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SUBMITTED 050113, REVISED 102513
As part of the PTM study of the screening method, inclusivity was assessed using a panel of 113 strains
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of
S
.
enterica
and
S
.
bongori
representing 108 serovars. With the single exception of one of two strains
2
of the rare serovar
S
. Weslaco, all strains were detected by the ANSR assay when tested at a
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concentration of approximately 1 x 10
5
cfu/mL, which is approximately 10-fold above the limit of
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detection of the assay [1]. Exclusivity testing was conducted using a panel of 38 strains of non-
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salmonellae, primarily closely related members of the Enterobacteriaceae and representing 15 genera
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and 37 species. Strains were tested after growth to high titer (~ 1 x 10
9
cfu/mL) in non-selective tryptic
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soy broth. All tests produced negative results. With regard to
S
. Weslaco, we have recently determined,
8
through PCR analysis, that the non-inclusive
S
. Weslaco strain does not contain the genetic target of the
9
ANSR assay.
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Here we describe results of a pre-collaborative study to validate the ANSR
Salmonella
assay for
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identification of presumptive
Salmonella
colonies taken from selective/differential agar plates specified
13
in the FDA
Bacteriological Analytical Manual
(BAM) [3] and USDA-FSIS
Microbiology Laboratory
14
Guidebook
(MLG) [4] reference culture procedures. An inter-laboratory collaborative study is planned.
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Method
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Caution
: Use of this test should be restricted to individuals with appropriate laboratory training in
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microbiology. Reagents are for laboratory use only. Refer to the Material Safety Data Sheet from
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Neogen Corp. for more information. Enrichment cultures, used agar plates, and ANSR assay lysates and
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reaction tubes should be handled and disposed of as potentially infectious material. The preferred
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method for disposal of contaminated materials, including cultures, pipette tips, tubes, etc. is
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autoclaving. Items that cannot be autoclaved should be decontaminated by treatment with disinfectant
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solution. ANSR reaction tubes should not be autoclaved in areas where they may open and possibly
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contaminate the laboratory environment with amplification products. Alternatively, they may be
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disposed of in a sealed container with a small amount of 10% household bleach added.
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A.
Principle
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ANSR
Salmonella
is a new isothermal nucleic acid amplification assay based on the nicking enzyme
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amplification reaction (NEAR
TM
) technology [5]. The amplification mechanism involves binding of an
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oligonucleotide “template” to a specific sequence of target DNA. The template contains a recognition
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