ERP Micro December 2019

to close. Visually inspect each tube to ensure the cap is securely sealed. (d) Invert the gel cooling block holding amplification tubes and shake with snapping motion to thoroughly mix tube contents. (e) Place amplification tubes into Assurance Rotor-Gene, B ( a ), in sequential order, beginning with position No. 1. Start Rotor- Gene cycle, B ( b ). Refer to Assurance GDS user manual for detailed instructions on operating the Rotor-Gene. Note : The Assurance GDS Rotor-Gene must be started within 15 min after addition of the samples to the amplification tubes. G. Assay Results (c) Interpretation and Test Results 1) To view a report on a completed run, select the desired completed test. The report can be viewed and printed from the report display screen. 2) Upon completion of the run, the Assurance® GDS Rotor- Gene® software will provide a results table. Each sample will be identified as “Positive”, “Negative”, or “No Amp”. If a “No Amp” result is obtained, repeat the test beginning from the Sample Preparation Protocol. If result continues to show “No Amp”, contact MilliporeSigma Technical Service. Caution: When the assay is completed, handle and dispose of all waste as a biohazard. Never, under any circumstances, open amplification tubes after the amplification process has started to prevent discharge of amplified materials into the laboratory which could cause contamination of the testing environment. Upon completion of the run, the Assurance Rotor-Gene program will provide a results table. Each sample will be identified as “positive,” indicating that the test sample is positive for E. coli O157:H7, “negative,” indicating that it is negative for E. coli O157:H7, or “no amp,” indicating that amplification did not occur. In the event of a “no amp” result, from the sample enrichment broth that has been returned to the incubator, E ( d ), remove 1 mL of broth and start at E ( a ). If result continues to show “no amp,” contact BioControl Systems Technical Service (800-245-0113). ( Caution : When the assay is completed, handle and dispose of all waste as a biohazard. Never, under any circumstances, open amplification tubes after the amplification process has started to prevent discharge of amplified materials into the laboratory, which could cause contamination of the testing environment.) H.F. Confirmation of Positive Results Following 8 – 18 h enrichment in mEHEC® at 42 °C (10 – 18 h for 375 g samples), samples can be confirmed from the retained mEHEC® enrichment via the following:Assurance GDS positive samples should be confirmed culturally, C ( f ), as described in current Bacteriological Analytical Manual , Chapter 4A “ Diarrheagenic Escherichia coli ,” http:// www.fda.gov/Food/ScienceResearch/LaboratoryMethods/

BacteriologicalAnalyticalManualBAM/default.htm or USDA/ FSIS Microbiology Laboratory Guidebook , Chapter MLG 5.05 “Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products , ” http://www.fsis.usda.gov/Science/ Microbiological_Lab_Guidebook/index.asp.

1)

Modified U.S. Department of Agriculture (USDA)

Microbiology Laboratory Guidebook (MLG 5C.00) 2) Bacteriological Analytical Manual (BAM Chapter 4A) 3)

Modified U.S. Food and Drug Administrative

Assurance® GDS Shiga Toxin Genes Tq kit (Cat. No.

71005-100) 4)

Assurance® GDS EHEC ID for E. coli O157:H7 Tq kit

(Cat. No. 71037-52)

Note: Enriched samples can be stored at 5 ± 3°C (refrigeration) for up to 24 h prior to confirmation. I. Precautions (a) If possible, maintain separate work zones and dedicated equipment and supplies for sample preparation, amplification, and detection. (b) Use of both positive and negative control samples is recommended. (c) Do not use test kit beyond the expiration date on the product box label. (d) Decontaminate and dispose of materials in accordance with good laboratory practices and in accordance with local, state, and federal regulations. (e) Do not open used amplification tubes. Do not autoclave used amplification tubes. Prior to disposal, immerse used amplification tubes in a sealed container with 20% bleach or 1.2% sodium hypochlorite solution. (f) Waste may be contaminated with E. coli O157:H7, which is potentially hazardous to human health. All biohazard waste should be disposed of appropriately (1). References: (1) Feldsine, P.T., Green, S.T., Lienau, A.H., Stephens, J., Jucker, M.T., & Kerr, D.E. (2005)

J. AOAC Int . 88 , 1334–1348 J. AOAC Int . 96 , 776(2013) DOI: 10.5740/jaoacint.CS2005_04

Formatted: Space Before: 6.75 pt

Revised 2011 to include procedural revisions; and December 2012 to add an increased test portion size (375 g) ; December 2019 to include new matrices, use of alternate automated sample processing and alternate smaller amplification tube size.

© 2012 AOAC INTERNATIONAL