AOAC-RI ERP Book Micro Jan 19 2017

OMAMAN-35 A : Collaborative Study Manuscript For ERP Use Only January 2017

intervals, were 0.29 CFU/test portion(0.09,0.55) for the low inoculum level and 1.92 CFU/test portion (1.00, 3.42) 1 for the high inoculum level. 2 For the low inoculum level, 32 out of 120 test portions (POD CP of 0.27) were reported as presumptive positive 3 by the 3M MDA 2 – E. coli O157 (including H7)method with 31 test portions (POD CC of 0.26) confirming 4 positive. For samples that produced presumptive positive results on the 3M MDA 2 – E. coli O157 (including H7) 5 method, 29 samples confirmed positive (POD C of 0.24). For test portions evaluated by the USDA/FSIS MLG 6 reference method, 42 out of 120 test portions produced positive results (POD R of 0.35). A dLPOD C value of -0.11 7 with 95% confidence intervals of (-0.22, 0.01) were obtained between the candidate and reference method, 8 indicating no statistical significant difference between the two methods. A dLPOD CP value of 0.01 with 95% 9 confidence intervals of (-0.10, 0.12) were obtained between presumptive and confirmed results indicating no 0 statistically significant difference between the presumptive and confirmed results. 1 For the high inoculum level, 97 out of 120 test portions (POD CP of 0.81) were reported as presumptive positive 2 by the 3M MDA 2 – E. coli O157 (including H7) method with 96test portions (POD CC of 0.80) confirming 3 positive.For samples that produced presumptive positive results on the 3M MDA 2 – E. coli O157 (including 4 H7)method, 92 samples confirmed positive (POD C of 0.77). For test portions evaluated by the USDA/FSIS MLG 5 reference method, 95 out of 120 test portions produced positive results (POD R of 0.79). A dLPOD C value of -0.025 6 with 95% confidence intervals of (-0.129, 0.080) wasobtained between the candidate and reference method, 7 indicating no statistical significant difference between the two methods. A dLPOD CP value of 0.008 with 95% 8 confidence intervals of (-0.092, 0.109) wasobtained between presumptive and confirmed results indicating no 9 statistically significant difference between the presumptive and confirmed results. 0 For the un-inoculated controls, zero out of 120 samples (POD CP of 0.00) produced a presumptive positive result by 1 the 3M MDA 2 - E. coli O157 (including H7) method with 2test portions (POD CC of 0.02) confirming positive. For 2 samples that produced presumptive positive results on the 3M MDA 2 – E. coli O157 (including H7)method, 3 zerosamples confirmed positive (POD C of 0.00). For test portions evaluated by the USDA/FSIS MLG reference 4 method, 3 out of 120 test portions produced positive results. Colonies isolated from the three test portions were 5 further characterized utilizing partial sequencing of the 16S rRNA gene; isolates were not identified as E. coli 6 O157:H7. A dLPOD C value of 0.017with 95% confidence intervals of (-0.059, 0.017) wasobtained between the 7 confirmed candidate and reference method, indicating no statistical significant difference between the two 8 methods. A dLPOD CP value of 0.00 with 95% confidence intervals of (-0.031, 0.031) wasobtained between 9 presumptive and confirmed results indicating no statistically significant difference between the presumptive and 0 confirmed results. POD statistical analysis for the un-inoculated test samples were conducted on the revised 1 results (See Table 2016.2A) from laboratory 11 and 12 after 16S rRNA sequencing. 2 3 Detailed results of the POD statistical analysis are presented in Table 2016.2A and Figures 1A-1B. 6 7 While three laboratories experienced technical issues during the evaluation, no negative feedback was provided by 8 the collaborating laboratories in regard to the performance of the 3M MDA 2- E. coli O157 (including H7) 9 method.During the evaluation, three laboratories culturally confirmed E. coli O157:H7in their (blinded) un- 0 inoculated control samples, see Table 2016.2A.The two test portions from Laboratory 5 were unavailable for 1 further identification as the collaborative study plates had been disposed.However, laboratories 11 and 12 2 submitted their samples directly to a third party laboratory capable of performing 16S rRNA sequencing for 3 additional identification.The inoculating strain E. coli O157:H7 ATCC 43895 was also analyzed as the control. 4 Theprotocol and results for 16S rRNA sequencing can be found in Supplementary Material. Utilizing the Basic 5 Local Alignment Search Tool (BLAST ® ) for nucleotides [10], the isolates were determined to 1) not be the 6 inoculating strain E. coli O157:H7 ATCC 43895 and 2) not E. coli . Serological cross reactivity of other members 7 of the microbial community for raw beef products has been documented due to the close relationship among 8 species [11, 12]. This indicates that the 3M Molecular Detection Assay 2 – E. coli O157 (including H7) is more 9 AOAC Research Institute Expert Review Pa el Use Only 4 5 Discussion

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