Validation of the ANSR[R] E. coli O157:H7 method for detection of E. coli O157:H7: AOAC Performance Tested Method[SM] 111502

• Published in: Journal of AOAC International Vol. 99; no. 3; p. 705
• Main Authors: Viator, Ryan, Alles, Susan, Le, Quyhn-Nhi, Hosking, Edan, Meister, Evan, Pinkava, Lisa, Tovar, Eric, Mozola, Mark, Rice, Jennifer
• Format: Journal Article
• Language: English
• Published: Oxford University Press 01.05.2016
• Subjects: Biochemical assays; Equipment and supplies; Escherichia coli; Evaluation; Food testing equipment; Measurement; Methods
• ISSN: 1060-3271
• DOI: 10.5740/jaoacint.15-0309

A performance validation of the ANSR[R] for E. coli O157:H7 method was conducted in selected food matrixes. This assay uses selective nicking enzyme amplification technology to amplify target genes. Samples are enriched for 12-24 h and then lysed. The assay is completed within 40 min using real-time detection in a combination incubator/fluorescence detector and software. When 44 distinct strains of Escherichia coli O157:H7 and 6 strains of E. coli O157:NM were tested for inclusivity, all 50 strains produced positive results. In exclusivity testing, 57 strains representing 33 species of closely related Gram-negative bacteria belonging to the Enterobacteriaceae family, including 11 non-H7 O157 strains and shiga toxin-producing E. coli other than O157:H7, were evaluated. All 57 nontarget strains generated negative ANSR assay results. Using 80% lean ground beef and beef trim (approximately 20% fat), ANSR method performance was compared to the U.S. Department of Agriculture, Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedure. ANSR performance with baby spinach and sprout irrigation water was measured against the U.S. Food and Drug Administration Bacteriological Analytical Manual reference method. ANSR method performance was not statistically different to that of the reference methods using two different enrichment options. For ground beef and beef trim, the standard enrichment in modified Tryptone Soya Broth can be analyzed using the ANSR assay with a 1:10 dilution of the enrichment in phosphate-buffered saline and produces equivalent results to the reference method. Additionally, in most matrixes tested (exception is spinach which required 24 h enrichment) the assay offers great efficiency and flexibility over the reference method with a 12-24 h single-step enrichment. Equivalent results were observed at both time points (12 and 24 h) to reference methods. Small changes to the assay parameters minimally affected ANSR method performance. Finally, accelerated stability results from three independently manufactured lots support a shelf-life of 6 months when stored at 4[degrees]C.