541 / 1195
and the qualitative VIDAS LPTresults were compared to the reference method for statistical
1
analysis. Data for each test portion size was analyzed using the probability of detection (POD)
2
statistical model [5, 8]. A confidence interval of a dLPOD not containingthe point zero would
3
indicate a statistically significant difference between the VIDAS LPTmethod and the AOAC
4
993.12 reference method at the 5 % probability level [9].
5
6
7
AOAC Official Method 2013.xxx
8
Listeria
speciesin a Variety of Foods and Environmental Surfaces
9
VIDAS
®
UP
Listeria
(LPT) Method
10
First Action 2013
11
12
(Applicable to detection of
Listeria
in deli ham (25g& 125 g),pepperoni (25 g), beef hot dogs (25
13
g), chicken nuggets (25 g), chicken liver pate (25 gram), ground beef (125 g), deli turkey (125 g),
14
cooked shrimp (25 g), smoked salmon (25 g), cantaloupe melon, bagged mixed salad (25 g),
15
peanut butter (25 g), black pepper (25 g), vanilla ice cream (25 g), queso fresco (25 g &125 g),
16
stainless steel, plastic, ceramic and concrete environmental surfaces.)
17
See table
2013.1A-B
for a summary of results of the collaborative study. See table
18
2013.2A-D
in Appendix 1 for detailed results of the collaborative study
19
20
A.
Principle
21
22
VIDAS
®
UP
Listeria
(LPT) method is for use on the automated VIDASinstrument for the
23
detection of
Listeria
antigens using the ELFA method (Enzyme Linked Fluorescent Assay). The
24
assay also incorporates phage proteins allowing an increase in sensitivity and specificity
25
compared to traditional immunoassay. The Solid Phase Receptacle (SPR
®
) serves as the solid
26
phase as well as the pipetting device. The interior of the SPR is coated with proteins specific for
27
Listeria
receptors. Reagents for the assay are ready-to-use and pre-dispensed in the sealed
28
reagent strips. All of the assay steps are performed automatically by the instrument. The
29
reaction medium is cycled in and out of the SPR several times. An aliquot of enrichment broth is
30
dispensed into the reagent strip. The
Listeria
receptors present will bind to the interior of the
31
SPR. Unbound components are eliminated during the washing steps. The proteins conjugated to
32
the alkaline phosphatase are cycled in and out of the SPR and will bind to any
Listeria
receptors
33
which are themselves bound to the SPR wall. A final wash step removes unbound conjugate.
34
During the final detection step, the substrate (4-Methyl-umbelliferyl phosphate) is cycled in and
35
out of the SPR. The conjugate enzyme catalyzes the hydrolysis of the substrate into a
36
fluorescent product (4-Methyl-umbellliferone) the fluorescence of which is measured at 450 nm.
37
At the end of the assay, results are automatically analyzed by the instrument which calculates a
38
test value for each sample. This value is then compared to internal references (thresholds) and
39
each result is interpreted as positive or negative.
40
B.
Apparatus and Reagents
41
42
Items (a)-(h) are available as the VIDAS UP
Listeria
(LPT) assay kit from bioMérieux
43
(595 Anglum Road, Hazelwood, MO 63042-2330, USA).
44
5