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SUBMITTED 050113, REVISED 102513
site for a specific endonuclease. The nicked strand is recognized as damaged and repaired by the action
1
of a thermostable DNA polymerase, displacing the original strand with the newly-synthesized repaired
2
portion. This displaced DNA “product” then binds to a second template and the same reactions lead to
3
formation of a second product. Amplification products are detected using a specific molecular beacon
4
probe. Fluorescent signal is generated in real time, with amplification and detection complete within 10
5
minutes. The entire assay is conducted at a constant temperature of 56
o
C using a temperature-
6
controlled fluorescence detection instrument. Assay software analyzes the fluorescent signal over time;
7
a data interpretation algorithm interprets results as negative, positive, or invalid based on baseline,
8
rate-of-change, and other criteria. Each tube of ANSR reagents also contains an internal positive
9
control, signaling in a second fluorescence channel irrespective of the presence of target DNA, and
10
indicating proper functioning of the amplification reagents.
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12
B.
Media and Reagents
13
(a)
ANSR
®
for Salmonella test kit. –
Available from Neogen Corp. (Lansing, MI
, www.neogen.com ).
14
Contains: – Lyophilized reagents in capped strip tubes, 8 tubes per strip, 12 strips (96 tests) per kit, in 2
15
sealed foil pouches with desiccant packs; cluster tubes, 8 tubes per strip, 12 strips per kit; permanent
16
caps, 8 caps per strip, 12 strips per kit; lysis buffer, 1 bottle, 60 mL; lysis reagent, 3 vials, lyophilized; kit
17
insert. Store reagent tubes at 2-8
o
C, in sealed foil pouches with desiccant. Store lysis buffer at 2-8
o
C.
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(b)
Phosphate-buffered saline (PBS)
. – Per liter: 8.0 g NaCl, 0.2 g KCl, 1.44 g Na
2
HPO
4
, 0.24 g
19
KH
2
PO
4
.
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(c)
Hektoen enteric agar
. – Available from Neogen Corp. and other suppliers. Follow
21
manufacturer’s instructions for preparation.
22
(d)
Xylose lysine deoxycholate (XLD) agar
. – Available from Neogen Corp. and other suppliers.
23
Follow manufacturer’s instructions for preparation.
24
(e)
Bismuth sulfite agar
. – Available from Neogen Corp. and other suppliers. Follow manufacturer’s
25
instructions for preparation.
26
(f)
Brilliant green sulfa agar
. – Available from Neogen Corp. and other suppliers. Follow
27
manufacturer’s instructions for preparation.
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