AOAC 2019 First Action Methods

Table 2019.03A. Summary of results for detection of E. coli O157:H7 in raw milk cheese (Comté, 34% fat, 0.8% salt) by GENE-UP® E. coli O157:H7 2 (ECO 2) a Extraction method Inoculation level Noninoculated Low High Candidate presumptive positive/total No. of samples analyzed 1/88 79/88 87/88 Candidate presumptive LPOD (CP) 0.01 (0.00, 0.06) 0.90 (0.82, 0.95) 0.99 (0.94, 1.00) s r b 0.11 (0.09, 0.20) 0.22 (0.19, 0.27) 0.11 (0.09, 0.20) s L c 0.00 (0.00, 0.05) 0.22 (0.14, 0.39) 0.00 (0.00, 0.05) s R d 0.11 (0.09, 0.13) 0.31 (0.26, 0.46) 0.11 (0.09, 0.13) P value e 0.4305 0.0000 0.4305 Candidate confirmed positive/total No. of samples analyzed 0/88 79/88 87/88 Candidate confirmed LPOD (CC) 0.00 (0.00, 0.04) 0.90 (0.82, 0.95) 0.99 (0.94, 1.00) s r 0.00 (0.00, 0.20) 0.22 (0.19, 0.27) 0.11 (0.09, 0.20) s L 0.00 (0.00, 0.20) 0.22 (0.14, 0.39) 0.00 (0.00, 0.05) s R 0.00 (0.00, 0.28) 0.31 (0.26, 0.46) 0.11 (0.09, 0.13) P value 1.0000 0.0000 0.4305 Candidate confirmed positive/total No. of samples analyzed 0/88 79/88 87/88 Candidate presumptive positive that confirmed LPOD (C) 0.00 (0.00, 0.04) 0.90 (0.82, 0.95) 0.99 (0.94, 1.00) s r 0.00 (0.00, 0.20) 0.22 (0.19, 0.27) 0.11 (0.09, 0.20) s L 0.00 (0.00, 0.20) 0.22 (0.14, 0.39) 0.00 (0.00, 0.05) s R 0.00 (0.00, 0.28) 0.31 (0.26, 0.46) 0.11 (0.09, 0.13) P value 1.0000 0.0000 0.4305 Positive reference samples/total No. of samples analyzed 0/88 55/88 72/88 Reference LPOD 0.00 (0.00, 0.04) 0.63 (0.41, 0.84) 0.82 (0.66, 0.98) s r 0.00 (0.00, 0.20) 0.39 (0.34, 0.46) 0.33 (0.28, 0.39) s L 0.00 (0.00, 0.20) 0.31 (0.19, 0.52) 0.22 (0.12, 0.42) s R 0.00 (0.00, 0.28) 0.49 (0.42, 0.53) 0.39 (0.34, 0.53) P value 1.0000 0.0000 0.0003 dLPOD (candidate vs. reference) f 0.00 (–0.04, 0.04) 0.27 (0.04, 0.49) 0.17 (0.01, 0.33) dLPOD (candidate presumptive vs. candidate confirmed) f 0.01 (–0.03, 0.06) 0.00 (–0.09, 0.09) 0.00 (–0.05, 0.05) a  Results include 95% confidence Intervals. b s r = Repeatability standard deviation. c s L = Among-laboratory standard deviation. d s R = Reproducibility standard deviation. e P value = Homogeneity test of laboratory PODs. f  A confidence interval for dLPOD that does not contain the value 0 indicates a statistically significant difference between the two methods.

Additional items.— Available from major laboratory suppliers. (a)  Incubators.— Capable of maintaining 41.5 ± 1°C or 42 ± 1°C. (b)  Adjustable, variable volume pipets.— Capable of sampling and delivering 10–20 µL. (c)  Adjustable, variable volumepipets (singleormultichannel).— Capable of sampling and delivering 0.5–10 µL. (d)  Compatible sterile, filter pipet tips.— 20 µL. (e)  Refrigerator.— Capable of maintaining 2–8°C. (f)  Vortex-Genie Pulse.— Scientific Industries, SKU: SI-P236.

C. General Instructions (a)  GENE-UP® ECO 2 kit is for professional use only with the GENE-UP® Lysis kit. (b)  Comply with Good Laboratory Practices. (c)  Do not use reagents after the expiration date indicated on the label. (d)  Visually inspect vials before testing. Do not use vials with evidence of damage, leakage, or deterioration. (e)  Do not mix reagents (or disposables) from different lots. (f)  Powder-free latex or nitrile gloves are recommended for all PCR steps.

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