AOAC Official Method 2013.11 B.

  Apparatus and Reagents

Listeria monocytogenes in a Variety of Foods Items (a)–(h) are available as the VIDAS Listeria monocytogenes
VIDAS® Listeria monocytogenes Xpress (LMX) Method (LMX) assay kit from bioMérieux (Hazelwood, MO, USA).
First Action 2013 (a)  VIDAS or miniVIDAS automated immunoassay system.
[Applicable to detection of Listeria monocytogenes in deli (b)  LMX reagent strips.—Sixty polypropylene strips of 10 wells,
ham (25 and 125 g), fermented sausage (25 g), liver pâté (25 g), each strip covered with a foil seal and label. The 10 wells contain
processed cheese (25 g), vanilla ice cream (25 g), cooked shrimp the reagents in Table 2013.11C.
(25  g), smoked white fish (25  g), frozen spinach (25  g), peanut (c)  SPR.—Sixty SPRs coated with proteins specific for Listeria
butter (25 g), deli turkey (25 and 125 g), queso fresco (125 g), and receptors.
ground beef (125 g).] (d)  Standard.—One vial (1 × 6 mL). Ready-to-use. Contains
See Tables 2013.11A and B for a summary of results of the purified and inactivated L. monocytogenes receptors + preservative
collaborative study. See supplemental data, Tables 2A–D, for + protein stabilizer.
detailed results of the collaborative study on J. AOAC Int. website, (e)  Positive control solution.—1 × 3 mL. Contains purified and
http://aoac.publisher.ingentaconnect.com/content/aoac/jaoac. inactivated L. monocytogenes antigen + preservative + protein
Caution:  Listeria monocytogenes is of particular concern for stabilizer.
(f)  Negative control solution.—1 × 6 mL. Contains Tris-buffered
pregnant women, the aged, and the infirmed. It is
saline (TBS; 150 mmol/l) – Tween pH 7.6 + preservative.
recommended that these concerned groups avoid (g)  Master lot entry (MLE) card.—One card providing

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handling this organism. Dispose of all reagents and other

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specifications for the factory master data required to calibrate the

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contaminated materials by acceptable procedures for test. To read the MLE data, refer to the Operator’s Manual.
potentially biohazardous materials. Some reagents in the (h)  Package insert.

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kit contain 1 g/L concentrations of sodium azide. Check (i)  Pipet.—Disposable to dispense appropriate volumes.
local regulations prior to disposal. Disposal of these (j)  VIDAS Heat and Go.—Available from bioMérieux, Inc.

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reagents into sinks with copper or lead plumbing should (k)  Water bath.—95–100°C or equivalent system.
(l)  Bag with filter.
be followed immediately with large quantities of water
(m)  SmasherTM Blender/Homogenizer.—Available from
Pa h
to prevent potential hazards. This kit contains products bioMérieux, Inc., or equivalent.
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of animal origin. Certified knowledge of the origin and/ (n)  LMX broth.—Available from bioMérieux, Inc.
or sanitary state of the animals does not totally guarantee (o)  Supplement for LMX broth.
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the absence of transmissible pathogenic agents. It is (p)  LPT broth.—Available from bioMérieux, Inc.
therefore recommended that these products be treated as (q)  Incubators.—Capable of maintaining 37  ± 1°C, 35  ±  1°C,
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potentially infectious and handled observing the usual and 30 ±1°C.

safety precautions (do not ingest or inhale). (r)  Diagnostic reagents.—Necessary for culture confirmation of
R R

assays.
A. Principle (s)  ALOA Chromogenic Agar.—Necessary for cultural
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confirmation as an alternative to selective agar required by

The VIDAS® Listeria monocytogenes Xpress (LMX) method is appropriate reference method. Available from bioMérieux, Inc.
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for use on the automated VIDAS instrument for the detection of (t)  Tryptic Soy Agar with yeast additive.
L.  monocytogenes antigens using the enzyme-linked fluorescent
C.  General Instructions
assay (ELFA) method. The Solid Phase Receptacle (SPR®) serves
Ex A

as the solid phase as well as the pipetting device. The interior of the (a)  Components of the kit are intended for use as integral unit.
SPR is coated with proteins specific for L. monocytogenes receptors. Do not mix reagents or disposables of different lot numbers.
Reagents for the assay are ready-to-use and predispensed in the sealed (b)  Store VIDAS LMX kits at 2–8°C.
reagent strips. All of the assay steps are performed automatically (c)  Do not freeze reagents.
by the instrument. The reaction medium is cycled in and out of the (d)  Bring reagents to room temperature before inserting them
SPR several times. An aliquot of enrichment broth is dispensed into into the VIDAS instrument.
the reagent strip. The L. monocytogenes receptors present will bind (e)  Standard, controls and heated test portions are mixed well
to the interior of the SPR. Unbound components are eliminated before using.
during the washing steps. The proteins conjugated to the alkaline (f)  Include one positive and one negative control with each
group of tests.
phosphatase are cycled in and out of the SPR and will bind to any
(g)  Return unused components to 2–8°C immediately after use.
Listeria monocytogenes receptors which are themselves bound to the
(h)  See Safety Precautions in the VIDAS LMX package insert
SPR wall. A final wash step removes unbound conjugate. During the
(refer to the following sections in the package insert: Warnings and
final detection step, the substrate (4-methyl-umbelliferyl phosphate)
Precautions and Waste Disposal).
is cycled in and out of the SPR. The conjugate enzyme catalyzes
(i)  Review the policies recommended by the Centers for Disease
the hydrolysis of the substrate into a fluorescent product (4-methyl-
Control and Prevention (CDC) on dealing with pathogens. http://
umbelliferone), the fluorescence of which is measured at 450  nm. www.cdc.gov/biosafety/publications/bmb15/index.htm/.
At the end of the assay, results are automatically analyzed by the
D.  Preparation of Test Suspension
instrument which calculates a test value for each sample. This value
is then compared to internal references (thresholds) and each result is (a)  Pre-enrichment.—Pre-enrich test portion using filter
interpreted as positive or negative. Stomacher bags to initiate growth of L. monocytogenes. For

© 2014 AOAC INTERNATIONAL

Table  2013.11A.  Summary of results for the detection of Listeria monocytogenes in queso fresco (25 g)a
VIDAS LMX with OXA VIDAS LMX with ALOA
Inoculation level
Uninoculated Low High Uninoculated Low High
Candidate presumptive positive/total 0/156 77/156 156/156 0/156 77/156 156/156
  No. samples analyzed
Candidate presumptive POD (CP) 0.00 0.49 1.00 0.00 0.49 1.00
(0.00, 0.02) (0.41, 0.58) (0.98, 1.00) (0.00, 0.02) (0.41, 0.58) (0.98, 1.00)
srb 0.00 0.51 0.00 0.00 0.51 0.00
(0.00, 0.15) (0.46, 0.52) (0.00, 0.15) (0.00, 0.15) (0.46, 0.52) (0.00, 0.15)
sLc 0.00 0.00 0.00 0.00 0.00 0.00
(0.00, 0.15) (0.00, 0.12) (0.00, 0.15) (0.00, 0.15) (0.00, 0.12) (0.00, 0.15)
sRd 0.00 0.51 0.00 0.00 0.51 0.00
(0.00, 0.21) (0.47, 0.52) (0.00, 0.21) (0.00, 0.21) (0.47, 0.52) (0.00, 0.21)

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P valuee 1.0000 0.9772 1.0000 1.0000 0.9772 1.0000

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Candidate confirmed positive/
0/156 75/156 156/156 0/156 75/156 156/156
  total No. samples analyzed

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Candidate confirmed POD (CC) 0.00 0.48 1.00 0.00 0.48 1.00

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(0.00, 0.02) (0.40, 0.56) (0.98, 1.00) (0.00, 0.02) (0.40, 0.56) (0.98, 1.00)
sr 0.00 0.51 0.00 0.00 0.51 0.00
(0.00, 0.15) (0.46, 0.52) (0.00, 0.15) (0.00, 0.15) (0.46, 0.52) (0.00, 0.15)
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sL 0.00 0.00 0.00 0.00 0.00 0.00
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(0.00, 0.15) (0.00, 0.14) (0.00, 0.15) (0.00, 0.15) (0.00, 0.14) (0.00, 0.15)
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sR 0.00 0.51 0.00 0.00 0.51 0.00

(0.00, 0.21) (0.46, 0.52) (0.00, 0.21) (0.00, 0.21) (0.46, 0.52) (0.00, 0.21)
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P value 1.0000 0.8718 1.0000 1.0000 0.8718 1.0000

Positive reference samples/
R R

0/156 69/156 153/156 0/156 69/156 153/156

  total No. samples analyzed
rt C

Reference POD 0.00 0.44 0.98 0.00 0.44 0.98

(0.00, 0.02) (0.36, 0.52) (0.94, 0.99) (0.00, 0.02) (0.36, 0.52) (0.94, 0.99)
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sr 0.00 0.51 0.13 0.00 0.51 0.13

(0.00, 0.15) (0.46, 0.52) (0.12, 0.15) (0.00, 0.15) (0.46, 0.52) (0.12, 0.15)
Ex A

sL 0.00 0.00 0.03 0.00 0.00 0.03

(0.00, 0.15) (0.00, 0.13) (0.00, 0.07) (0.00, 0.15) (0.00, 0.13) (0.00, 0.07)
sR 0.00 0.51 0.14 0.00 0.51 0.14
(0.00, 0.21) (0.46, 0.52) (0.12, 0.16) (0.00, 0.21) (0.46, 0.52) (0.12, 0.16)
P value 1.0000 0.9320 0.0877 1.0000 0.9320 0.0877
dLPOD (candidate vs reference) 0.00 0.04 0.02 0.00 0.04 0.02
(–0.02, 0.02) (–0.08, 0.15) (–0.01, 0.06) (–0.02, 0.02) (–0.08, 0.15) (–0.01, 0.06)
dLPOD (candidate presumptive vs
0.00 0.01 0.00 0.00 0.01 0.00
  candidate confirmed)
(–0.02, 0.02) (–0.10, 0.13) (–0.02, 0.02) (–0.02, 0.02) (–0.10, 0.13) (–0.02, 0.02)
a
 Results include 95% confidence intervals.
b
 Repeatability standard deviation.
c
 Among-laboratory standard deviation.
d
 Reproducibility standard deviation.
e
 
P value = Homogeneity test of laboratory PODs.

© 2014 AOAC INTERNATIONAL

Table  2013.11B.  Summary of results for the detection of Listeria monocytogenes in queso fresco (125 g)a
VIDAS LMX with OXA VIDAS LMX with ALOA
Inoculation level
Uninoculated Low High Uninoculated Low High
Candidate presumptive positive/ 0/144 70/144 144/144 0/144 70/144 144/144
  total No. samples analyzed
Candidate presumptive POD (CP) 0.00 0.49 1.00 0.00 0.49 1.00
(0.00, 0.03) (0.40, 0.57) (0.97, 1.00) (0.00, 0.03) (0.40, 0.57) (0.97, 1.00)
srb 0.00 0.51 0.00 0.00 0.51 0.00
(0.00, 0.16) (0.46, 0.52) (0.00, 0.16) (0.00, 0.16) (0.46, 0.52) (0.00, 0.16)
sLc 0.00 0.00 0.00 0.00 0.00 0.00
(0.00, 0.16) (0.00, 0.12) (0.00, 0.16) (0.00, 0.16) (0.00, 0.12) (0.00, 0.16)
sRd 0.00 0.51 0.00 0.00 0.51 0.00
(0.00, 0.22) (0.46, 0.52) (0.00, 0.22) (0.00, 0.22) (0.46, 0.52) (0.00, 0.22)

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P valuee 1.0000 0.9730 1.0000 1.0000 0.9730 1.0000

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Candidate confirmed positive/ 0/144 70/144 144/144 0/144 70/144 144/144
  total No. samples analyzed

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Candidate confirmed POD (CC) 0.00 0.49 1.00 0.00 0.49 1.00

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(0.00, 0.03) (0.40, 0.57) (0.97, 1.00) (0.00, 0.03) (0.40, 0.57) (0.97, 1.00)
sr 0.00 0.51 0.00 0.00 0.51 0.00
(0.00, 0.16) (0.46, 0.52) (0.00, 0.16) (0.00, 0.16) (0.46, 0.52) (0.00, 0.16)
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sL 0.00 0.00 0.00 0.00 0.00 0.00
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(0.00, 0.16) (0.00, 0.12) (0.00, 0.16) (0.00, 0.16) (0.00, 0.12) (0.00, 0.16)
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sR 0.00 0.51 0.00 0.00 0.51 0.00

(0.00, 0.22) (0.46, 0.52) (0.00, 0.22) (0.00, 0.22) (0.46, 0.52) (0.00, 0.22)
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P value 1.0000 0.9730 1.0000 1.0000 0.9730 1.0000

Positive reference samples/total No. samples analyzed 0/144 69/144 144/144 0/144 69/144 144/144
R R

Reference POD 0.00 0.48 1.00 0.00 0.48 1.00

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(0.00, 0.03) (0.39, 0.56) (0.97, 1.00) (0.00, 0.03) (0.39, 0.56) (0.97, 1.00)
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sr 0.00 0.51 0.00 0.00 0.51 0.00

(0.00, 0.16) (0.46, 0.52) (0.00, 0.16) (0.00, 0.16) (0.46, 0.52) (0.00, 0.16)
sL 0.00 0.00 0.00 0.00 0.00 0.00
Ex A

(0.00, 0.16) (0.00, 0.12) (0.00, 0.16) (0.00, 0.16) (0.00, 0.12) (0.00, 0.16)
sR 0.00 0.51 0.00 0.00 0.51 0.00
(0.00, 0.22) (0.46, 0.52) (0.00, 0.22) (0.00, 0.22) (0.46, 0.52) (0.00, 0.22)
P value 1.0000 0.9672 1.0000 1.0000 0.9672 1.0000
dLPOD (C vs R) 0.00 0.01 0.00 0.00 0.01 0.00
(–0.03, 0.03) (–0.10, 0.13) (–0.03, 0.03) (–0.03, 0.03) (–0.10, 0.13) (–0.03, 0.03)
dLPOD (CP vs CC) 0.00 0.00 0.00 0.00 0.00 0.00
(–0.03, 0.03) (–0.12, 0.12) (–0.03, 0.03) (–0.03, 0.03) (–0.12, 0.12) (–0.03, 0.03)
a
 Results include 95% confidence intervals.
b
 Repeatability standard deviation.
c
 Among-laboratory standard deviation.
d
 Reproducibility standard deviation.
e
 
P value = Homogeneity test of laboratory PODs.

© 2014 AOAC INTERNATIONAL

Table  2013.11C.  Reagents included in 10-well reagent strip Table  2013.11D.  Interpretation of test

Wells Reagents (LMX) Test value threshold Interpretation

1 Sample well: 0.25 mL of enrichment broth, <0.05 Negative
standard or control
≥0.05 Positive
2 Prewash solution (600 µL): TRIS-NaCl
(150 mmol/L) – Triton X100 pH 7.6 + preservative
Note: The standard must be tested upon receipt of a new lot of
3, 4, 7–9 Wash buffer (600 µL): TRIS-NaCl (150 mmol/L) -
reagents and then every 14 days. The relative fluorescence value (RFV)
Tween pH 7.6 + preservative
of the standard must fall within the set range provided with the kit.
5 Conjugate (400 µL): biotin-labeled anti-Listeria
(e)  Load the LMX reagents strips and SPRs into the positions
monocytogenes antibodies + preservative
that correspond to the VIDAS section indicated by the work list.
6 Streptavidin – ALP (400 µL)
Verify that the color labels with the assay code on the SPRs and
10 Reading cuvette with substrate (300 µL): reagent strips match.
4-methyl-umbelliferyl phosphate (0.6 mmol/L) +
(f)  Initiate the assay processing as directed in the VIDAS
diethanolaminea (DEA; 0.62 mol/L or 6.6%, pH 9.2) +
preservative operator’s manual.
(g)  After the assay is completed, remove the SPRs and reagent
a
Irritant reagent: See VIDAS LPT package insert for more information.
 
strips from the instrument and dispose of properly.

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F.  Results and Interpretation
25  g test portions, add 225  mL pre-LMX broth brought to room

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temperature (18–25°C) and 500 µL LMX broth supplement to each The results are analyzed automatically by the VIDAS system.

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test portion and homogenize thoroughly for 2 min. For 125 g test A report is printed which records the type of test performed, the

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portions, add 375 mL LPT broth brought to room temperature (18– test sample identification, the date and time, the lot number, and

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25°C) to each test portion and homogenize thoroughly for 2 min. expiration date of the reagent kit being used, and each sample’s
(b)  25 g Test portions.—After homogenization, incubate for RFV, test value, and interpreted result (positive or negative).
26–30 h at 37 ± 1°C. Fluorescence is measured twice in the reagent strip’s reading cuvette
125 g Test portions.—After homogenization, incubate for 24–
Pa h
for each sample tested. The first reading is a background reading of
30 h at 30 ± 1°C.
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the substrate cuvette before the SPR is introduced into the substrate.
After the primary enrichment, transfer a 1 mL aliquot into 10 mL The second reading is taken after incubating the substrate with the
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LPT broth brought to room temperature (18–25°C) and incubate enzyme remaining on the interior of the SPR. The test value is
for 22–26 h at 30 ± 1°C. calculated by the instrument and is equal to the difference between
(c)  After incubation, homogenize samples manually and prepare
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the background reading and the final reading. The calculation

samples for assay according the following procedures (based on appears on the result sheet. A “negative” result has a test value
sample size):
R R

less than the threshold (0.05) and indicates that the sample does
125 g Test portions.—No heating is necessary for method not contain L.  monocytogenes or contains L.  monocytogenes at a
performance. Load 0.25 mL of enrichment into the VIDAS LMX
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concentration below the detection limit. A “positive” result has a

reagent strip and perform the VIDAS test.
test value equal to or greater than the threshold (≥0.05) and indicates
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25 g Test portions.—Follow appropriate instructions based on

that the sample may be contaminated with L. monocytogenes. If the
heating method.
background reading is above a predetermined cutoff, then the result
(1) Boiling.—Transfer 2–3 mL of the enrichment broth into a tube.
is reported as invalid (Table 2013.11D).
Ex A

Seal the tube. Heat in a water bath for 5 ± 1 min at 95–100°C. Cool the
tube. Mix the boiled broth and transfer 0.25 mL into the sample well of G.  Confirmation
the VIDAS LMX reagent strip. Perform the VIDAS test. All positive VIDAS LMX results must be culturally confirmed.
(2)  Heat and Go.—Transfer 0.25 mL of the enrichment broth into Confirmation should be performed using the nonheated enrichment
the sample well of the VIDAS LMX reagent strip. Heat for 5 ± 1 min broth (the LMX primary enrichment broth for 25 g test portions and
(see VIDAS Heat and Go User’s Manual). Remove the strip and allow
the LPT secondary enrichment broth for 125 g test portions) stored
to cool for 10 min prior to test initiation. Perform the VIDAS test.
between 2–8°C, and should be initiated within 72 h following the
E.  Enzyme Immunoassay end of incubation (AFNOR Certificate No. BIO 12/33-05/12).
(a) Enter factory master calibration curve data into the Presumptive positive results may be confirmed by isolating on
instrument using the MLE card. selective agar plates such as ALOA or on the appropriate reference
(b)  Remove the kit reagents and materials from refrigerated storage method selective agar plates. Typical or suspect colonies from
and allow them to come to room temperature for at least 30 min. each plate are confirmed as described in appropriate reference
(c)  Use one VIDAS LMX reagent strip and one VIDAS LMX method. As an alternative to the conventional confirmation for
SPR for each sample, control or standard to be tested. Reseal the L.  monocytogenes, VITEK  2 GP Biochemical Identification
storage pouch after removing the required number of SPRs. (see 2012.02) or API Listeria biochemical kits may be used for
(d)  Enter the appropriate assay information to create a work list. presumptive generic identification of foodborne L. monocytogenes.
Enter the test code by typing or selecting “LMX”, and number of tests Reference: J. AOAC Int. 97, 442(2014)
to be run. If the standard is to be tested, identify the standard by “S1” DOI: 10.5740/jaoacint.13-368
and test in duplicate. If the positive control is to be tested, identify it by
“C1”. If the negative control is to be tested, identify it by “C2”. Posted: May 2014

© 2014 AOAC INTERNATIONAL