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Mozola et al.: J ournal of AOAC International Vol. 97, No. 3, 2014 829
Evaluation of the ANSR® for Salmonella Assay for
Identification of Salmonella spp. from Colony Picks from
Selective/Differential Agar Media: First Action 2013.14
Mark M ozola, Maximilian Botimer, Carolyn Jagadics, Paul Norton, Oscar Caballero, Nicole Enslin ,
Preetha Biswas, and Jennifer R ice
Neogen Corp., 620 Lesher Pl, Lansing, MI 48912
Collaborators: E.S. Adams, H. Alnughaymishi, J.B. Barrett, J. Benzinger, M.E. Berrang, M. Boyle, J. Cannon, D. Clark,
A. Copeland, M. Corebello, D.E. Cosby, N.A. Cox, N. Cuthbert, H. Dammann, J. Dyszel, E. Feldpausch, J. Flannery, C. Flores,
J.G. Frye, R. Fuller, V. Gill, L.M. Hiott, B. Howard, M. Hudgens, C.R. Jackson, W. Jones, S.W. Knapp, L. Kuepfer, P. Kulkarni,
B. Kupski, C. Pidgeon, A. Quenneville, L.L. Rigsby, N. Rogman, E. Sai, A. Scollon, M. Sisemore, J. Stepnitz, J. Van Brunt,
M. Vross, D. Waltman, H. Wang, G. Whitbeck, S. York, L. Zhang
Received December 18, 2013.
The method was approved by the Expert Review Panel for Food
Biological Contaminants as First Action.
The Expert Review Panel for Food Biological Contaminants
invites method users to provide feedback on the First Action methods.
Feedback from method users will help verify that the methods are
fit for purpose and are critical to gaining global recognition and
acceptance of the methods. Comments can be sent directly to the
corresponding author or
Corresponding author’s e-mail:
An appendix is available on the J. AOAC Int. website,
DOI: 10.5740/jaoacint.14-004
FOOD BIOLOGICAL CONTAMINANTS
A collaborative study was conducted to evaluate
performance of the ANSR® for Salmonella assay for
identification of Salmonella spp. from colony picks
taken from selective/differential agar media. The
ANSR Salmonella assay is an isothermal nucleic
acid amplification test based on the nicking enzyme
amplification reaction chemistry. The test can be
completed in less than 40 min including sample
preparation. A total of 18 laboratories representing
industry, government, academic, and commercial
testing laboratories participated in the study. Each
collaborator tested up to 84 samples, comprised of
colony picks of six Salmonella spp. and six non-
salmonellae taken from six selective/differential
agar media as well as tryptic soy agar. A total of
1441 analyses were performed, 1416 of which gave
the correct identification, for overall accuracy of
98.3%. For identification of Salmonella spp., 755 of
756 tests (99.9%) produced the correct result. For
identification of non-salmonellae as such, 661 of 685
assays (96.5%) produced the correct result. Of the
18 laboratories, 15 produced data sets with 99–100%
accuracy. The majority of false-positive results were
clustered in three laboratories; analysis of raw data
suggests procedural difficulties in at least two cases,
which may explain the atypical data from these
collaborators. The ANSR Salmonella assay can be
used as a rapid, accurate adjunct or alternative to
biochemical testing for identification of presumptive
Salmonella spp. isolates.
Identification of presumptive Salmonella colonies from
selective/differential agar media as Salmonella spp. has
historically been achieved using a variety of biochemical and
serological procedures. In the case of food and environmental
sample analysis, these procedures are specified in reference
methods such as those in the U.S. Food and DrugAdministration’s
Bacteriological Analytical Manual (BAM; 1) and the U.S.
Department ofAgriculture’s Microbiology LaboratoryGuidebook
(MLG; 2). These methods include conventional biochemical tests,
miniaturized biochemical test devices, automated biochemical
identification platforms, and serological agglutination tests using
Salmonella-specific antisera. The biochemical identification
procedures, although accurate and reliable, generally require
6–24 h to obtain results. The serological procedures may be
rapid, but often require subculture to enhance antigen expression,
especially in the case of flagellar (H) antigen typing.
As an adjunct or alternative to biochemical and serological
procedures, nucleic acid-based identification methods hold
promise for providing timely and accurate results. This has been
acknowledged, for example, by reference in both BAM and MLG
to use of nucleic acid-based methods for identification of Listeria
monocytogenes(3, 4).
The ANSR® Salmonella assay was originally developed for
rapid screening of enriched food and environmental samples.
The assay is an isothermal nucleic acid amplification procedure,
based on the nicking enzyme amplification reaction (NEAR)
technology (5). The ANSR method has been evaluated in three
AOAC Performance Tested MethodSM (PTM) validation studies,
leading to certification as PTM 061203, with claims for a variety
of food and environmental sample types (6–7). In these studies,
the ANSR method exhibited sensitivity comparable to that of
the BAM and MLG reference culture methods by probability
of detection statistical analysis, as well as >99% inclusivity and
100% exclusivity in testing of target and nontarget bacteria.
This method performance, coupled with the simplicity and
rapidity of the assay (less than 40 min), suggested that the method
could also serve as a useful tool for identification of presumptive
Salmonella spp. isolates from selective/differential agar plating
media. A precollaborative study has been completed in which
colonies of 113 Salmonella spp. strains and 37 non-Salmonella
strains were picked from tryptic soy agar (TSA) and six selective/
differential agar media [Hektoen enteric agar (HE), xylose lysine
Candidates for 2016 Method of the Year
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