Cert if Report

8/3/2019 Cert if Report

http://slidepdf.com/reader/full/cert-if-report 1/4

AOAC Research Institute481 N. Frederick Ave, Suite 500

Gaithersburg, MD 20877-2417Telephone: +1 (301) 924-7090

Fax: +1 (301) 924-7089

Certification Report

Performance Tested Method sm 001101

TECRA E.coli O157 Visual Immunoassayand

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 FDABAM (8 th edition) reference method in its performance in detecting E.coli O157 bacteriain a variety of foods including raw ground beef, cooked ground beef raw ground chickenand cooked ground chicken. The TECRA E.coli O157 Visual Immunoassay wasdemonstrated 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, andpepperoni was also collected by the AOAC Research Institute in cooperation with the

Canadian Food Inspection Agency. This data supports equivalency claims for theTECRA E.coli O157 Visual Immunoassay to the FDA BAM reference method in thesematrixes at the levels of E.coli O157 tested in the study. The data was inconclusive asto the minimum level of E.coli O157 that could be detected by the TECRA E.coli O157Visual Immunoassay.

The TECRA E.coli O157 Visual Immunoassay is an enzyme-linked immunoassay(ELISA) performed in the sandwich configuration. The TECRA E.coli O157 VisualImmunoassay detects all E.coli with the O157 antigen, not just the O157:H7 strains. TheTECRA 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



AOAC Research Institute TECRA Diagnostics1 November, 2000 E.coli O157 Visual Immunoassay

2.3 Ordering Information

2.3.1 USA - International BioProducts, 21312 38 th Avenue SE, Bothell, WA98021, USA. Phone: 425-398-7993. Fax: 425-398-7973.

2.3.2 Outside the US - TECRA International Pty Ltd., PO Box 788,Willoughby, NSW, 2068, Australia. Phone: 61-2-9928-8900. Fax: 61-2-9417-7858.

2.4 Test Kit Reagents

2.4.1 TECRA E.coli O157 Visual Immunoassay (ECOVIA)

2.4.1.1 Reagent 1 – Wash concentrate containing 1.45g TRIS, 7.03gNaCl, 1.0g Tween and 0.0025g thimerosal.

2.4.1.2 Reagent 2 – Positive control containing purified E.coli O157antigen.

2.4.1.3 Reagent 3 – Control diluent (negative control) containing 0.42gTRIS, 0.044g NaCl, and 0.0025g Tween 20.

2.4.1.4 Reagent 4 - Conjugate containing conjugated anti- E.coli O157antibodies coupled to horseradish peroxidase.

2.4.1.5 Reagent 5 - Conjugate diluent containing 0.42g Na 2B4O7·10H2O, 0.193g NaCl, 0.22g hydrolyzed gelatin, and 0.0022gthimerosal.

2.4.1.6 Reagent 6 – Substrate containing 0.111g 2,2’-azino-di(3-ethylbenzthizoline sulfonate) and 0.123 NaH 2PO 4·2H 20

2.4.1.7 Reagent 7 - Substrate diluent contain 0.116g citric acid,0.0011g H 2O 2, and 0.0185g NaOH.

2.4.1.8 Reagent 8 – Stop Solution containing 0.15g NaF.2.4.1.9 Reagent 9 – Sample additive containing Tween 20.

2.4.2 TECRA E.coli O157 Immunocapture™ System (ECOICM)

2.4.2.1 Reagent 1 – buffer 2.4.2.2 Reagent 2 – wash solution 2.4.2.3 Reagent 3 – Enrichment broth

2.5 Additional Supplies and Reagents

2.5.1 Modified EC medium with novobiocin (mEC + N). 2.5.2 Buffered tryptone soy broth with novobiocin (BTSB + N).2.5.3 Sterile saline, butterfield’s phosphate buffer, or 0.1% peptone solution.

2.5.4 CT-SMAC culture media plates

2.6 Apparatus

2.6.1 35-37°C incubator 2.6.2 42°C incubator 2.6.3 serological pipette 2.6.4 plastic squeeze bottle 2.6.5 pipette (20 ц L, 50 ц L, and 200 ц L)

2




2.6.6 plastic wrap 2.6.7 waterbath (100°C) 2.6.8 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- weresubmitted 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, weretested in 3 different studies. All isolates tested negative using the ECOVIA indicatingthat 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 avariety of matrixes including raw ground beef, cooked ground beef, raw groundchicken and cooked ground chicken.

Additional data for apple juice, bean sprouts, lettuce, raw ground pork, salami, andpepperoni was also collected by the AOAC Research Institute in cooperation with theCanadian Food Inspection Agency. Analytical results between the TECRA E.coli O157 Visual Immunoassay and the FDA BAM (8 th edition) reference method wereequivalent. 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 methodparameters including temperature of reagents; capture temperate; conjugate

temperature; substrate temperature; substrate solution age; absorbance readingwavelength variations, and age of wash solution; were studied. No significant effectswere observed.

3




3.5 Stability

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

4

Cert if Report

Documents

Transcript of Cert if Report