buffered Listeria enrichment broth (BLEB).17.0 g
pancreatic digest of casein, 3.0 g papaic digest of soybean meal,
2.5 g dextrose, 5.0 g NaCl, 2.5 g K2HPO4, 6.0 g yeast extract,
0.05 g cycloheximide, 0.015 g acriflavine HCl, 0.04 g nalidixic
acid, 9.6 g Na2HPO4, 1.35 g KH2PO4. Suspend ingredients in
1 L H2O. Heat, if necessary, to dissolve components. Dispense
10 mL portions into 16 150 mm screw-cap tubes. Cap tubes
loosely and autoclave 15 min at 121C. Store medium away
from light.
Note: Acriflavine can photo-oxidize to form inhibitory compounds against Listeria.
(j) Modified Oxford medium (MOX).39.0 g Columbia
blood agar base (10 g peptone, 10 g bitone, 3 g tryptic digest of
beef heart, 1 g corn starch, 5 g NaCl, 15 g agar), 1.0 g esculin,
0.5 g ferric ammonium chloride, 15.0 g LiCl, 2.0 g agar. Suspend ingredients in 1 L H2O, and heat to boiling to dissolve
agar completely. Autoclave 10 min at 121C. Swirl and cool to
50C. Rehydrate vial of Modified Oxford Antimicrobic Supplement (10 mg colistin sulfate and 20 mg moxalactam; available from Difco) with 10 mL sterile distilled H2O. Rotate the
vial in end-over-end motion to dissolve ingredients completely.
Add vial contents to 1 L tempered sterile Oxford Medium Base.
Mix well and pour into sterile Petri dishes.
(k) Lithium chloride-phenylethanol-moxalactam (LPM)
agar.35.5 g phenylethanol agar (10 g tryptone, 3 g beef extract, 5 g NaCl, 2.5 g phenylethanol, 15 g agar), 10.0 g glycine
anhydride, 5.0 g LiCl. Suspend ingredients in 1 L H2O and boil
1 min to dissolve agar completely. Autoclave for 15 min at
121C and cool to 46C. Aseptically add 2 mL filter-sterilized
moxalactam stock solution to 1 L tempered LPM agar base (final concentration 20 mg/L base). Moxalactam stock solution
consists of 1 g moxalactam salt (ammonium or sodium) in
100 mL 0.1M potassium phosphate buffer, pH 6.0. After adding
moxalactam solution, mix well and pour into sterile Petri dishes.
Items B(a)(c) and C(a)(g) are available as Listeria-Tek
ELISA test system (Organon Teknika Corp., 100 Akzo Ave,
Durham, NC 27704).

D. General Instructions
Store components at 28C when not in use. All kit components used in an assay must be same kit lot number. Do not
use materials after expiration date.
Bring foil packs to room temperature (2025C) before
opening to prevent condensation on strips. Remove strips as
needed. Reseal pack with clamp and rod provided; store at 2
8C. Bring components and test samples to room temperature
(2025C) before testing. Return reagents to 28C after use.

Handle strip holders carefully; no strip should be dislodged

during testing. Do not touch bottom surface of wells.
Before use mix samples and reagents well. Use separate
pipet tips for each sample and kit reagent to avoid cross contamination. Keep plastic troughs well separated to dispense
conjugate and substrate. Upon assay completion, flush the aspiration/wash system with H2O.
Do not reuse microtiter strips.

E. Preparation of Test Samples

(1) Primary enrichment.Transfer 25 g sample and
225 mL modified Fraser broth, C(h), into blender jar. Blend 2
min at ca 12 000 rpm or stomach 2 min as required for thorough mixing. Incubate 2426 h (raw shrimp, 2628 h) at 30C.
(2) Secondary enrichment.Mix incubated enriched sample well. Transfer 0.1 mL (raw shrimp, 1.0 mL) mixture into
10 mL BLEB, C(i). Incubate BLEB tube 2426 h (raw shrimp,
2628 h) at 30C.
(3) Preparation of sample for EIA analysis.Mix incubated mixture well. Remove 1 mL from BLEB tube and place
in clean glass screw-cap tube. Heat tube in boiling water bath
or in flowing steam 20 min. Cool heated BLEB tubes to room
temperature (2025C) prior to EIA analysis.

F. Enzyme Immunoassay
Perform following steps prior to commencing assay:
(1) Reconstitute control antigens: Add 2 mL sterile H2O to
lyophilized negative control vial and 1 mL sterile H2O to positive control vial. Reconstituted antigens stored at 28C are
stable 60 days.
(2) Calculate volume (mL) TMB substrate used in assay:
Multiply number of wells used by 0.12. Immediately after last
wash prepare substrate by mixing (v/v) TMB solutions A and
B (50 + 50) in clean glass tube.
(3) Prepare 1 wash solution by diluting 10 mL 50 wash
concentrate with 490 mL distilled or deionized H2O. Wash solution is stable 60 days.
(4) Assemble strip holder. Use one microtiter well for each
sample. Include 2 negative controls and one positive control
per assay.
(5) Transfer a 100-L aliquot of each sample and control
into the assigned wells.
(6) Transfer a 100-L aliquot conjugate into each well containing samples or controls. Do not touch well contents with
pipet tip.
(7) Cover plate with plate sealer. Mix by gently tapping
strip. Incubate strip 1 h at 37C.
(8) Aspirate contents from wells using manual or automated wash system. Aspirate excess conjugate solution. Immediately refill wells with ca 0.20.3 mL wash solution. Aspirate
and refill wells 6 with wash solution. Note: Incomplete washing will adversely affect results. Do not overflow wash solution
into adjacent wells. Do not allow strips to dry. Go immediately
to the next step.
(9) Add a 100-L aliquot mixed TMB substrate into each
well. Incubate 30 min at ambient temperature (20-25C). Discard any unused TMB substrate.

(10) Add a 100-L aliquot stop solution into each well.

Read plate immediately.

G. Reading
Select 450 nm wavelength on plate reader. Zero reader on
air (without strip holder and strips) and read absorbance, A, of
solution in each well.
Calculate mean A for negative control wells (should be
<0.30) and positive control (should be >0.70). Cutoff value is
calculated by adding 0.15 to mean A for negative controls. Test
sample is presumed positive if sample A is cutoff value.

H. Confirmation of Positive EIA Samples

Positive EIA reading indicates Listeria spp. may be present.
Because antibodies may cross-react with species of Listeria
other than L. monocytogenes, culture confirmations must be
performed by streaking BLEB culture onto MOX and LPM
agar plates. Incubate MOX plates 2448 h at 35 1C, and
LPM plates 2448 h at 30 1C. Presumptive Listeria isolates
must be biochemically confirmed as in 992.18, 992.19 or in
Bacteriological Analytical Manual, Chapter 10, 7th Ed., 1992.
Ref.: J. AOAC Int. 77, 1472 (1994)
Statistical Evaluation
The study results were subjected to the outlier test recommended by McClure (6) for qualitative methods. The outlier
test was applied to ELISA and culture results. For the outlier
test, inoculated and naturally contaminated samples were classified as positive samples and control samples without L.
monocytogenes were classified as negative. Laboratory results
identified as outliers for either method were not eliminated for
reasons presented in the section Results and Discussion.
Sensitivity and specificity rates for the culture and ELISA
methods were calculated according to the method of McClure
(6). Sensitivity is the number of method-positive samples divided by the total number of positive samples. Specificity is the
number of method-negative samples divided by the total number of negative samples. The incidence of false negatives
among positive samples is defined as 1 minus the sensitivity,
and the incidence of false presumptive positive assays among
negative tests is defined as 1 minus the specificity.
Agreement between the ELISA and culture methods was the
fraction of the test samples with the same test results.
Results and Discussion
The 1350 test samples were prepared and shipped to
27 laboratories for analysis by the ELISA and a reference culture method (U.S. FDA or USDA). Each laboratory tested one
or more food types depending on individual interest and time
constraints (Table 2). Data for sample sets not analyzed according to the study protocol were excluded from the evaluation.
The U.S. FDA method was used as a reference method for
seafood and dairy products, because these food types are regulated by the U.S. FDA. Meat and meat products are regulated
by the USDA, so the appropriate USDA reference method for

meat was used. Identification procedures were performed using

U.S. FDA procedure, because the USDA method identifies
only hemolytic species of Listeria. Because the monoclonal antibodies in Listeria-Tek recognize all species of Listeria, all isolates were identified to species to make the study more complete. The AOAC Official Method 992.18, MICRO-ID
Listeria, was used for characterization of the Listeria isolates
from the third set of shrimp.
Following the analyses of the dairy, meat, and crab samples
and prior to the analyses of the shrimp, the change in the form
of the conjugate solution (from lyophilized to liquid) was made
in the AOAC Official Method 992.18.
The outliers were identified according to the procedure of
McClure (6) and consisted of laboratories reporting proportionally fewer L. monocytogenes isolations compared to the other
collaborators. Two types of outliers were observed. Type I was
characterized by a general failure to isolate Listeria. This observation did not appear to be laboratory, food, or method
(ELISA or culture) related and has been observed in another
collaborative study of a Listeria method (4) and in a precollaborative study (Robison and Cunningham, personal communication). These findings suggest that the available Listeria methods may fail occasionally to detect Listeria. Type II outliers
consisted of isolations of Listeria species other than L. monocytogenes. These data were the result of a laboratory problem
to identify Listeria to species. Type II outliers were excluded
from the determination of performance statistics. However,
data from type I outliers have not been excluded from this
study, because deleting the results would overestimate the
specificity and sensitivity of the methods.

Frankfurters
Sample sets were shipped to 13 laboratories (Table 3); however, one set arrived after the test date and was not analyzed.
The Listeria-Tek method detected L. monocytogenes in
100.0 and 98.3% of the samples containing high and low inocula (Table 3), respectively. In comparison, 96.7% of the high
and 90% of the low inoculum samples were positive by the
USDA method. Interestingly, cultural analysis of both the negative Listeria-Tek assay broths and the negative Fraser broth
tubes revealed all high and low inoculum samples as containing
Listeria. Because the immunoassay and Fraser broths are negative screening tests for L. monocytogenes, culture broths from
negative samples normally would not be examined further.
However, in this study all negative assay broths were streaked
for Listeria isolation. L. monocytogenes was recovered from
one ELISA-negative broth (Laboratory 6, sample 3) and
8 USDA Fraser broth tubes (Laboratory 4, samples 2, 4, and 9;
Laboratory 6, samples 1, 3, 4, 7, and 14). These are false negative results because L. monocytogenes was present in the broth
but not detected by the assay. It is likely that the Listeria cell
count was below the detection limit of the assay.
L. monocytogenes was detected in 2 different uninoculated
control samples using the ELISA and the USDA culture methods. Presence of L. monocytogenes in these samples was within
the confidence limits of the MPN determination made by the
reference laboratory. Moreover, presence of Listeria in this

product type is not unusual (7). Eight additional control samples were immunoassay positive; however, Listeria could not
be cultured from the enrichment broths. These findings represent false presumptive positive reactions. One ELISA-negative
broth contained L. innocua after culturing. This isolation was
not classified as either a false presumptive positive or a false
negative result, because the method was under evaluation for
the detection of L. monocytogenes only and not other Listeria
species. Although the immunoassay is reactive with many L.
innocua isolates, failure of the assay to detect this isolate likely
resulted from insufficient numbers in the enrichment broth. It
is possible that the isolate was nonreactive.
The blackening of the Fraser enrichment broth in the USDA
procedure did not correlate with the confirmed results. While
listeriae were recovered from all esculin-positive tubes, L.
monocytogenes was recovered from all esculin-negative tubes
for the high inoculum level (2 samples), the low inoculum level
(6 samples), and the control level (2 samples). These results
demonstrate the potential for false negative reactions when the
24 h esculin reaction is used as a negative screening test for
Listeria species.

Roast Beef
Samples of roast beef were sent to 12 laboratories for comparative analysis (Table 4). Listeria-Tek results from Laboratories 7, 12, and 23 were outliers for the ELISA procedure when
compared to results submitted by the other laboratories. These
type I outliers were not excluded from statistical analyses.
L. monocytogenes was detected by the ELISA method in
48.3% and 53.3% of the high and low inoculum samples, respectively. Based on the culture method recovery from the
ELISA enrichment broths, 61.7% of the high and 61.7% of the
low inoculum samples contained L. monocytogenes. These results were comparable to those obtained using the USDA procedure: 55.0% for the high and 50.0% for the low inoculum
sets. Better results of L. monocytogenes were obtained when all
Fraser broths were streaked for isolation: 60.0% for the high
and 61.7% for the low inoculum sets.
L. monocytogenes was found in 5% and 11.7% of the control
samples using the ELISA and the culture methods, respectively.
L. monocytogenes was found in 14.0% of the immunoassaynegative enrichment broths and 3.8% of the negative USDA
Fraser broth tubes. These broths likely contained Listeria cell
counts below detectable limits of the Listeria-Tek assay and the
esculin test.
False presumptive positive results for the ELISA assay
(positive assay, no L. monocytogenes recovered after culturing)
were observed at each of the 3 test levels. Of the 60 samples
examined at each inoculum level, there were 3 high level, 4 low
level, and 6 control samples that were falsely positive by immunoassay. False presumptive positive results (suspect colonies on isolation plates that were not L. monocytogenes) were
also obtained by the USDA plate assay. Following the same
scoring procedure for the ELISA method, these samples were
recorded as negative for L. monocytogenes.
L. monocytogenes was recovered from 65.8% of the blackened (esculin-positive) Fraser broths and from 18.2% of the

yellow (esculin-negative) Fraser broths when the results for the

3 inoculation levels were combined. According to these results, the incidence of Listeria was underestimated by the esculin test and was not a reliable screening procedure for detection of Listeria.

Brie Cheese
Samples of Brie cheese were sent to 13 collaborators for
analysis (Table 5). The ELISA and U.S. FDA culture results
from Laboratory 10 were significantly different (P <0.05)
when compared to data from the other laboratories. While this
laboratory isolated Listeria from all inoculated samples, only
L. welshimeri was identified in the ELISA enrichments and
both L. welshimeri and L. seeligeri were present in the U.S.
FDA culture broths. The fact that no other laboratory reported
the presence of these species suggests an error in identification.
Consequently, the data from Laboratory 10 were excluded
from statistical analysis.
Using the Listeria-Tek method, L. monocytogenes was
found in 95.0% of the high inoculum samples, 86.7% of low
inoculum samples and 3.3% of control samples. These results
compare favorably to the U.S. FDA method which identified L.
monocytogenes in 93.3% of high inoculum samples, 86.7% of
low level samples and 0% of controls. Slightly better recovery
was achieved by culture analysis of the ELISA enrichments,
independent of ELISA result; L. monocytogenes was detected
in 98.3% of the high and 95.0% of the low inoculum samples.
Presence of Listeria in several control samples was unexpected, because this product generally tested negative and establishing a stable inoculum appeared impossible. However,
the incidence of the organism among the collaborators samples
was within the expected confidence interval of the MPN determination reported by the reference laboratory.
Collaborators were unable to confirm L. monocytogenes in
6 immunoassay-positive control samples or in 7 U.S. FDApositive enrichments with Listeria-like colonies. The false presumptive positive results by the U.S. FDA culture method were
all found by one laboratory and may have been the result of a
more cautious selection of colonies for identification.

Milk
Milk samples were sent to 12 laboratories for analysis (Table 1). One laboratory withdrew from the study. A second laboratory accidentally discarded assay materials and was unable to
complete the analyses. Consequently, only 10 data sets were
available for the study (Table 6).
Three sets of results for the ELISA method and 5 sets of
results for the U.S. FDA method were statistically different (P
<0.05) from results generated at other laboratories. The outliers
detected Listeria in either none or one of the 10 inoculated samples as compared to 9 or 10 isolations obtained by the other
laboratories. The failure of either the ELISA method or the culture method to detect Listeria has been discussed previously for
the roast beef samples in the present study.
L. monocytogenes was detected in 70.0% of high inoculum
samples, 62.0% of low inoculum samples, and 0.0% of the control samples using the ELISA procedure. No ELISA negative

enrichment broths were positive for L. monocytogenes after

streaking for isolation. Using the U.S. FDA culture method L.
monocytogenes was identified in 48.0, 30.0, and 0.0% of high
inoculum, low inoculum, and control samples, respectively. No
other Listeria species were detected in the milk.
One false presumptive positive immunoassay and 7 false
presumptive positive U.S. FDA culture results occurred. Because the false presumptive positives cultures were confined to
one laboratory, it appears this was a laboratory-related event.

Shrimp
Shrimp sample sets were sent to the collaborators 3 times.
In the first set, L. monocytogenes was found by the ELISA
method in 27.7% of high inoculum, 29.2% of low inoculum,
and 7.7% of control samples. In comparison, L. monocytogenes
was recovered by the U.S. FDA method from 38.5% of high
inoculum, 24.6% of low inoculum, and 4.6% of control samples (Table 7). This sample set also contained a high background of naturally occurring Listeria, with L. innocua as the
predominant species. With the background Listeria, over 70%
of the inoculated and 40% of the controls contained Listeria. Because of potential interference caused by species other than monocytogenes, the AOAC General Referee for Non-Dairy Food Microbiology requested that another sample of shrimp be analyzed.
Shipping problems were encountered with the second set of
shrimp. Some collaborators received product overnight with
the samples still frozen. Other laboratories received product after 48 h, with the samples arriving in different conditions, from
partially frozen to thawed.
Because the samples were not uniform to all collaborators,
a third set of samples was prepared. Before the third set of samples was analyzed, in-house studies of raw shrimp showed that
better results were obtained when 1.0 mL of the modified
Fraser broth enrichment was transferred to the buffered Listeria
enrichment broth (BLEB) instead of 0.1 mL, and incubation of
enrichments was extended to 2628 h rather than 2426 h. Collaborators were instructed to modify the method for raw shrimp
to include these changes.
The third set of shrimp samples was shipped to 15 laboratories. All collaborators returned the test results (Table 8). Samples inoculated with high and low levels of L. monocytogenes
as well as uninoculated control samples were naturally contaminated with L. innocua and L. monocytogenes. The high and
low levels contained the naturally occurring Listeria plus L.
monocytogenes introduced by inoculation.
L. monocytogenes was found in 100.0% of high inoculum,
93.3% of low inoculum, and 2.7% of control samples by the
ELISA method. In comparison, L. monocytogenes was recovered from 98.7% of high inoculum, 98.7% of low inoculum,
and 4.0% of control samples by the U.S. FDA method. Presence of L. monocytogenes in the control samples was probably
the result of natural contamination.

Crab Meat
Samples of crab meat were sent to 12 laboratories for analysis (Table 9). ELISA results from 4 laboratories and U.S. FDA
culture results from 2 laboratories contained proportionally

fewer agreements (P <0.05) with the expected results when

compared to the other laboratories. Results from Laboratory 10
fell into this category because all Listeria isolations were reported as L. seeligeri. Therefore, data from this laboratory were
omitted from statistical evaluations.
Using the ELISA method, L. monocytogenes was recovered
from 76.3% of the high inoculum, 74.5% of the low inoculum,
and 1.8% of the control samples. Recoveries using the U.S.
FDA method were slightly higher: 83.6% at the high and low
inoculum levels and 3.6% for uninoculated controls. The methods would have agreed if L. monocytogenes had been detected
in all ELISA-negative, culture-positive ELISA broths. These
failures by the immunoassay represent false negative results.
False negative readings were reported by 5 of the 12 laboratories; 46% of the false negative results were from one laboratory.
Method Performance
A summary of the data used to calculate the performance
statistics is shown in Table 10. Sensitivity and specificity rates
for the ELISA and U.S. FDA/USDA culture methods are
shown in Table 994.03. The sensitivities of the methods were
within 10% for inoculated samples with the exception of milk.
The ELISA method was more sensitive for frankfurters, high
inoculum level Brie cheese, milk, and shrimp. The cultural procedure exhibited greater sensitivity in the analysis of roast beef,
low inoculum shrimp and crab. Differences in sensitivity were
likely a function of the particular food lot and not the food type
in general, because similar sensitivities were not observed in
precollaborative trials.
The true status of many inoculated and naturally contaminated food samples in this study was not known. Many of the
positive test samples contained very low levels of Listeria, one
or less than one cell/25 g test portion. Different 25 g portions of
each sample were analyzed by the ELISA and culture methods,
such that one portion might or might not contain Listeria. This
could lead to differences in numbers of Listeria positive samples by either method. For samples containing higher levels of
Listeria, differences would be expected if the samples had not
been rendered totally homogeneous. Low levels of analyte and
nonhomogeneous distribution of analyte are both common
problems in food analysis. Neither of these situations are adequately addressed by test kit statistical methods.
The method agreement statistic and the incidence of false
negatives among the positive sample value are derived from
numerical methods that do not require previous knowledge or
inference of test sample status. Values calculated for this study
are presented in Table 994.03. Because different portions of
each test sample were analyzed by the 2 methods and the levels
of Listeria in the samples were generally low, Listeria may
have been unevenly distributed in the sample. Consequently,
the Listeria populations in the subsample used for one method
might not be equivalent to those in the subsample used for the
other method. However, even with unequal distribution within
products, similar results would be expected using either
method. Agreement rates for inoculated samples ranged from
58% for high inoculum level milk to 98.7% for high inoculum

level shrimp. The high agreement rates and low incidence of

false negatives samples (i.e., frankfurters, Brie cheese, and
shrimp) suggest that subsamples tested by each method contained detectable levels of Listeria. Lower agreement rates and
higher incidences of false negatives for the other foods (roast
beef, milk and crab) suggest that the Listeria level was very low
such that L. monocytogenes was absent from one of 2 subsamples. Lower false negative results for roast beef, low inoculum
shrimp and crab meat favor the culture method; whereas results
for frankfurters, high inoculum Brie, milk, and shrimp favor the
ELISA procedure. Differences between numbers of positives
for the Listeria-Tek and cultural methods, but not always for the
same foods observed here, were seen in the precollaborative
study (Robison and Cunningham, personal communication).
Such differences may be partly related to laboratory performance or the particular sample that was analyzed.
In the present study, collaborators frequently reported all
samples negative by only one of the 2 methods; however, other
laboratories reportedly detected Listeria. These results suggest
that one or the other method failed. While no obvious failure
pattern could be discerned, an enrichment failure such as a temperature variation or improper antibiotic or inhibitor concentration could possibly account for these discrepancies.
The ELISA and U.S. FDA/USDA culture methods exhibited comparable incidences of false presumptive positives for
all foods (Table 994.03). Both methods performed well when
only L. monocytogenes was present in the sample. However,
when other Listeria species were present, as seen in the first set
of shrimp (Table 7), fewer suspect samples were found to contain L. monocytogenes. This result was not unexpected, because
the Listeria-Tek assay reacts with other Listeria species and the
selective plating media used in the U.S. FDA/USDA culture
procedures are not designed to differentiate Listeria species.
Recommendation
On the basis of the results of this study it is recommended
that the enzyme-linked immunoassay for detection of Listeria
monocytogenes in dairy products, seafoods, and meats be
adopted first action.
Acknowledgments
We greatly appreciate the efforts of the following collaborators:
David A. Blank, Michael R. Lankford, and Mark Pratt, U.S.
Department of Agriculture, Food Safety and Inspection Service
Midwestern Laboratory, Saint Louis, MO
James L. Bryant, U.S. Food and Drug Administration,
Bothell, WA
Henry S. Commager, Conagra Analytical Laboratory, Columbia, MO
Tim Donlevy, Silliker Laboratories of Illinois, Chicago
Heights, IL
A. Wayne Edwards, U.S. Food and Drug Administration,
Cincinnati, OH

Luanne Fanning, Silliker Laboratories Group, Chicago

Heights, IL
Edna Hu, Strasburger and Siegel, Inc., Baltimore, MD
Jennifer L. Johnson, U.S. Department of Agriculture,
Beltsville, MD
Kathy Jost Keating, Silliker Laboratories of New Jersey,
Garwood, NJ
Cathy Lord, Karen Warner, Gary Fisher, and Chuck Kasper,
Cargill Analytical Services, Cedar Rapids, IA
Curtis J. Hagen, U.S. Food and Drug Administration, Denver, CO
Don Higgins and Rebecca Durham, Organon Teknika, Durham, NC
Andrea Hildenbrand, James Faison, and Yvonne Visier,
Marshall Durbin Co., Jackson, MS
Charles D. Hoffman, U.S. Food and Drug Administration,
San Francisco, CA
Jim Madden and Norma Corristan, Oregon Department of
Agriculture, Salem, OR
S. McNally and Cliff Coles, DelMonte, Walnut Creek, CA
Charles W. Noah, U.S. Food and Drug Administration, Dallas, TX
Corliss OBryan, Hudson Food Laboratory, Rogers, AR
Michael J. Palmieri and Alice Cohen, U.S. Food and Drug
Administration, Brooklyn, NY
Shital V. Patel and Sheri L. Richardson, Lipton, Englewood, NJ
Rebecca Pfundheller, Raksha Parmar, and Fran Moller, Silliker Laboratories of Texas, Grand Prairie, TX
Dan Potel and Greg Miklovic, Stouffer Frozen Foods,
Solon, OH
Larry Restaino and E.W. Frampton, R+F Laboratories,
Bridgeview, IL
Michelle Thompson, Linda Tilton, and Argie Codd, Foster
Farms, Livingston, CA
Edward Richter, Maria Thomas, and Steven Pao, Silliker
Laboratories of Ohio, OH
Peggy Cook and Jerri Lynn Witt, Tyson Foods, Inc., AR
References
(1) Mattingly, J.A., Butman, B.T., Plank, M.C., Durham, R.J., &
Robison, B.J. (1988) J. Assoc. Off. Anal. Chem. 71, 679681
(2) Hitchins, A.D. (1992) in Bacteriological Analytical Manual,
7th Ed., AOAC Arlington, VA, Chapter 10
(3) McClain, D., & Lee, W.H. (1989) FSIS Method for the Isolation and Identification of Listeria monocytogenes from
Processed Meat and Poultry Products, U.S. Department of
Agriculture, Food Safety and Inspection Service, Microbiology Division Laboratory Communication No. 57.
(4) Curiale, M.S., Sons, T., Fanning, L., Lepper, W., McIver, D.,
Garramone, S., & Mozola, M.A. (1994) J. Assoc. Off. Anal.
Chem. 77, 602
(5) Dangerous Goods Regulations (1990), 31st Ed., International
Air Transport Association, Montreal, Canada
(6) McClure, F.D. (1990) J. Assoc. Off. Anal. Chem. 73, 953
(7) Ryser, E.T., & Marth, E.H. (1991) Listeria, Listeriosis, and
Food Safety, Marcel Dekker, Inc., New York, NY

Table 994.03. Method performance for colorimetric monoclonal enzyme-linked immunosorbent assay for Listeria
monocytogenes in dairy products, seafoods, and meats

Food
Frankfurters

L. monocytogenes Methods
agreelevel,
MPN/g ment,a %

Incidence of false
negative results
among total positive
samples, %

Sensitivityc, %

Incidence of false
presumptive positives
among negative
samples, %

Specificityd, %

EIA

Culture

EIA

Culture

EIA

Culture

EIA

Culture

1.1
0.46
<0.003
0.043
0.023
<0.003

96.7
88.3
96.7
53.2
66.7
83.3

0.0
1.7
b
35.6
22.0

3.3
10.0

26.7
26.8

100.0
98.3

64.4
78.0

96.7
90.0

73.3
73.2

13.6

10.5

0.0

2.0

86.4

89.5

100.0

98.0

Brie cheese

>0.66
0.264
<0.005

88.3
86.7
96.7

5.0
7.1

6.7
7.1

95.0
92.9

93.3
92.9

5.2

8.3

94.8

91.7

2% milk

1.1
0.043
<0.003

58.0
60.0
100.0

12.5
6.1

40.0
54.5

87.5
93.9

60.0
45.5

2.0

4.0

98.0

96.0

Shrimp

>2.4
0.15
<0.003

98.7
94.7
96.0

0.0
5.4

1.3
0.0

100.0
94.6

98.7
100.0

2.8

10.0

97.2

90.0

Crab

0.66
0.28
<0.003

81.8
83.6
98.2

14.3
14.6

6.1
4.2

85.7
85.4

93.9
95.8

1.9

10.4

98.1

89.6

Roast beef

b
c
d

Agreement between EIA and USDA culture method (for frankfurters and roast beef), and U.S. FDA culture method (for milk, cheese, shrimp,
and crab).
Undefined for that inoculation level.
Percent positive samples detected of total number of positive samples tested.
Percent negative samples detected of total number of negative samples tested.

Table 1. Strains of Listeria monocytogenes and

contamination levels of the test samples used in the
collaborative study
Food type

Strain

Frankfurters

1/2c

Roast beef

3b

Milk, 2%

4b

Brie cheese

3a

Shrimp, set 1

1/2a

Shrimp, set 3

1/2a

Crab meat

1/2b

Level
high
low
control
high
low
control
high
low
control
high
low
control
high
low
control
high
low
control
high
low
control

MPN/g
1.1
0.46
<0.003
0.043
0.023
<0.003
1.1
0.043
<0.003
>0.66
0.264
<0.005
0.023
0.015
0.003
>2.4
0.15
<0.003
0.66
0.28
<0.003

Table 2. Laboratory participation in the collaborative study for detection of Listeria monocytogenes in dairy
products, seafoods, and meats
Sample seta
Raw shrimp
Laboratory

Frankfurter

Roast beef

Brie cheese

Milk, 2%

set 1

set 3

Crab

1
2
3
4
5
6
7
8
9
10
11
12
13
14
16
17
18
19
20
21
22
23
24
25
26
27

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

+
+

+
+
+

+
+
+
+

+
+

+
+
+
+
+

+
+
+

+
+

b b
+

+
c c
+

+
+

+
+
+

+
+

+
+

+
+

+
+

+
+

+
+
+

+
+

+
+
+
+

+
+

+
+

+
+

+
+

No. of sets sent

Data sets received

13
12

12
12

13
13

12
10

11
11

15
15

12
12

a
b
c
d

+ indicates samples were sent to collaborator; indicates samples were not sent.
Collaborator was unable to complete the analyses, no results were submitted.
Laboratory error, results excluded from the statistical evaluation.
Sample shipment delayed by the carrier; analyses were not performed.

Table 3. Collaborative study results for detection of Listeria monocytogenes in frankfurters

High inoculum samples
Laboratory

11

Low inoculum samples

13

Control samples

14

10

12

15

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

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

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

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

+
+
+
+
+
+
b b

+
+
+
+
+

+
+
+
+
+
+
b b

+
+
+
+
+

Listeria-Tek assay
1
2
3
4
5
6
7
8
9
11
12
23

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

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

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

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

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

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

+
+
+
+
+

+
+
+
+
+
+

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

Listeria-Tek assay confirmationa

1
2
3
4
5
6
7
8
9
11
12
23

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

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

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

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

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

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

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

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

USDA culture method

1
2
3
4
5
6
7
8
9
11
12
23
a
b

+
+
+
b b

+
+
+
+
+
+
+
+

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

+
+
+
b b

+
+
+
+
+
+
+
+

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

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

+
+
+
+
+
+
b b

+
+
+
+
+

+
+
+
+
+
+
b b

+
+
+
+
+

+
+
+
b b

+
+
b b

+
+
+
+
+

L. monocytogenes isolated from ELISA culture broth.

USDA culture method false negative result; Fraser tube was not black and L. monocytogenes was isolated from Fraser tube.

Table 4. Collaborative study results for detection of Listeria monocytogenes in roast beef
High inoculum samples
Laboratory

Low inoculum samples

10

Control samples

11

13

12

14

15

+
+

+
+
+

+
+
+
+

+
+

+
+

+
b b

+
b b

+
b b

+
b b

+
b b

+
+

+
b b

b b

Listeria-Tek assay
1
2
4
6
7
8
10
11
12
13
16
23

+
+
+

+
+

+
+
+

+
+

+
+
+
+

+
+
+

+
+
+

+
+
+
+

+
+
+

+
+
+
+

+
+
+

+
+

+
+
+

+
+

Listeria-Tek assay confirmationa

1
2
4
6
7
8
10
11
12
13
16
23

+
+
+
+
+

+
+
+
+

+
+

+
+
+

+
+

+
+
+

+
+
+

+
+
+
+
+

+
+
+
+

+
+
+
+
+
+
+
+

+
+
+
+
+
+
+

+
+

+
+
+
+

USDA culture method

1
2
4
6
7
8
10
11
12
13
16
23
a
b

+
+
b b

+
b b

+
+
+

+
+
+
+

+
+
+
+

+
+

b b

+
+

+
+

+
+

+
+
+
+
+

+
+

+
+
b b

+
b b

L. monocytogenes isolated from ELISA culture broth.

USDA culture method false negative result; Fraser tube was not black and L. monocytogenes was isolated from Fraser tube.

Table 5. Collaborative study results for detection of Listeria monocytogenes in Brie cheese
High inoculum samples
Laboratory

11

Low inoculum samples

12

Control samples

10

15

13

14

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

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

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+

+
+
+

+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

Listeria-Tek assay
1
2
4
7
8
9
a
10
11
14
17
18
19
21

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

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

+
+
+
+

+
+
+
+
+
+

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

+
+
+
+

+
+
+
+
+
+
+
+

+
+
+
+

+
+
+
+
+
+

+
+
+
+

+
+
+
+
+

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

Listeria-Tek assay confirmationb

1
2
4
7
8
9
a
10
11
14
17
18
19
21

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+

+
+

+
+
+

+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

U.S. FDA culture method

1
2
4
7
8
9
a
10
11
14
17
18
19
21
a
b

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
+
+

+
+
+

+
+

+
+
+
+
+
+

+
+
+
+

+
+

+
+
+
+
+

+
+

+
+

Data excluded from statistical evaluation; systematic speciation error.

L. monocytogenes isolated from ELISA culture broth.

+
+
+
+
+
+

+
+
+
+
+
+

Table 6. Collaborative study results for detection of Listeria monocytogenes in milk

High inoculum samples
Laboratory

14

Low inoculum samples

15

Control samples

11

12

10

13

+
+

+
+
+
+
+

+
+

+
+
+

+
+

+
+
+
+
+

+
+

+
+
+

+
+

+
+
+

Listeria-Tek assay
1
2
7
10
11
14
17
18
19
21

+
+

+
+
+
+
+

+
+

+
+
+
+
+

+
+

+
+
+
+
+

+
+

+
+
+
+
+

+
+

+
+
+
+
+

+
+
+

+
+

+
+
+
+
+

+
+
+
+
+

Listeria-Tek assay confirmationa

1
2
7
10
11
14
17
18
19
21

+
+

+
+
+
+
+

+
+

+
+
+
+
+

+
+

+
+
+
+
+

+
+

+
+
+
+
+

+
+

+
+
+
+
+

+
+
+

+
+

+
+
+
+
+

+
+
+
+
+

U.S. FDA culture method

1
2
7
10
11
14
17
18
19
21
a

+
+

+
+

+
+

+
+

+
+

+
+
+

+
+
+

+
+

+
+
+

L. monocytogenes isolated from ELISA culture broth.

+
+

+
+
+

Table 7. Collaborative study results for detection of Listeria monocytogenes in the first set of shrimps
High inoculum samples
Laboratory

Low inoculum samples

11

Control samples

10

15

12

13

14

+
+

+
+

+
+
+
+

+
+

+
+

+
+

+
+

+
+
+

+
+
+

+
+

+
+

+
+

+
+

Listeria-Tek assay
1
2
7
8
9
10
11
14
16
20
22
24
25

+
+

+
+
+
+

+
+
+
+

+
+

+
+
+
+

+
+
+

+
+

+
+
+

+
+
+
+

+
+

+
+
+
+

+
+
+
+

+
+

+
+
+
+
+

+
+
+
+

+
+

+
+

+
+

+
+

Listeria-Tek assay confirmationb

1
2
7
8
9
10
11
14
16
20
22
24
25

+
+

+
+

+
+

+
+

+
+

+
+

+
+

+
+

+
+

U.S. FDA culture method

1
2
7
8
9
10
11
14
16
20
22
24
25
a
b

+
+

+
+

+
+

+
+

+
+

+
+

+
+

+
+

2424 h samples enrichment; the second enrichment was inoculated with 0.1 mL of the primary enrichment broth.
L. monocytogenes isolated from ELISA culture broth.

Table 8. Collaborative study results for detection of Listeria monocytogenes in the third set of shrimps
High inoculum samples
Laboratory

10

Low inoculum samples

12

Control samples

14

15

11

13

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

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

+
+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+

+
+
+
+
+
+
+
+
+
+

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

+
+
+
+

+
+
+
+
+
+
+
+
+
+

Listeria-Tek assay
1
2
4
7
8
9
11
14
18
21
22
24
25
26
27

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

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

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

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

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

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

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

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

Listeria-Tek assay confirmationb

1
2
4
7
8
9
11
14
18
21
22
24
25
26
27

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

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

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

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

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

+
+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+

+
+
+
+
+
+
+
+
+
+

+
+
+
+

+
+
+
+
+
+
+
+
+
+

U.S. FDA culture method

1
2
4
7
8
9
11
14
18
21
22
24
25
26
27
a
b

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

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

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

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

+
+
+
+
+
+

+
+
+
+
+
+
+
+

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

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

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

2628 h samples enrichment; the second enrichment was inoculated with 1 mL of the first enrichment broth.
L. monocytogenes isolated from ELISA culture broth.

Table 9. Collaborative study results for detection of Listeria monocytogenes in crab meat
High inoculum samples
Laboratory

10

Low inoculum samples

15

Control samples

11

13

12

14

+
+
+
+
+
+

+
+
+
+

+
+
+
+

+
+

+
+
+

+
+
+
+
+
+
+

+
+
+

+
+

+
+
+
+

+
+
+

+
+
+
+
+
+
+

+
+
+

+
+

+
+
+
+

Listeria-Tek assay
1
2
7
8
a
10
11
14
16
20
22
24
25

+
+

+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+

+
+

+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+
+
+
+
+
+
+
+

+
+
+
+

+
+

+
+

+
+

+
+
+
+

Listeria-Tek assay confirmationb

1
2
7
8
a
10
11
14
16
20
22
24
25

+
+
+
+

+
+

+
+
+
+

+
+
+

+
+

+
+
+
+

+
+

+
+

+
+
+
+

+
+
+

+
+
+
+
+
+
+

+
+
+

+
+

+
+
+
+

+
+
+

+
+
+
+
+
+
+

+
+

+
+
+
+

+
+
+
+

+
+

+
+
+
+

U.S. FDA culture method

1
2
7
8
a
10
11
14
16
20
22
24
25
a
b

+
+
+

+
+

+
+
+
+

+
+
+

+
+

+
+

+
+
+
+

+
+

+
+
+
+

+
+
+

+
+
+
+
+
+
+

+
+
+

+
+

+
+
+
+

Data not used; systematic speciation error.

L. monocytogenes isolated from ELISA culture broth.

+
+

+
+
+
+
+
+
+

+
+
+

+
+

+
+
+
+

+
+
+

+
+

+
+
+
+

Table 10. Summary of the collaborative study results for detection of Listeria monocytogenes in dairy products,
seafoods, and meats

Food
Frankfurters

Roast beef

Brie cheese

2% milk

Shrimp

Crab

Number of results in
agreement

Number of false negative

results

Number of false presumptive

positive assaysa

L. monocytogenes,
MPN/g

Number of
samples

Positive

Negative

ELISA

Culture
method

ELISA

Culture
method

1.1
0.46
<0.003
0.043
0.023
<0.003
>0.66
0.264
<0.005
1.1
0.043
<0.003
>2.4
0.15
<0.003
0.66
0.28
<0.003

60
60
60
60
60
60
60
60
60
50
50
50
75
75
75
55
55
55

58
53
0
17
21
0
53
48
0
19
13
0
74
70
1
39
39
1

0
0
58
15
19
50
0
4
58
10
17
50
0
1
71
6
7
53

0
1
1
16
9
7
3
4
0
5
2
0
0
4
1
7
7
1

2
6
1
12
11
3
4
4
2
16
18
0
1
0
2
3
2
0

0
0
8
3
4
6
0
3
3
0
0
1
0
5
2
4
0
1

0
0
0
2
3
1
0
2
5
2
3
2
1
1
8
5
0
5

Unconfirmed positive assays refers to the negative screening assay for each method and is the first step in the method which eliminates
negative samples. The immunoassay in the ELISA, the Fraser broth tubes in the USDA culture method, and the plate reading procedure in
the FDA culture method constitute negative screening assays.

Figure 1. Collaborative study process for analysis of test samples.