AOAC Official Method 2013.01 (g)  Master lot entry (MLE) card.

—One card providing

Salmonella in a Variety of Foods specifications for the factory master data required to calibrate the
VIDAS® UP Salmonella (SPT) Method test.
First Action 2013 (h)  Package insert.
(i)  Disposable pipet to dispense appropriate volumes.
[Applicable to detection of Salmonella in raw ground beef (25 (j)  VIDAS Heat and Go.—Available from bioMérieux, Inc.
and 375 g), processed American cheese (25 g), deli roast beef (k)  Water bath (95–100°C) or equivalent system.
(25  g), liquid egg (25 g), peanut butter (25 g), vanilla ice cream (l)  Stomacher®-type bag with filter.
(25 g), cooked shrimp (25 g), raw cod (25 g), bagged lettuce (25 (m)  Stomacher.—Stomacher Lab Blender 400, available from
and 375 g), dark chocolate (375 g), powdered eggs (25 g), instant Seward Medical (London, UK); Smasher, bioMérieux, Inc., or
nonfat dry milk (25 and 375 g), ground black pepper (25 g), dry dog equivalent.
food (375 g), raw ground turkey (375 g), almonds (375 g), chicken (n)  BPW.—Available from bioMérieux, Inc.
carcass rinsates (30 mL), and stainless steel, plastic, and ceramic (o)  Salmonella supplement.—Available from bioMérieux, Inc.
environmental surfaces.] (p)  Incubators.—Capable of maintaining 42 ± 1°C and 35 ± 1°C.
See Tables 2013.01A and B for a summary of results of the (q)  Diagnostic reagents.—Necessary for culture confirmation of
interlaboratory study. For detailed results of the interlaboratory assays. See 967.27 (see 17.9.03).
study, see Tables A–F in Appendix  1 on J. AOAC Int. website, (r)  IBISA chromogenic agar.—Necessary for cultural
http://aoac.publisher.ingentaconnect.com/content/aoac/jaoac). confirmation as an alternative to selective agar required by

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A.  Principle appropriate reference method. Available from bioMérieux, Inc.

nl
(s)  ASAP chromogenic agar.—Necessary for cultural
The VIDAS SPT method is for use on the automated VIDAS confirmation as an alternative to selective agar required by

O
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instrument for the detection of Salmonella receptors using the appropriate reference method. Available from bioMérieux, Inc.
enzyme-linked fluorescent assay. The solid-phase receptacle (SPR) (t)  Vancomycin.—Available from bioMérieux, Inc.

ne Ins
serves as the solid phase, as well as the pipetting device. The
C.  General Instructions
interior of the SPR is coated with proteins specific for Salmonella
receptors. Reagents for the assay are ready-to-use and predispensed (a)  Components of the kit are intended for use as integral unit.
Pa h
in the sealed reagent strips. The instrument performs all the assay Do not mix reagents or disposables of different lot numbers.
w rc

steps automatically. The reaction medium is cycled in and out of the (b)  Store VIDAS SPT kits at 2–8°C.
SPR several times. An aliquot of enrichment broth is dispensed into (c)  Do not freeze reagents.
ie ea

the reagent strip. The Salmonella receptors present will bind to the (d)  Bring reagents to room temperature before inserting them
interior of the SPR. Unbound components are eliminated during the into the VIDAS instrument.
ev es

washing steps. The proteins conjugated to the alkaline phosphatase (e)  Mix standard, controls, and heated test portions well before
are cycled in and out of the SPR and will bind to any Salmonella using.
R R

receptors, which are themselves bound to the SPR wall. A final (f)  Include one positive and one negative control with each
wash step removes unbound conjugate. During the final detection group of tests.
rt C

step, the substrate (4-methylumbelliferyl phosphate) is cycled in (g)  Return unused components to 2–8°C immediately after use.
and out of the SPR. The conjugate enzyme catalyzes the hydrolysis (h)  See safety precautions in the VIDAS SPT package insert
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of the substrate into a fluorescent product (4-methylumbelliferone), (refer to the following sections in the package insert: Warnings and
the fluorescence of which is measured at 450 nm. At the end of the Precautions and Waste Disposal).
Ex A

assay, results are automatically analyzed by the instrument which D.  Preparation of Test Suspension
calculates a test value for each sample. This value is then compared
to internal references (thresholds) and each result is interpreted as (a)  Pre-enrichment.—Pre-enrich test portion in BPW using
positive or negative. filter Stomacher bags to initiate growth of Salmonella. For 25 g test
portions, add 225 mL BPW to each test portion and homogenize
B.  Apparatus and Reagents
thoroughly for 2 min. For 375 g test portions, prewarm BPW to
Items (a)–(h) are available as the VIDAS SPT assay kit from 42 ± 1°C, add 1125 mL to each test portion, and homogenize
bioMérieux Inc., Hazelwood, MO. thoroughly for 2 min.
(a)  VIDAS or miniVIDAS automated immunoassay system. (b)  After homogenization add Salmonella supplement to
(b) SPT reagent strips.—60 polypropylene strips of 10  wells, each test portion. For 25 g test portions, add 1 mL of Salmonella
each strip covered with a foil seal and label. The 10 wells contain supplement, mix samples manually, and incubate for 18–24  h
the reagents in Table 2013.01C. at 42 ± 1°C. For 375 g test portions, add 5 mL of Salmonella
(c)  SPR.—60 SPRs coated with proteins specific for Salmonella supplement, mix samples manually, and incubate for 22–26  h at
receptors. 42 ± 1°C.
(d)  Standard.—One vial (6 mL). Contains purified and (c)  After incubation, homogenize samples manually. If a water
inactivated Salmonella receptors + preservative + protein stabilizer. bath is used, transfer 2–3 mL enrichment broth into a tube. Seal the
(e)  Positive control solution.—One vial (6 mL). Contains tube. Heat for 5 ± 1 min at 95–100°C. Cool the tube. Mix the boiled
purified and inactivated Salmonella receptors + preservative + broth and transfer 0.5 mL into the sample well of the VIDAS SPT
protein stabilizer. reagent strip. If the VIDAS Heat and Go is used, transfer 0.5 mL
(f)  Negative control solution.—One vial (6 mL). Contains Tris- of the enrichment broth into the sample well of the VIDAS SPT
buffered saline (150 mmol/L)–Tween pH 7.6 + preservative. reagent strip. Heat for 5 ± 1 min (see VIDAS Heat and Go User’s

© 2013 AOAC INTERNATIONAL

 Table  2013.01A.  Summary of results for the detection of Salmonella spp. in raw ground beef (25 g)
VIDAS SPT with alternative confirmation on IBISA and
Methoda VIDAS SPT with traditional confirmation on BGSA and XLT4 VIDAS SPT with traditional confirmation on IBISA and ASAPb ASAPc

Inoculation level Uninoculated Low High Uninoculated Low High Uninoculated Low High

Candidate presumptive positive/total samples analyzed 0/144 144/144 144/144 0/144 144/144 144/144 0/144 144/144 144/144

Candidate presumptive POD (CP) 0.00 1.00 1.00 0.00 1.00 1.00 0.00 1.00 1.00
(0.00, +0.03) (+0.97, +1.00) (+0.97, +1.00) (0.00, +0.03) (+0.97, +1.00) (+0.97, +1.00) (0.00, +0.03) (+0.97, +1.00) (+0.97, +1.00)

srd 0.00 0.00 0.00 0.00 0.00 (0.00, +0.16) 0.00 0.00 0.00 0.00
(0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16)

sLe 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
(0.00 +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16) (0.00, +0.16)

sRf 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
(0.00, +0.22) (0.00, +0.22) (0.00, +0.22) (0.00, +0.22) (0.00, +0.22) (0.00, +0.22) (0.00, +0.22) (0.00, +0.22) (0.00, +0.22)
Ex A
P-value 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000 1.0000

Candidate confirmed positive/total samples analyzed 0/144 143/144 144/144 0/144 143/144 144/144 0/144 143/144 144/144
pe OA
Candidate confirmed POD (CC) 0.00 0.99 1.00 0.00 0.99 1.00 0.00 0.99 1.00
(0.00, +0.03) (+0.96, +1.00) (+0.97, +1.00) (0.00, +0.03) (+0.96, +1.00) (+0.97, +1.00) (0.00, +0.03) (+0.96, +1.00) (+0.97, +1.00)
rt C
sr 0.00 0.08 0.00 0.00 0.08 0.00 0.00 0.08 0.00
R R
(0.00, +0.16) (+0.07, +0.16) (0.00, +0.16) (0.00, +0.16) (+0.07, +0.16) (0.00, +0.16) (0.00, +0.16) (+0.07, +0.16) (0.00, +0.16)

sL 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

(0.00, +0.16) (0.00, +0.03) (0.00, +0.16) (0.00, +0.16) (0.00, +0.03) (0.00, +0.16) (0.00, +0.16) (0.00, +0.03) (0.00, +0.16)
ev es
sR 0.00 0.08 0.00 0.00 0.08 0.00 0.00 0.08 0.00
(0.00, +0.22) (+0.08, +0.10) (0.00, +0.22) (0.00, +0.22) (+0.08, +0.10) (0.00, +0.22) (0.00, +0.22) (+0.08, +0.10) (0.00, +0.22)
ie ea
P-value 1.0000 0.4368 1.0000 1.0000 0.4368 1.0000 1.0000 0.4368 1.0000

Positive reference samples/total samples analyzed 0/144 84/144 138/144 0/144 84/144 138/144 0/144 84/144 138/144
w rc
Reference POD 0.00 0.58 0.96 0.00 0.58 0.96 0.00 0.58 0.96
(0.00, +0.03) (+0.50, +0.67) (+0.91, +0.98) (0.00, +0.03) (+0.50, +0.67) (+0.91, +0.98) (0.00, +0.03) (+0.50, +0.67) (+0.91, +0.98)
Pa h
sr 0.00 0.50 0.19 0.00 0.50 0.19 0.00 0.50 0.19
(0.00, +0.16) (+0.45, +0.52) (+0.17, +0.22) (0.00, +0.16) (+0.45, +0.52) (+0.17, +0.22) (0.00, +0.16) (+0.45, +0.52) (+0.17, +0.22)

sL 0.00 0.00 0.06 0.00 0.00 0.06 0.00 0.00 0.06

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(0.00, +0.16) (0.00, +0.18) (+0.02, +0.13) (0.00, +0.16) (0.00, +0.18) (+0.02, +0.13) (0.00, +0.16) (0.00, +0.18) (+0.02, +0.13)

sR 0.00 0.50 0.20 0.00 0.50 0.20 0.00 0.50 (+0.45, +0.52) 0.20
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(0.00, +0.22) (+0.45, +0.52) (+0.18, +0.24) (0.00, +0.22) (+0.45, +0.52) (+0.18, +0.24) (0.00, +0.22) (+0.18, +0.24)

P-value 1.0000 0.6298 0.0179 1.0000 0.6298 0.0179 1.0000 0.6298 0.0179

dLPOD (candidate vs reference) 0.00 0.41 0.04 0.00 0.41 0.04 0.00 0.41 0.04
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(–0.03, +0.03) (+0.32, +0.49) (0.01, +0.09) (–0.03, +0.03) (+0.32, +0.49) (+0.01, +0.09) (–0.03, +0.03) (+0.32, +0.49) (+0.01, +0.09)

dLPOD (candidate presumptive vs candidate confirmed) 0.00 0.01 0.00 0.00 0.01 0.00 0.00 0.01 0.00
(–0.03, +0.03)
O(–0.02, +0.04) (–0.03, +0.03) (–0.03, +0.03) (–0.02, +0.04) (–0.03, +0.03) (–0.03, +0.03) (–0.02, +0.04) (–0.03, +0.03)

a
 Results include 95% confidence intervals.
b
nl
 Traditional confirmation on ASAP/IBISA = secondary enrichments streaked onto IBISA and ASAP.
y
c
 Alternative confirmation = direct streak of the primary enrichment onto IBISA and ASAP.
d
 Repeatability standard deviation.
e
 Among-laboratory standard deviation.
f
 Reproducibility standard deviation.

© 2013 AOAC INTERNATIONAL

Table  2013.01B.  Summary of results for the detection of Salmonella spp. in raw ground beef (375 g)
VIDAS SPT with traditional confirmation on VIDAS SPT with traditional confirmation on VIDAS SPT with alternative confirmation on
Methoda BGSA and XLT4 IBISA and ASAPb IBISA and ASAPc

Inoculation level Uninoculated Low High Uninoculated Low High Uninoculated Low High

Candidate 0/132 58/131 130/132 0/132 58/131 130/132 0/132 57/131 130/132
presumptive
positive/total
samples
analyzed
Candidate 0.00 (0.00, 0.44 (+0.34, 0.98 (+0.95, 0.00 (0.00, 0.44 (+0.34, 0.98 (+0.95, 0.00 (0.00, 0.44 (+0.33, 0.98 (+0.965,
presumptive +0.03) +0.55) +1.00) +0.03) +0.55) +1.00) +0.03) +0.54) +1.00)
POD (CP)
srd 0.00 (0.00, 0.49 (+0.43, 0.12 (+0.11, 0.00 (0.00, 0.49 (+0.43, 0.12 (+0.11, 0.00 (0.00, 0.49 (+0.44. 0.12 (+0.11,
+0.16) +0.52) +0.16) +0.16) +0.52) +0.16) +0.16) +0.52) +0.16)
sLe 0.00 (0.00, 0.10 (0.00, 0.00 (0.00, 0.00 (0.00, 0.10 (0.00, 0.00 (0.00, 0.00 (0.00, 0.09 (0.00, 0.00 (0.00,
+0.16) +0.27) +0.05) +0.16) +0.27) +0.05) +0.16) +0.26) +0.05)
sRf 0.00 (0.00, 0.50 (+0.44, 0.12 (+0.11, 0.00 (0.00, 0.50 (+0.44, 0.12 (+0.11, 0.00 (0.00, 0.50 (+0.45, 0.12 (+0.11,
+0.23) +0.52) +0.14) +0.23) +0.52) +0.14) +0.23) +0.52) +0.14)

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P-value 1.0000 0.1551 0.5190 1.0000 0.1551 0.5190 1.0000 0.1906 0.5190

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Candidate 0/132 58/131 130/132 0/132 59/131 130/132 0/132 58/131 130/132

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confirmed
positive/total

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samples
analyzed
Candidate 0.00 (0.00, 0.44 (+0.34, 0.98 (+0.95, 0.00 (0.00, 0.45 (+0.35, 0.98 (+0.95, 0.00 (0.00, 0.44 (+0.34, 0.98 (+0.95,
confirmed POD +0.03) +0.55) +1.00) +0.03) +0.55) +1.00) +0.03) +0.55) +1.00)
Pa h
(CC)
w rc

sr 0.00 (0.00, 0.49 (+0.43, 0.12 (+0.11, 0.00 (0.00, 0.49 (+0.44, 0.12 (+0.11, 0.00 (0.00, 0.49 (+0.43, 0.12 (+0.11,
+0.16) +0.52) +0.16) +0.16) +0.52) +0.16) +0.16) +0.52) +0.16)
ie ea

sL 0.00 (0.00, 0.10 (0.00, 0.00 (0.00, 0.00 (0.00, 0.09 (0.00, 0.00 (0.00, 0.00 (0.00, 0.10 (0.00, 0.00 (0.00,
+0.16) +0.27) +0.05) +0.16) +0.25) +0.05) +0.16) +0.27) +0.05)
ev es

sR 0.00 (0.00, 0.50 (+0.45, 0.12 (0.11, 0.00 (0.00, 0.50 (+0.45, 0.12 (+0.11, 0.00 (0.00, 0.50 (+0.45, 0.12 (+0.11,
+0.23) +0.52) +0.14) +0.23) +0.52) +0.14) +0.23) +0.52) +0.14)
R R

P-value 1.0000 0.1551 0.5190 1.0000 0.2060 0.5190 1.0000 0.1551 0.5190
Positive 0/132 57/132 132/132 0/132 57/132 132/132 0/132 54/132 131/132
rt C

reference
samples/total
pe OA

samples
analyzed
Reference POD 0.00 (0.00, 0.43 (+0.35, 1.00 (+0.97, 0.00 (0.00, 0.43 (+0.35, 1.00 (+0.97, 0.00 (0.00, 0.41 (+0.32, 0.99 (+0.96,
Ex A

+0.03) +0.52) +1.00) +0.03) +0.52) +1.00) +0.03) +0.50) +1.00)

sr 0.00 (0.00, 0.50 (+0.45, 0.00 (0.00, 0.00 (0.00, 0.50 (+0.45, 0.00 (0.00, 0.00 (0.00, 0.49 (+0.44, 0.09 (+0.08,
+0.16) +0.52) +0.17) +0.16) +0.52) +0.17) +0.16) +0.52) +0.16)
sL 0.00 (0.00, 0.00 (0.00, 0.00 (0.00, 0.00 (0.00, 0.00 (0.00, 0.00 (0.00, 0.00 (0.00, 0.05 (0.00, 0.00 (0.00,
+0.16) +0.18) +0.17) +0.16) +0.18) +0.17) +0.16) +0.22) +0.04)
sR 0.00 (0.00, 0.50 (+0.45, 0.00 (0.00, 0.00 (0.00, 0.50 (+0.45, 0.00 (0.00, 0.00 (0.00, 0.49 (+0.44, 0.09 (+0.08,
+0.23) +0.52) +0.23) +0.23) +0.52) +0.23) +0.23) +0.52) +0.10)
P-value 1.0000 0.6261 1.0000 1.0000 0.6261 1.0000 1.0000 0.3313 0.4338
dLPOD (C vs R) 0.00 (–0.03, 0.01 (–0.12, –0.02 (–0.05, 0.00 (–0.03, 0.02 (–0.18, –0.02 (–0.05, 0.00 (–0.03, 0.03 (–0.18, –0.01 (–0.05,
+0.03) +0.15) +0.02) +0.03) +0.22) +0.02) +0.03) +0.24) +0.03)
dLPOD (CP vs 0.00 (–0.03, 0.00 (–0.15, 0.00 (–0.04, 0.00 (–0.03, –0.01 (–0.15, 0.00 (–0.04, 0.00 (–0.03, –0.01 (–0.21, 0.00 (–0.04,
CC) +0.03) +0.15) +0.04) +0.03) +0.14) +0.04) +0.03) +0.23) +0.04)
a
 Results include 95% confidence intervals.
b
 Traditional confirmation on ASAP/IBISA = secondary enrichments streaked onto IBISA and ASAP.
c
 Alternative confirmation = direct streak of the primary enrichment onto IBISA and ASAP.
d
 Repeatability standard deviation.
e
 Among-laboratory standard deviation.
 Reproducibility standard deviation.
f

© 2013 AOAC INTERNATIONAL

Table  2013.01C.  Reagents included in 10-well reagent strip Table  2013.01D.  Interpretation of test
Wells Reagents (SPT) Test value threshold Interpretation

1 Sample well: 0.5 mL of enrichment broth, standard or control <0.25 Negative

2 Prewash solution (400 µL): Buffer pH 7.8 + preservative ≥0.25 Positive
3–5, 7–9 Wash buffer (600 µL): TRIS-buffered saline (150 mmol/L) –
Tween pH 7.6 + preservative
6 Conjugate (400 µL): alkaline phosphatase-labeled proteins F.  Results and Interpretation
specific for Salmonella receptors + preservative
10 Reading cuvette with substrate (300 µL): 4-methyl-umbelliferyl
The results are analyzed automatically by the VIDAS system.
phosphate (0.6 mmol/L) + diethanolaminea A report is printed which records the type of test performed, test
(DEA; 0.62 mol/L or 6.6%, pH 9.2) + preservative sample identification, date and time, lot number, and expiration date
a
 Irritant reagent; see VIDAS SPT package insert for more information.
of the reagent kit being used, each sample’s RFV, test value, and
interpreted result (positive or negative). Fluorescence is measured
twice in the reagent strip’s reading cuvette for each sample tested.
Manual). Remove the strip and allow to cool for 10 min prior to test The first reading is a background reading of the substrate cuvette
initiation. Perform the VIDAS test. before the SPR is introduced into the substrate. The second reading
is taken after incubating the substrate with the enzyme remaining
E.  Enzyme Immunoassay on the interior of the SPR. The test value is calculated by the

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instrument and is equal to the difference between the background

nl
(a)  Enter factory master calibration curve data into the
reading and the final reading. The calculation appears on the result
instrument using the MLE card.

O
sheet. A negative result has a test value less than the threshold

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(b)  Remove the kit reagents and materials from refrigerated
(0.25) and indicates that the sample does not contain Salmonella

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storage and allow them to come to room temperature. spp. or contains Salmonella spp. at a concentration below the
(c)  Use one VIDAS SPT reagent strip and one VIDAS SPT SPR detection limit. A positive result has a test value equal to or greater
for each sample, control, or standard to be tested. Reseal the storage than the threshold (≥0.25) and indicates that the sample may be
Pa h
pouch after removing the required number of SPRs. contaminated with Salmonella spp. If the background reading is
above a predetermined cutoff, then the result is reported as invalid
w rc

(d)  Enter the appropriate assay information to create a work list.

Enter the test code by typing or selecting “SPT,” and number of (Table 2012.01D).
ie ea

tests to be run. If the standard is to be tested, identify the standard G.  Confirmation
by “S1” and test in duplicate. If the positive control is to be tested, All positive VIDAS SPT results must be culturally confirmed.
ev es

identify it by “C1.” If the negative control is to be tested, identify Confirmation should be performed using the non-heated enrichment
it by “C2.” broth stored between 2 and 8°C, and should be initiated within 72
R R

Note: The standard must be tested upon receipt of a new lot of h after the end of incubation at 42 ± 1°C. Presumptive positive
reagents and then every 14 days. The relative fluorescence value results may be confirmed by isolating on selective agar plates
rt C

(RFV) of the standard must fall within the set range provided with such as IBISA or ASAP, or on the appropriate reference method
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the kit. selective agar plates. Typical or suspect colonies from each plate are
(e)  Load the SPT reagents strips and SPRs into the positions that confirmed as described in 967.27 (see 17.9.03). As an alternative to
the conventional tube system for Salmonella, any AOAC-approved
correspond to the VIDAS section indicated by the work list. Verify
Ex A

commercial biochemical kits may be used for presumptive generic

that the color labels with the assay code on the SPRs and reagent
identification of foodborne Salmonella as described in 978.24 (see
strips match. 17.9.04), 989.12 (see 17.9.05), 991.13 (see 17.9.06), and 2011.17
(f)  Initiate the assay processing as directed in the VIDAS (see 17.15.01).
operator’s manual.
Reference: J. AOAC Int. 96, 808(2013)
(g)  After the assay is completed, remove the SPRs and reagent DOI: 10.5740/jaoacint.CS2013_01
strips from the instrument and dispose of properly.

© 2013 AOAC INTERNATIONAL