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© 2014 AOAC INTERNATIONAL
C. Apparatus
(
a
)
Incubator/reader.—
Available from Neogen Corp. Incubator/
reader capable of operating at 56 ± 1°C and reading fluorescence in
real time in two channels (485/535 nm and 540/590 nm).
(
b
)
Computer and ANSR software.—
Available from Neogen
Corp. For connection to incubator/reader. Minimum requirements
for computer: Intel
®
Core i3 processor, 1 GB RAM, Windows
®
7,
Ethernet, and USB connections.
(
c
)
Heater block.—
With insert for 1.2 mL cluster tubes, 80 ± 2°C.
(
d
)
Micropipettor.—
50 µL, fixed or adjustable volume.
(
e
)
Pipettor.—
100–1000 µL, adjustable volume.
(
f
)
8-Channel micropipettor.—
20–200 µL, adjustable volume.
(
g
)
Pipet tips.—
100 µL, with filter.
(
h
)
Pipet tips.—
1000 µL.
(
i
)
Tubes.—
Glass or plastic, 12 × 75 mm or similar, sterile, with
caps.
(
j
)
Inoculating loops or needles.—
Sterile.
D. Preparation of Test Samples
Pick an isolated colony from nonselective or selective/differential
agar medium (one of the media listed in
B
) with an inoculating loop
or needle and resuspend (vortex or otherwise thoroughly mix) in
0.5 mL PBS in a sterile, capped tube.
E. Test Procedure
(
a
)
General preparation.—
(
1
) This assay should be performed in
a controlled laboratory environment.
(
2
) Do not use culture media or ANSR reagents beyond their
expiration dates. Do not interchange reagents betweenANSR kit lots.
(
3
) Remove ANSR reaction tubes from the foil pouch just before
use. Avoid prolonged exposure to light. Tap reaction tubes on bench
top to make sure that lyophilized reagents are at the bottom of the
tube prior to adding the lysed sample.
(
4
) Complete all assay steps in sequence, avoiding delays between
steps.
(
5
) Exercise care in pipetting steps to avoid cross-contamination
of samples.
(
6
) Do not remove caps from reaction tubes at any point after the
assay is started; this will prevent accidental contamination of the
environment with amplification products.
(
7
)
Prior to starting the assay.—
(
i
) Preheat the lysis heater block
to 80 ± 2°C. (
ii
) Start the ANSR software using the computer
connected to the ANSR reader. Select “
Salmonella
” as the test type.
Enter sample identifications and other experiment information. The
reader will preheat to 56 ± 1°C.
(
b
)
Assay procedure.—
(
1
) Add 50 µL of colony resuspension to a
1.2 mL cluster tube. Use a new pipet tip for each sample.
(
2
) Add 450 µL lysis buffer to the cluster tube.
Note
: It is not
necessary to use the lysis reagent provided with the test kit for this
application.
(
3
) Transfer the cluster tubes to the 80°C heater block and
incubate for 20 min.
Note:
The incubation time may be extended to
a maximum of 60 min for the purpose of managing staggered assay
start times.
(
4
) Approximately 3 min before the end of the lysis step, preheat
the ANSR reaction tubes to 56°C by placing the tubes in the
incubator/reader.
Note:
The strip of tubes may be cut to provide the
number of tubes needed.
(
5
) At the end of the 20 min lysis incubation, remove and discard
the caps from the reaction tubes.
Note
: Steps (
6
)–(
8
) should be completed without delay (within
1 min).
(
6
) Using an 8-channel micropipettor and 100 µL tips with filters,
carefully transfer 50 µL of the lysed samples to the reaction tubes.
Mix by rapidly pipetting up and down at least 10 times until the
sample appears homogenous in the pipet tip. Avoid excessive bubble
formation by not depressing the pipettor plunger beyond the first
stop.
(
7
) Place the permanent caps on the reaction tubes and close the
lid of the incubator/reader.
(
8
) Click START in the ANSR software to begin the assay.
(
9
) The assaywill complete in 10min and results will be displayed.
F. Interpretation of Results
The ANSR software will indicate the test results as POSITIVE,
NEGATIVE, or INVALID. Apositive result indicates that the colony
tested contains
Salmonella
spp. A negative result indicates that the
colony tested does not contain
Salmonella
spp. Assays producing
invalid results must be repeated. The real-time fluorescence curves
for both the test and positive control channel can be viewed using the
ANSR software.
G. Limitations
The assay detects serovars of both
S. enterica
and
S. bongori
,
including all genetic subgroups. In testing of 113 strains of
Salmonella
spp., representing 108 serovars, only a single strain of
S
. Weslaco was not detected.
References: (1) Van Ness, J., Van Ness, L.K., & Galas, D. (2003)
Proc. Natl. Acad. Sci. USA
100
, 4504–4509
J. AOAC Int . 97 , 829(2014)DOI: 10.5740/jaoacint.14-004
Posted: June 2014