4. AOACRIMicroMethods-2018Awards

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M ozola et al .: J ournal of AOAC I nternational V ol . 97, N o . 3, 2014  829

FOOD BIOLOGICAL CONTAMINANTS

Evaluation of the ANSR ® for Salmonella Assay for Identification of Salmonella spp. from Colony Picks from Selective/Differential Agar Media: First Action 2013.14 M ark M ozola , M aximilian B otimer , C arolyn J agadics , P aul N orton , O scar C aballero , N icole E nslin , P reetha B iswas , and J ennifer 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

I dentification 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 Method SM (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

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 methodfeedback@aoac.org. Corresponding author’s e-mail: mmozola@neogen.com An appendix is available on the J. AOAC Int. website, http://aoac. publisher.ingentaconnect.com/content/aoac/jaoac DOI: 10.5740/jaoacint.14-004 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. 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,

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