ERP Micro December 2019

commercially. (a) Assurance® GDS Rotor-Gene® Q thermocycler – (Cat. No. 73070BC) (b) Laptop computer with Assurance GDS Rotor-Gene software – (Cat. No. 73071BC), version 2.3.103 (c) Assurance GDS Rotor-Gene Q Rotor-Disc – (Cat. No. 73039BC), 36-well (d) Assurance GDS Rotor-Gene Q locking ring (Cat. No. 73040BC), 36-well, to lock the Rotor-Disc (e) Adhesive film strips – (Cat. No. 73026BC), 200/case (f) Sample wells and sample wells base – samples wells (Cat. No. 73044BC), 200/case and sample wells base (Cat. No. 73043BC), 10/case (g) Resuspension plate – (Cat. No. 73006BC), 120/case (h) Gel cooling block – (Cat. No. 73083BC), box of 2 units (i) PickPen TM device – (Cat. No. 73091BC), 8 magnets (j) PickPen TM tips – (Cat. No. 73087BC), racked, 96/box (k) Adjustable micropipette – capable of accurately dispensing 1000 µL (l) 8-channel micropipette – capable of dispensing 30 µL (m) Filter barrier micropipette tips – (Cat. No. 73034BC), 10 – 50 µL, 960/case, or equivalent and (Cat. No. 73030BC), 200–1000 µL, 800/case, or equivalent (n) Adjustable repeat pipette – capable of dispensing 20 µL, 45 µL, 500 µL, and 1000 µL (o) Repeat pipette tips – (Cat. No. 73001BC), 0.5 mL, 100/case, or equivalent and (Cat. No. 73036BC), 10 mL, 100/case, or equivalent (p) Incubator – capable of maintaining and 42 ± 1oC (q) Freezer – capable of maintaining -20 ± 2oC (r) Refrigerator – capable of maintaining 5 ± 3oC, for storing GDS kits and enriched samples (s) Microtiter plate vortex mixer – (Cat. No. 73063BC), IKA® MS 3 or equivalent, capable of maintaining 900 RPM (t) Laboratory paddle blender – Stomacher® 400 and 3500 or equivalent, for sample homogenization (u) Sterile laboratory filtered Stomacher bags – Nasco or equivalent (v) Top-loading balance – minimum 4,000 g capacity, sensitivity of 0.1 g (w) Digital countdown UP timer (a) Amplification and detection instrument. —Assurance GDS Rotor-Gene ® , or equivalent. (b) Computer and software. (c) Sample concentration device. —PickPen ® . (d) Vortex mixer. (e) Sample wells and sample wells base.

AOAC Official Method 2005.04 Escherichia coli O157:H7 in Selected Foods Assurance ® GDS E. coli O157:H7 Tq

First Action 2005 Final Action 2008

Revised First Action2011 Revised First Action2012 Revised First Action 2019

(Applicable to detection of E. coli O157:H7 in raw ground beef, beef trim, finely textured beef, carcass cloths, orange juice, apple juice, fresh vegetables, green onions and sprout process water . ) Caution : E. coli O157:H7 are pathogenic bacteria. Symptoms of infection include bloody diarrhea and cramping, little to no fever, and hemolytic uremic syndrome. Decontaminate all spent media and equipment used in test prior to disposal of media or re-use of equipment. Note : Use proper environmental and procedural precautions for the handling of reagents and equipment when performing genetic- based assays. See Table 2005.04 (original study, 2005) for the results of the interlaboratory study supporting acceptance of the method. A. Principle The Assurance ® GDS E. coli O157:H7 Tq method is a gene- based assay that uses specific primers and proprietary probes directed against a highly conserved DNA sequence in the target organism. The method uses enrichment in modified EHEC ( mEHEC ® ) broth. After enrichment, populations of target microorganisms are concentrated by using a proprietary concentration (PickPen TM ) device and reagents or processed with an automated concentration instrument. The concentrate is transferred to a conical reaction vessel containing amplification reagents (Amplification tube) . The vessel is sealed and placed in a rotary thermocycler PCR instrument (Rotor-Gene®) which uses proprietary software placed in an instrument which allows for simultaneous amplification and detection. All tests, positive and negative, are indicated at the end of analysis, as well as the results of a procedural control which is contained in every Amplification tubereaction vessel . Assurance ® GDS E. coli O157:H7 Tq detects strains of E. coli O157:H7 and toxigenic E. coli O157:NM. This method is designed to be highly selective, and does not detect microorganisms that are potential cross-reactors in antibody-based assays, including E. coli O157 that are not H7 or NM, and other microorganisms that express O157 antigen but are not E. coli O157:H7. B. Apparatus Items are available from MilliporeSigma (www.sigmaaldrich.com). Ordering information—SigmaAldrich, 12822 SE 32nd St, Bellevue, WA 98005, Tel: 425-586-3300, email: bcs_cust_svc_bellevue@milliporesigma.comItems ( a )–( l ) are available from BioControl Systems (Bellevue, WA, USA; www.biocontrolsys.com). Items ( m )–( n ) are available

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(f) Resuspension plate. (g) Gel cooling block. (h) PickPen tips in box. (i) Sealing film with adhesive backing.

(j) Dedicated pipets.— Eight-channel pipettor, capable of dispensing 30 µL; repeater pipettor; and micropipettors capable of accurately dispensing 10 and 35 µL. (k) Repeat pipettor tips.— 0.5 and 10 mL.

© 2012 AOAC INTERNATIONAL