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rowley
et al
.
:
J
ournal of
AOAC I
nternational
V
ol
. 97, N
o
. 2, 2014
F. Results and Interpretation
The results are analyzed automatically by the VIDAS system.
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 reagent kit being used, and 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. The first reading is a background
reading of the substrate cuvette before the SPR is introduced
into the substrate. The second reading is taken after incubating
the substrate with the enzyme remaining on the interior of the
SPR. The test value is calculated by the instrument and is equal
to the difference between the background reading and the final
reading. The calculation appears on the result sheet. A “negative”
result has a test value less than the threshold (0.05) and indicates
that the sample does not contain
L. monocytogenes
or contains
L. monocytogenes
at a concentration below the detection limit.
A “positive” result has a test value equal to or greater than
the threshold (≥0.05) and indicates that the sample may be
contaminated with
L. monocytogenes.
If the background reading
is above a predetermined cutoff, then the result is reported as
invalid (Table
2013.11D
).
G. Confirmation
All positive VIDAS LMX results must be culturally
confirmed. Confirmation should be performed using the non-
heated enrichment broth (the LMX primary enrichment broth
for 25 g test portions and the LPT secondary enrichment broth
for 125 g test portions) stored between 2–8°C, and should be
initiated within 72 h following the end of incubation (AFNOR
Certificate No. BIO 12/33-05/12). Presumptive positive results
may be confirmed by isolating on selective agar plates such as
ALOA or on the appropriate reference method selective agar
plates. Typical or suspect colonies from each plate are confirmed
as described in appropriate reference method. As an alternative to
the conventional confirmation for
L. monocytogenes,
VITEK 2
GP Biochemical Identification (AOAC
2012.02
) or API
Listeria
biochemical kits may be used for presumptive generic
identification of foodborne
L. monocytogenes
.
Results of Collaborative Study
In this collaborative study, the VIDAS
Listeria monocytogenes
(LMX) method was compared to the to AOAC
993.12
for one
food product, queso fresco, at two test portion sizes, 25 and
125 g. A total of 14 laboratories throughout the United States
participated in this study, with 14 laboratories submitting data
for the 25 g test portions and 13 laboratories submitting data for
the 125 g test portions as presented in Table 1. Each laboratory
analyzed 36 test portions for each method 12 inoculated with a
high level of
L. monocytogenes,
12 inoculated with a low level of
L. monocytogenes,
and 12 uninoculated controls. A background
screen of the matrix indicated an absence of indigenous
Listeria
species. As per criteria outlined in Appendix J, fractional positive
results were obtained for both the 25 and 125 g test portions sizes.
For each test portion size, the actual level of
L. monocytogenes
was determined by MPN determination on the day of initiation
of analysis. The results of the inoculum heat-stress protocol
are presented in Table 2. The individual laboratory and sample
results are presented in Tables 3 and 4. Tables
2013.11A
and
B
summarize the collaborative study results for all foods tested,
including POD statistical analysis (10). Detailed results for each
laboratory are presented in Tables 2A–D, and Figures 1A–D and
2A–D as supplemental data on the
J. AOAC Int.
website.
Queso Fresco (25 g Test Portions)
Queso fresco test portions were inoculated at a low and
high level and were analyzed (Table 3) for the detection of
L. monocytogenes
. Uninoculated controls were included in each
analysis. Fourteen laboratories participated in the analysis of
this matrix and the results of 13 laboratories were included in
the statistical analysis. Laboratory 11 reported 11 test portions
(including four uninoculated test portions) that produced
non-
L. monocytogenes
profiles. Colonies on these plates
contained one or more of the following biochemical reactions
not typically associated with
L. monocytogenes
: Gram-negative,
Gram-positive with spores, non-beta-hemolytic, and catalase
negative. Based on the preliminary biochemical tests conducted,
the test portions should not have been carried through for
final biochemical identification on API
Listeria
strips which
resulted in the misidentification of the test portion as
Listeria
spp. The selective agar plates for these test portions were sent
to the coordinating laboratory for further examination. The
coordinating laboratory confirmed the supplementary results
(Gram stain, hemolysis, and catalase reaction) reported by
Table 2013.11D. Interpretation of test
Test value threshold
Interpretation
<0.05
Negative
≥0.05
Positive
Table 1. Participation of each collaborating laboratory
a
Queso fresco
Lab
25 g test portions
125 g test portions
1
Y
Y
2
Y
b
Y
3
Y
Y
4
Y
Y
5
Y
Y
6
Y
Y
7
Y
Y
8
Y
Y
9
Y
Y
10
Y
Y
11
Y
c
Y
c
12
Y
Y
13
Y
Y
14
Y
Y
a
Y= Collaborator analyzed the food type.
b
Results were not submitted to the coordinating laboratory.
c
Results were not used in statistical analysis due to laboratory error.
Candidates for 2016 Method of the Year
349