4. AOACRIMicroMethods-2018Awards

180

448  C rowley et al . : J ournal of AOAC I nternational V ol . 97, N o . 2, 2014

screen of the matrix indicated an absence of indigenous Listeria species. As per criteria outlined in Appendix J, fractional positive results were obtained for both the 25 and 125 g test portions sizes. For each test portion size, the actual level of L. monocytogenes was determined by MPN determination on the day of initiation of analysis. The results of the inoculum heat-stress protocol are presented in Table 2. The individual laboratory and sample results are presented in Tables  3 and 4. Tables 2013.11A and B summarize the collaborative study results for all foods tested, including POD statistical analysis (10). Detailed results for each laboratory are presented in Tables  2A–D, and Figures 1A–D and 2A–D as supplemental data on the J. AOAC Int. website. Queso fresco test portions were inoculated at a low and high level and were analyzed (Table 3) for the detection of L. monocytogenes . Uninoculated controls were included in each analysis. Fourteen laboratories participated in the analysis of this matrix and the results of 13 laboratories were included in the statistical analysis. Laboratory 11 reported 11 test portions (including four uninoculated test portions) that produced non- L. monocytogenes profiles. Colonies on these plates contained one or more of the following biochemical reactions not typically associated with L. monocytogenes : Gram-negative, Gram-positive with spores, non-beta-hemolytic, and catalase negative. Based on the preliminary biochemical tests conducted, the test portions should not have been carried through for final biochemical identification on API Listeria strips which resulted in the misidentification of the test portion as Listeria spp. The selective agar plates for these test portions were sent to the coordinating laboratory for further examination. The coordinating laboratory confirmed the supplementary results (Gram stain, hemolysis, and catalase reaction) reported by Table 1. Participation of each collaborating laboratory a Queso fresco Lab 25 g test portions 125 g test portions 1 Y Y 2 Y b Y 3 Y Y 4 Y Y 5 Y Y 6 Y Y 7 Y Y 8 Y Y 9 Y Y 10 Y Y 11 Y c Y c 12 Y Y 13 Y Y 14 Y Y a  Y= Collaborator analyzed the food type. b  Results were not submitted to the coordinating laboratory. c  Results were not used in statistical analysis due to laboratory error. Queso Fresco (25 g Test Portions)

Table 2013.11D. Interpretation of test Test value threshold

Interpretation

<0.05 ≥0.05

Negative Positive

F. Results and Interpretation The results are analyzed automatically by the VIDAS system. A report is printed which records the type of test performed, the test sample identification, the date and time, the lot number, and expiration date of the reagent kit being used, and each sample’s RFV, test value, and interpreted result (positive or negative). Fluorescence is measured twice in the reagent strip’s reading cuvette for each sample tested. The first reading is a background reading of the substrate cuvette before the SPR is introduced into the substrate. The second reading is taken after incubating the substrate with the enzyme remaining on the interior of the SPR. The test value is calculated by the instrument and is equal to the difference between the background reading and the final reading. The calculation appears on the result sheet. A “negative” result has a test value less than the threshold (0.05) and indicates that the sample does not contain L. monocytogenes or contains L. monocytogenes at a concentration below the detection limit. A “positive” result has a test value equal to or greater than the threshold (≥0.05) and indicates that the sample may be contaminated with L. monocytogenes. If the background reading is above a predetermined cutoff, then the result is reported as invalid (Table 2013.11D ). G. Confirmation All positive VIDAS LMX results must be culturally confirmed. Confirmation should be performed using the non- heated enrichment broth (the LMX primary enrichment broth for 25 g test portions and the LPT secondary enrichment broth for 125 g test portions) stored between 2–8°C, and should be initiated within 72 h following the end of incubation (AFNOR Certificate No. BIO 12/33-05/12). Presumptive positive results may be confirmed by isolating on selective agar plates such as ALOA or on the appropriate reference method selective agar plates. Typical or suspect colonies from each plate are confirmed as described in appropriate reference method. As an alternative to the conventional confirmation for L. monocytogenes, VITEK 2 GP Biochemical Identification (AOAC 2012.02 ) or API Listeria biochemical kits may be used for presumptive generic identification of foodborne L. monocytogenes . In this collaborative study, the VIDAS Listeria monocytogenes (LMX) method was compared to the to AOAC 993.12 for one food product, queso fresco, at two test portion sizes, 25 and 125 g. A total of 14 laboratories throughout the United States participated in this study, with 14 laboratories submitting data for the 25 g test portions and 13 laboratories submitting data for the 125 g test portions as presented in Table 1. Each laboratory analyzed 36 test portions for each method 12 inoculated with a high level of L. monocytogenes, 12 inoculated with a low level of L. monocytogenes, and 12 uninoculated controls. A background 03/10/2019 Results of Collaborative Study