1057 / 1195
MozoLA
E1'
Ar-.:
JOURNAL
oF
AOAC
lNl'ERNAlloNAt-
Vol..
97.
No.
3.
20
l4
831
84. Completed Data
Iìecording
Forms were retulned to the Study
[)irector
by
ol
l̡x.
ANSIì
assay
law
data
were provided
to
the
Srud),
Director by email
as
..json
fìles,
This
rarv data included
the real-time llrrorescence curves
lor
each assay peltbr-rned.
AOAC
Official
Method
2013.14
ldentification
of Sa/monel/a spp.
from Colony
Picks
ANSR@
Sal
mon
el
la
Confirmation
Test
First
Action
2013
(Applicable
to
the
identilìcalion
of
Salmonella
spp.
fì'om
colony picks fì'orn
selective/difTerential
agar media:
Bismuth
sulfìte
agar'.
blilliant
gleen sirlfa
agar, double-nrodified
lysine
ilon
agar.
llektoen
enteric
agar',
tryplic
soy
agar.
xylose
l¡,sine
deoxvcholate
agar'.
and
xylose
lysine
tergitol
agat',)
,5Þe
'l'ables
2013,14Ä and
B
lbr
a summary
of
lesults
of
the
collabolative
stud),,
Safety precautir¡ns.-^
[Jse ol'
this test
should be restricted
to
individuals
u'ith
appropriate laboratory
training
in microbiology
and
molccular
techniques. Reagents are
for
laborator)'use
only.
Iìel'er to the Materiaì Safety Data
Sheet
fronr
Neogen Clorp.
fbr'
more
inf'onnaiion.
Ilnrichmeni
cultures,
usecl
agar plates,
and
ANSR
assay l),sates
and reaction tubes shouÌd be handled
and
disposed
of
as
potentially infèctious r¡ateli¿il
and
Biosafèty
l-evel
2
measures
enrployed.
The preferled method
f'or disposal
of
contaminated materials. including cultures, pipet tips,
tubes.
e1c..
is autoclaving. Iterns that cannot be autoclaved should
be
dccontaminated
by
tleatment
rvith
disinlÞctant solution. ANSR
reaction tubes should
not
be
autoclaved
in
aleas rvhere they may
open and
possibly
contar-ninate
the laboratory environment rvith
anrplilìcation
products. Alternative
ly, lhey
nay
be
dìsposed
of
in
a sealed
conlainer
rvith
a
srnall
anlount
of
l0%
household bleach
added.
A.
Principle
ANSR Sa/nonella is
an
isothelnlal nucleic
acid
amplification
assay
based
on
the
nicking
enzyme amplification
t'eaction
(NEAR)
technology (5). The anrplifìcalion
mechanism
involvcs
binding
of
an
oligonucleotide "templatc"
to
a
specific
sequence
o1-
target
DNA.
'ì'he
te
mplatc contains
a
recognition
site
fbl
a
specific
enclonuclease.'l'he
nicked
strand
is
recognizerJ
as
danraged
and repaired by the action
o1'a therrnostable
DNA
polymerase,
displacing
the
original
sh'and
with
the
nervh,-synthesized repaired
portion.
'T'his
displaced
DNA
"ploduct"
then binds
to
a
second
te
mplate and the
same
reactions
lead
to
fornlation
of
a
second
product.
Amplifìcation
products
are
detected
usir.rga
specilìc
nrolecular
beacon
probe.
Fluorescent
signal is
generated
in
real
tinle. rvifh
amplification
and
delection
cornplete
rvithin
l0
min,
'l'hc entire
assay is oonducted at
a
constant tempcratr¡re
of
56uC
using
a
temperatule-conlrolled
fl
uorescence
defection instrument.
Assal,
sollu,are
analyzes the fluorescent
signal over tirne;
a data
intcrpretalion
algorithm
intelprets
lcsults
as negative.
positivc',
ol
invalid
based
on
baselìne.
r¿ìte-of--change, ancl
othct'critelia.
Ilach
nbe oIANSR
reagents
also contains an internal positive
control. signaling
in
a
sccond
1ìuol'escence
channel inespective
ol'the
prcsence
of
targel
Dr'r--A,
ancl
inciicating proper
linctioning
o1-
the
arnpl
ilìcation
reagents.
Table
2013.14A,
Interlaboratory study results for
the
ANSR Sa/monel/a
test: lnclusive
isolates
Organism
Correct
M¡s¡dentif¡ed
Total
Salmonella enlerlca subsp. artzonae
Salmonella enter¡ca
subsp
enterica
Ser.
Typhimurium
Sal
mon ella
erlerlca
subsp. enlerlca
Ser
Cubana
Salmonella bongori
Salmon
ella enlerlca subsp.
ente,ca
Ser. Cerro
Salmonella enteríca subsp. enterica
Ser. Enterit¡dis
Total isolates
B.
Media
and
Reagents
(a)
¡lÀ¡SR@/or
SaÌntonella
test
k¡t.-'Available
liom
Neogen
Corp.,
Cat.
No.
9843 (Lansing,
Ml,
rvrvw.neogen.com). Contains:
l-yophilized
reagents
in
capped
strip
trrbes,
eight
tubes
pel
strip.
l2
strips
(96
tests)
per
kit. in
trvo
sealed
fbil
pouches
with
desiccant packs;
clustel
tubes.
eighl
tubes per
strip,
l2
strips
per
kitl
permanent caps.
eight
caps
per
strip,
l2
strips per
kit:
lysis
bul'ler; one
bottle, 60
rnl-;
l.vsis
reagenl, three
vials. l¡'ophilized:
kit
insert. Store leagenl tubes at
2-8oC,
in
sealed
lbil
pouches
rvith
desiccant. Store
l¡'5i5
bull-er
at
2-8'Cì.
(b)
Phos¡shate-buJþred
saline
(PBS).-Per liter:
8.0
g
NaCl,
0.2 g
KCl,
L44
g
Na2l-lPOa,
0.2'l
g
KH2PO4.
(c)
Hektoen
enferic
agar
(L{E).-Available fi'om
Neogen
Corp. and
othel
suppliers.
Irollotv manufacturer's
instructions
lor
preparation.
(d) Xl,lose lysine
deorycholafe
agar
(XLD)---Available
from
Neogen Corp. and other suppliers.
Follow
rnanufacturer's
instructions
lor
preparalion.
(e)
Bisnruth
sulfite agar
(Bó).-Available
fiorn
Neogen Corp.
and other
suppliels. F'ollorv manulacturer's instructions lor
preparation.
(f)
Brillianr
green
sulfa agar (llGS1.'-'Available
fi'om Neogen
Corp.
and
other suppliers. F'ollorv nranulàcturer's
instluctions
lbr'
preparation.
(E)
h,lose
l),sine
tergitol
agar
(XL7'-4).--Available fiom
Neogen
Corp. and othel
suppliers.
Follou,
m¿rnufàcturer''s
inslructions
f'ol prepalation.
(h)
Double-modtfed lvsine
irott ttgar
(D^,Íl,l¡1) 'Available
Table
2013.148.
lnterlaboratory study results for
the ANSR
Salmonella
test: Exclusive
isolates
Organism
Correct
Misidentified
Total
Enterobacter cloacae
Escher¡chra
coli
Proteus vulgaris
Prov
¡de
nc¡a
a
lca
ltÍac ¡ens
Citrobacler freund¡¡
Klebsiella pneumon¡ae
Total isolâtes
126
0
126
126
0
126
126
0
126
126
0
126
126
tzo
126
125
755
98
125
106
126
123
2
I
4
4
24
96
1't7
102
105
122
't't
I
ô61
685