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Certification Report Performance Tested Methodsm 001101 TECRA E.coli O157 Visual Immunoassay and TECRA E.coli 0157 Immunocapture System 1 November, 2000 1. General Comments The TECRA E.coli O157 Visual Immunoassay was validated as equivalent to the FDA BAM (8th edition) reference method in its performance in detecting E.coli O157 bacteria in a variety of foods including raw ground beef, cooked ground beef raw ground chicken and cooked ground chicken. The TECRA E.coli O157 Visual Immunoassay was demonstrated to be capable of detecting low numbers (MPN 0.9 to 3.75 cells) of E.coli O157 in 25 grams food samples. Additional data for apple juice, bean sprouts, lettuce, raw ground pork, salami, and pepperoni was also collected by the AOAC Research Institute in cooperation with the Canadian Food Inspection Agency. This data supports equivalency claims for the TECRA E.coli O157 Visual Immunoassay to the FDA BAM reference method in these matrixes at the levels of E.coli O157 tested in the study. The data was inconclusive as to the minimum level of E.coli O157 that could be detected by the TECRA E.coli O157 Visual Immunoassay. The TECRA E.coli O157 Visual Immunoassay is an enzyme-linked immunoassay (ELISA) performed in the sandwich configuration. The TECRA E.coli O157 Visual Immunoassay detects all E.coli with the O157 antigen, not just the O157:H7 strains. The TECRA E.coli O157 Visual Immunoassay provides a rapid method for detection of E.coli O157 requiring an 18-24 hour sample enrichment period and 2 hours for performance of the assay. Positive presumptive results are confirmed using the TECRA E.coli 0157 Immunocapture system or by conventional cultural assays. 2. Test Kit Information 2.1 Kit Name TECRA E.coli O157 Visual Immunoassay (ECOVIA) and TECRA E.coli O157 Immunocapture System (ECOICM). 2.2 Catalog Numbers ECOVIA48 (48 wells) and ECOVIA96 (96 wells); ECOICM08

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TECRA Diagnostics E.coli O157 Visual Immunoassay

2.3 Ordering Information 2.3.1 2.3.2 USA - International BioProducts, 21312 38th Avenue SE, Bothell, WA 98021, USA. Phone: 425-398-7993. Fax: 425-398-7973. Outside the US TECRA International Pty Ltd., PO Box 788, Willoughby, NSW, 2068, Australia. Phone: 61-2-9928-8900. Fax: 61-29417-7858.

2.4 Test Kit Reagents 2.4.1 TECRA E.coli O157 Visual Immunoassay (ECOVIA) 2.4.1.1 2.4.1.2 2.4.1.3 2.4.1.4 2.4.1.5 2.4.1.6 2.4.1.7 2.4.1.8 2.4.1.9 2.4.2 Reagent 1 Wash concentrate containing 1.45g TRIS, 7.03g NaCl, 1.0g Tween and 0.0025g thimerosal. Reagent 2 Positive control containing purified E.coli O157 antigen. Reagent 3 Control diluent (negative control) containing 0.42g TRIS, 0.044g NaCl, and 0.0025g Tween 20. Reagent 4 - Conjugate containing conjugated anti-E.coli O157 antibodies coupled to horseradish peroxidase. Reagent 5 - Conjugate diluent containing 0.42g Na2B4O710 H2O, 0.193g NaCl, 0.22g hydrolyzed gelatin, and 0.0022g thimerosal. Reagent 6 Substrate containing 0.111g 2,2-azino-di(3ethylbenzthizoline sulfonate) and 0.123 NaH2PO42H20 Reagent 7 - Substrate diluent contain 0.116g citric acid, 0.0011g H2O2, and 0.0185g NaOH. Reagent 8 Stop Solution containing 0.15g NaF. Reagent 9 Sample additive containing Tween 20.

TECRA E.coli O157 Immunocapture System (ECOICM) 2.4.2.1 2.4.2.2 2.4.2.3 Reagent 1 buffer Reagent 2 wash solution Reagent 3 Enrichment broth

2.5 Additional Supplies and Reagents 2.5.1 2.5.2 2.5.3 2.5.4 2.6 Apparatus 2.6.1 2.6.2 2.6.3 2.6.4 2.6.5 35-37C incubator 42C incubator serological pipette plastic squeeze bottle pipette (20L, 50 L, and 200 L) Modified EC medium with novobiocin (mEC + N). Buffered tryptone soy broth with novobiocin (BTSB + N). Sterile saline, butterfields phosphate buffer, or 0.1% peptone solution. CT-SMAC culture media plates

AOAC Research Institute 1 November, 2000

TECRA Diagnostics E.coli O157 Visual Immunoassay

2.6.6 2.6.7 2.6.8

plastic wrap waterbath (100C) small screw cap bottles or tubes capable of containing 5 mL and suitable for boiling. 2.6.9 Vortex mixer 2.6.10 Glass test tubes ( 15cm x 15cm) 2.6.11 Stomacher bags. 3. Validation Studies 3.1 Inclusivity Study Inclusivity data for 101 isolates of E.coli O157: H7 and E.coli O157 H- were submitted and reviewed. All 101 isolates were detected by the ECOVIA. 3.2 Exclusivity Study Fifty-one non-E.coli O157 isolates, including 11 non-O157 E.coli isolates, were tested in 3 different studies. All isolates tested negative using the ECOVIA indicating that the ECOVIA is highly specific for O157 strains. 3.3 Comparison to Reference Methods Results from studies comparing the ECOVIA system to the BAM method for E.coli O157 indicate that the ECOVIA gives comparable results to the BAM method in a variety of matrixes including raw ground beef, cooked ground beef, raw ground chicken and cooked ground chicken. Additional data for apple juice, bean sprouts, lettuce, raw ground pork, salami, and pepperoni was also collected by the AOAC Research Institute in cooperation with the Canadian Food Inspection Agency. Analytical results between the TECRA E.coli O157 Visual Immunoassay and the FDA BAM (8th edition) reference method were equivalent. However, the data was inconclusive as to the minimum level of E.coli O157 that could be detected by the TECRA E.coli O157 Visual Immunoassay. 3.4 Ruggedness The ECOVIA is a robust test method. The effects of variations in 7 method parameters including temperature of reagents; capture temperate; conjugate temperature; substrate temperature; substrate solution age; absorbance reading wavelength variations, and age of wash solution; were studied. No significant effects were observed.

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TECRA Diagnostics E.coli O157 Visual Immunoassay

3.5 Stability TECRA provided stability data indicating that ECOVIA kits are stable for at least 12 months when stored at 4C.