17.9.33
—Necessary for culture confirmation of
AOAC Official Method 2004.03
Salmonella in Foods
Enzyme-Linked Fluorescent Assay (ELFA)
Screening Method
First Action 2004
Final Action 2007
Caution: Some reagents in the kit contain 1 g/L concentrations
of sodium azide. Check local regulations prior to
disposal. Disposal of these reagents into sinks with
copper or lead plumbing should be followed
immediately with large quantities of water to prevent
potential hazards.
See Table 2004.03A for the results of the interlaboratory study
supporting acceptance of the method.
A. Principle
Identification of Salmonella antigens is based on enzyme-linked
fluorescent assay performed in automated VIDAS instrument. The
solid-phase receptacle (SPR) functions as both the solid phase as
well as the pipeting device. The SPR is coated with mixture of
highly specific monoclonal antibodies. All steps of the assay are
performed automatically. Following pre-enrichment, selective
enrichment, and post-enrichment of test portions, an aliquot of
boiled broth is placed into the reagent strip and the mixture is cycled
in and out of SPR for specific length of time. Salmonella antigens, if
present, bind to monoclonal antibodies coating the interior of the
SPR. All other unbound compounds are washed away. Antibodies
conjugated with alkaline phosphatase are cycled in and out of the
SPR, binding to any Salmonella antigen bound to the SPR wall. A
final wash step re moves unbound conjugate. The substrate,
4-methyl umbelliferyl phosphate, is converted by an enzyme on the
SPR wall to the fluorescent product, 4-methyl umbelliferone. The
intensity of fluorescence is measured by the optical scanner located
inside the VIDAS instrument. Results are analyzed automatically by
the computer. A test value based on the fluorescence measurement is
generated and compared to a standard. A report is printed for each
batch of tests performed.
B. Reagents
Items (a)–(f) are available as VIDAS Salmonella (SLM) assay kit
from bioMérieux, Inc. (100 Rodolphe St, Durham, NC 27712, USA;
www.biomerieux-usa.com). Store all materials at 2°–8°C. A kit is
sufficient for 60 tests.
(a) Reagent strips.—60 polypropylene strips of 10 wells, each
strip covered with a foil seal and label. The 10 wells contain the
reagents in Table 2004.03B.
(b) Solid-phase receptacle (SPR).—60 SPRs coated with
anti-Salmonella antibodies.
(c) Stan dard.—One vial (3 mL). Con tains pu ri fied and
inactivated Salmonella antigen with 1 g/L sodium azide and protein
stabilizers.
(d) Positive control solution.—One vial (6 mL). Contains
purified and inactivated Salmonella antigen with 1 g/L sodium azide
and protein stabilizers.
(e) Negative control solution.—One vial (6 mL).—Contains
Tris-buffered saline.
(f) Package insert.
(g) Diagnostic reagents.
assays. See 967.27 (see 17.9.03).
(h) M-broth.—5.0 g yeast extract, 12.5 g Tryptone, 2.0 g
D-mannose, 5.0 g sodium citrate, 5.0 g NaCl, 5.0 g K2PO4, 0.14 g
MnCl2, 0.8 MgSO4, 0.04 g FeSO4, and 0.75 g Tween 80. Suspend
ingredients in 1 L H2O and heat to boiling 1–2 min. Dispense 10 mL
portions into 16 ´ 125 mm screw-cap tubes. Cap tubes loosely and
autoclave 15 min at 121°C. Tighten caps securely for storage. Final
pH should be 7.0 ± 0.2.
C. Apparatus
(a) VIDAS or miniVIDAS automated immunoassay
system.—bioMérieux Inc.
(b) Incubators.—35° ± 1°C and 41°–42°C.
(c) Water bath.—Maintaining 95°–100°C.
D. General Instructions
(1) Components in kit are intended for use as integral unit. Do not
mix reagents or disposables of different lot numbers.
(2) Store VIDAS SLM kits at 2°–8°C.
(3) Bring reagents to room temperature (minimum 30 min)
before inserting them into the VIDAS.
(4) Mix well the standard, controls, and boiled test portions
before use.
(5) Include 1 positive and 1 negative control with each group of
tests.
(6) Return unused components to 2°–8°C immediately after use.
(7) Treat all materials in contact with bacterial cultures as
biohazards and decontaminate by autoclaving.
E. Preparation of Test Suspension
(a) Pre-enrichment.—Pre-enrich test portion in nonselective
medium to initiate growth of salmonellae. Pre-enrichment media
vary with product type and procedure must be performed as
described in 967.26 (see 17.9.02) or as in Bacteriological Analytical
Manual (current edition, www.cfsan.fda.gov/~ebam/bam-toc.html).
Enrich for 18–24 h at 35° ± 1°C.
(b) Se lec tive en rich ment.—Trans fer 0.1 mL in cu bated
pre-enrichment culture to tube containing 10 mL
Rappaport-Vassiliadis (RV) medium and 1 mL to tube containing
10 mL tetrathionate broth. For all foods other than raw foods or
foods with high microbial load, incubate selective enrichments for
6–8 h at 41°–42°C. For raw foods or foods with high microbial load,
incubate broths 18–24 h.
(c) Post-en rich ment.—Af ter in cu ba tion, trans fer 1 mL
tetrathionate culture to tube containing 10 mL M-broth. Transfer
1 mL RV culture medium to separate tube containing 10 mL
M-broth. For all foods other than raw foods or foods with high
microbial load, incubate M-broths 18–24 h at 41°–42°C. For raw
foods or foods with high microbial load, incubate M-broths 6–8 h at
41°–42°C. Reincubate selective enrichment broths at 41°–42°C for
total incubation time of 24 ± 2 h to be used for confirmation of
positive VIDAS results.
(d) After incubation, mix 1 mL of each M-broth into the same
tube. Recap the tube tightly and heat for 15 ± 1 min in a water bath at
95°–100°C. Allow tube to cool, and perform the VIDAS test. Store
M, RV, and tetrathionate broths at 2°–8°C for confirmation.
© 2007 AOAC INTERNATIONAL
 Table 2004.03A. Interlaboratory study results for Salmonella in foods (enzyme-linked fluorescent assay)

Incidence of false negatives Incidence of false positives

among total positive test among total
Sensitivity rateb portions, %c negative test portions, %e

Total Total Culture Agreement,

Product Level samples positive VIDAS method c2a VIDAS Culture VIDAS Culture Specificity rated VIDAS %f

Milk chocolate Controlg 78 0 0 0 —h — — — — 100 0 100

Low 78 39 38 39 0.00 97 100 2.6 0.0 100 0 99
High 78 51 51 51 — 100 100 0.0 0.0 100 0 100
Dried whole egg Control 84 0 0 0 — — — — — 100 0 100
Low 84 16 16 16 — 100 100 0.0 0.0 100 0 100
High 84 66 65 66 0.00 98 100 1.5 0.0 100 0 99
Nonfat dry milk Control 60 0 0 0 — — — — — 100 0 100
Low 60 26 26 26 — 100 100 0.0 0.0 100 0 100
High 60 54 54 54 — 100 100 0.0 0.0 100 0 100
Soy flour Control 60 0 0 0 — — — — — 98 2 100
Low 60 20 19 19 0.50 95 95 5.0 5.0 100 0 97
High 60 52 51 49 0.25 98 94 1.9 5.8 86 14 93
Raw turkey Control 90 3 3 2 0.00 — — — — 99 1 99
Low 90 32 30 29 0.00 94 91 6.3 9.4 83 17 94
High 90 84 83 83 0.50 99 99 1.2 1.2 83 17 98
Raw pork Control 66 0 0 0 — — — — — 100 0 100
Low 66 66 66 66 — 100 100 0.0 0.0 100 0 100
High 66 66 66 66 — 100 100 0.0 0.0 100 0 100
Raw peeled shrimp Control 78 0 0 0 — — — — — 100 0 100
Low 78 17 17 17 — 100 100 0.0 0.0 98 2 100
High 78 73 73 73 — 100 100 0.0 0.0 100 0 100
Lactic casein Control 72 0 0 0 — — — — — 97 3 100
Low 72 58 54 58 2.25 93 100 6.9 0.0 100 0 94
High 72 67 65 67 0.50 97 100 3.0 0.0 100 0 97
a
c2 is defined by McNemar as (|a – b| – 1)2/(a + b) where a = test portions positive by VIDAS and negative by culture method, and b = test portions negative by VIDAS and positive by culture method. A c2 value
greater than 3.84 indicates significance at p > 0.05.
b
Sensitivity rate is defined as 100 times the total number of analyzed positive test portions among “known” positive test portions divided by total number of “known” test portions, where “known” positive is defined
as test portions confirmed positive by the reference method.
c
Incidence of false negative is the number of misclassified known positives divided by the total number of positive test portions obtained with the method.
d
Specificity rate is defined as 100 times the total number of analyzed negative test portions among “known” negative test portions divided by the total number of “known” negative test portions, where “known”
negative is defined as samples confirmed negative by the reference method and negative controls.
e
Incidence of false positive is the number of misclassified known negatives divided by the total test portions obtained with the method.
f
Rate reflects the number of confirmed determinations that were equivalent between the VIDAS and culture methods.
g
Uninoculated control samples are by definition known negatives, sensitivity rates not calculated.
h
— = Statistical analysis not applicable.

© 2007 AOAC INTERNATIONAL

Table 2004.03B. Reagents included in 10-well reagent strip
Wells Reagents (SLM)
1 Sample well: 0.5 mL boiled enrichment
broth is placed into the well
2 Prewash solution: Tris-buffered saline/Tween 20
with 1 g/L sodium azide
3–5, 7–9 Wash solution (0.6 mL): Tris-buffered saline/Tween
20 with 1 g/L sodium azide
6 Conjugate (0.4 mL): alkaline phosphatase labeled
polyclonal antibodies with 1 g/L sodium azide
10 Substrate (0.3 mL): 4-methyl umbelliferyl
phosphate with 1 g/L sodium azide
F. Enzyme Immunoassay
(1) Label desired number of SLM reagent strips, 1 per test
sample. For each series of test samples, label SLM strips and as
needed for positive (C1), negative (C2), and standard (S1) control
solutions provided with the test kit. Allow strips to come to room
temperature.
(2) Mix positive, negative, and standard solutions.
(3) Pipet 0.5 mL standard, controls, and heated M-broth into each
SLM strip.
(4) Enter the appropriate assay information to create a work list.
Type “SLM” to enter the assay code, and number of tests to be run.
Type “S” for standard and “Pos" and “Neg” for positive and negative
control samples, respectively.
(5) Load the SLM reagent strips and the SPRs into the positions
that correspond to the VIDAS section indicated by the work list.
(6) Initiate the assay processing as directed in the VIDAS
operator’s manual.
Table 2004.03C. Interpretation of test
Test value threshold Interpretation
<0.23 Negative
³0.23 Positive
G. Assay Results
The results are analyzed automatically by the computer. A report
is printed which records the type of test performed, the test sample
identification, the date and time, the lot number and expiration date
of the re agent kit be ing used, and each sam ple’s rel a tive
fluorescence value (RFV), test value, and interpreted result. The
RFV is the final reading of the test sample minus the background
reading, and the test value is the ratio of RFV and the standard.
Results are interpreted after the test values and control samples are
compared to thresholds stored in the computer (Table 2004.03C).
A positive result must be confirmed by using enrichment and
post-enrichment broths according to standard cultural procedures.
Typical or suspect colonies from each plate are confirmed as in
967.26C (see 17.9.02), 967.27 (see 17.9.03), and 967.28 (see
17.9.07). As an alternative to conventional tube sys tem for
Salmonella, any AOAC-approved commercial biochemical kits
may be used for presumptive generic identification of foodborne
Salmonella as in 978.24 (see 17.9.04), 989.12 (see 17.9.05), and
991.13 (see 17.9.06).
Reference: J. AOAC Int. 87, 867(2004).
© 2007 AOAC INTERNATIONAL