358 / 363
832
M
ozola
et al
.
:
J
ournal of
AOAC I
nternational
V
ol
. 97, N
o
. 3, 2014
from various suppliers. Follow manufacturer’s instructions for
preparation.
(
i
)
Tryptic soy agar (TSA).—
Available from Neogen Corp.
and other suppliers. Follow manufacturer’s instructions for
preparation.
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 section
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 between ANSR 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 assay will complete in 10 min and results will be
displayed.
F. Interpretation of Results
TheANSR software will indicate the test results as POSITIVE,
NEGATIVE, or INVALID. A positive 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.
Results
A summary of results for inclusive and exclusive isolates is
shown in Tables
2013.14A
and
B
, respectively. Detailed results,
by collaborating laboratory, are shown in Tables 2 and 3. For
inclusive strains, all collaborators reported that all six strains
grew on all media and a total of 756 ANSR analyses were
performed. There were 755 positive results, for accuracy of
99.9% in identification of presumptive
Salmonella
spp. colonies.
Laboratory 2 reported a negative result for
S
. Enteritidis on HE
agar. There is no obvious explanation for this result.
There were a maximum of 756 possible results on exclusive
strains. There were 65 cases of reported no growth or lack of
distinct isolated colonies. A detailed analysis of results showed
that three collaborators (laboratories 3, 12, and 13) reported no
growth for the
Enterobacter cloacae
culture on all seven media,
accounting for 21 of the no-growth results. Most collaborators
reported that neither
Providencia alcalifaciens
nor
Proteus
Candidates for 2016 Method of the Year
357