1058 / 1195
832
Moz-or.^ [:r'AI-.t
JoijItNAt-
oF
AOAC--
lNI'UìN,{]'loNAl.
VoL.
97,
No.
3,2014
lì'om
various supplicrs.
Follorv
manufactr¡ret''s
instruclions for
pre
pirration.
(i)
'tfupric
sort
agar
(1'SA).-Available
fì'om
t"\eogen Cotp.
and
othel
suppliels. Irollort, manulàcturer's
instt'uctions
lbr'
preparalion.
C.
Apparatus
(a)
lncubaÍorit'e¿rrle¡:-
Âr,¿rilable
fiom
Neogen
Corp.
Incubatolireader
capablc
of
opelating
at
56
+
loC
and
reading
1ìuoresccnce
in
leal time
in
ttvo
channels
(485/535
nm
and
540/590 nm).
(b)
()ontpurer
und
ANSII
softvare.--Available fi'onl
Neogen
Corp.
l-or conncction to incubator/rcader'.
lvlinirnum
requit'ctnc'ttts
lbl
conrputcr:
lnte
ls
Core i3 proccssor.
I CB RAM.
Windorvse
7.
Dthernet,
¿ind
LJSB
connections.
(c) lleuter
óbck.-With
insert
for
1.2
mL. cluster
tubes.
80
+
2oC.
(d)
trlicropipettot',--50 pL,
1ìxed
or
adiustable volurrle.
(e)
Pipettot-.-I00-1000 pl,.
adjustable volume.
(f)
8
-
C
h
a
n n e
I
m
i
c
rop i
p
e
t Í
or.
-20¿00
¡rL,
ad.i
ustable volume.
(g)
Piper
rips.
I
00
pL, rvith
fìlter.
(h)
Piper tips,---1000 ¡rL.
(i)'l-ubes.
Glass
or
plastic,
l2
x
75
mm
or
sinlilar,
sterile.
rvith
caps.
(j)
lnoculating
loops
or
needles.
Sterile.
D.
Preparation
of
Test Sarnp/es
Pick
an
isolated
colony
l-rom nonselective
or
selectivc/
dilÌerential
agal
nrediunr
(one
of
the media listed
in
section B)
*'ith
an inoculating loop
or
needle
and
resuspend
(vorlex
or
other$'ise thoroughly
mix) in
0.5
ml.
PBS
in
a
steriìe,
capped
fube.
E.
i'esf Procedure
(a)
General
pt'eparation.-f
l)
This
assay shoulcl be per'l'ormed
in
a
controlìed
labot'atory
environnlent.
(2)
Do not
use
culture
nrediâ or
ANSR
reagents beyoncl
their'
expiration
dates.
Do
not interchange reagents between
ANSIì
kit
lots.
(3)
Renrove
ANSIì
reaction tubes
lrom
the
loil
pouch
.iust
before
r¡se.
Avoicl prolonged exposure
to light.
Tap reaction
tubes
on
bench
top
to
rlake
sure
that
l¡,ophilized
reagents
are at
the
botlom of
the tubc prior'1o adding
the
lysed
sample.
(l)
Conplete
all
assay
steps
in
sequence,
avoiding
delays
benveen
steps.
(5) Iixelcise
care
in
pipetting steps
to
avoid
cross-
conlamination of
santples.
(ó)
Do not
remove
caps
lront
reaction tubes at any
point after
the
assay
is started: this
rvill
prevent accidental
contamination
of
tlrc:
en vi
ronrnerrt
u'i
th
ampl
i
lìcation
prod ucts.
(7)
Prior
to sturÍing the ossov.'-(i)
Plcheat the
lysis
heater
block
to
80
*
2oC.
(ir)
Start
thcr
ANSR
solirvale using
the
conrputcl
connected to
the
ANSR
leadel'. Select "Sr¿lntoneIIct'as
thc
test
t!'pc.
lintcr
sarlpìe
identìfìcations
¿rnd
othel
experitnent
inlbrnration.
I-he re¿ider
rvill
pt'eheat
to
56
*
|oC.
(b)
Assalt
procedure.--{Ì) Add
50
pL
olcolony
resuspension
to
a
1.2
nrl- clusler
tube.
Use
¿r
neiv pipet
tip
f'or
e¿rch
sample.
(2)
Add
450 ¡rL lysis buffer to
the
cluster
tube.
No/e:
lt
is not
nccessaìy
to
use
the lysis
rcagent
provided
with
the
test
kit
lbr
this application.
(-i)
Translbr the cluster tubes
to
the
80'C
heater
block
and
incubatc
lbr
20
rrin.
À,o¡e.
'lhe
incubation
time
mal'be
exlencled
to
a
ma¡iirnunl
o1'60
min
fol
the purpose
of
managing
staggereti
assay starf
tirres.
(l)
Approximately
3
min
beibre
the end
of
the l¡'sis
stcp.
preheal the
ANSR
reaclion tubcs
to 56'C
by
placing the
tttbes
in
the
incubator/reader. .\¡o¡e. 'l-he
strip
o1
tubes may
be
cul
to
provide
ther
number
oi'n¡bes
necdcci.
(i)
At
the end
of
the 20
min
lysis
incubation, remove
and
discard the
caps
front
the reaclion
trrbes.
Nole:
Steps
(óf{8)
should
be
cornpleted
without dela¡'(rvithin
I
rnin).
(ó)
Using an 8-channel
micropipettor ancl 100
¡rL tips
rvith
fìlters.
carelilly
transfèr
50
¡rL
ol
the
lysed
saniples
to
the
reaotion
tubes.
Mix
by rapidly pipetting up
and
down at
least
l0
times
until
the
sample appears hotnogenous
in
the pipet
tip.
Avoid
excessive bubble
formation by
not depressing the
pipettor
plunger beyond the
first
stop.
(Z)
Place the permanent caps
on the leaction
tubes and close
the
lid
ofthe
incubator/reader.
(S) Click
Sl
AIìT
in
the
ANSR soiìware
to begin the
assay
(9)
The
assav
rviii
cotnplete
in i0
lnin
ancl
results
rvill
be
displayed.
F.
lnterpretation
of
Results
The
ANSR
softrvarc
rvill
indicate
the test
resulls
as
POSITIVE.
NEC,{l'lvll,
or
INVALID.
A
positive result
indicates
that
the
colon¡, tested contains Salntonella spp.
A
negative
result
indicates that the
colony
tested does
nof
conTain
Salntonella
spp.
Assays
producing invalid
results must
be repeated.
The real-time
fluorescence curves
fot'both
the
test
and
positive
contl'ol channel
can be
vierved using
the
ANSR softwale.
G.
Limitations
'l'he
assay
detects serovars
ol
both
S.
enlerica
and S.
bottgori.
including
all
genetic subgroups.
In
testing
of
ll3
strains
ol'
Salmonella
spp.,
representing lOfl
serovars,
onlv
a
single
strain
of'S.
Weslaco was not
detected.
Results
A
surnmary
of
results
lor
inclusive
and exclusive
isolates
is
shorvn
in'lables
2013.144
and
B, respectively. Detailed
results.
by collaborating
laboratorl', arc shorvn
in'Iables
2
and
3.
Iror
inclusive
strains,
all
collaborators
repor-ted
that
all
six
strains
grcw on
all
mcdia and
a total
ol
756
ANSIì
analvses
rvere
perfolrrred.
There
rvere
755 positive
results,
for
accuract' ol'
99.9Yo
nt identifi
calion
of
plesurnptive
Sa
I
nt
one
I
I
a spp.
colonies.
I-abolator,v 2
reported
a
negative result for,S. Enteritidis
on
I-[ij
agar'.'fhele
is no
obvious explanation
lor
this
result.
Thele
rvere
a
r.n¿Ltill-tull'l
of
756 possible resulls on
excltlsive
stlains. l-here
rvere
65
ca-ses
o1'reported
no
gro"vlh
or
lack
ol
distinct
isolated
colonies.
A
detailed analvsis
of
results
shorved
that
three
collaborators (laboratories
3.
12,
and
l3)
reported
no
grorvth
for
lhe
EnÍerobacter
clr¡ttccte
cultut'e on
¿ill seven
media,
accounting
lor
2l
ol'the
no-grorvth results.
Most
collaborators
reporled
that
neither
Providencia ulcalifaciens
nor
Ptoleus