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SUBMITTED 050113
AOAC International Collaborative Study Protocol
ANSR
TM
Salmonella
for Identification of
Salmonella
spp. from Colony Picks
from Selective/Differential Agar Media
Oscar Caballero, Maximilian Botimer, Carolyn Jagadics, Paul Norton, and Mark Mozola*
Neogen Corporation, 620 Lesher Place, Lansing, MI 48912
*Study Director
Phone: 517-372-9200
mmozola@neogen.comIntroduction
The ANSR
Salmonella
isothermal nucleic acid amplification assay was originally developed as a screening
test for food and environmental samples following broth culture enrichment. The method has been
granted Performance Tested Method
SM
status by the AOAC Research Institute for testing of a wide
variety of food and environmental samples (certificate no. 061203; [1, 2]). While useful as a screening
method, the potential advantages of the assay as a confirmatory test for presumptive colonies taken
from selective/differential agar plates are compelling. First, a presumptive colony can be definitively
identified as
Salmonella
spp. in 30 min., compared with 24-48 h required by typical biochemical
identification methods. Second, there is no requirement that the colony pick be pure, i.e., unlike
biochemical identification methods, the contaminating presence of non-
Salmonella
bacteria will not
interfere with the ability of the assay to identify the presence of
Salmonella
spp. in the sample. Finally,
the method is cost effective, with minimal equipment requirements and an assay platform scaled for 1-
16 determinations per experimental run.
A pre-collaborative study has been completed and a study report submitted for review. In this study,
113 strains of
S. enterica
and
S. bongori
(representing 108 serovars) and 38 non-
Salmonella
strains
(primarily closely related Enterobacteriaceae) were tested as colonies picked from tryptic soy agar (TSA)
and the six selective/differential agar media specified in the FDA
Bacteriological Analytical Manual
(BAM, [3]) or USDA-FSIS
Microbiology Laboratory Guidebook
(MLG, [4]). Single colonies were
resuspended in 0.5 mL phosphate-buffered saline (PBS) and tested in the ANSR assay. The ANSR assay
was able to correctly identify all but one of the 113
Salmonella
strains as “
Salmonella
spp.” from all agar
media. One of two strains of
S
. Weslacowas consistently negative in the ANSR assay. This strain was
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