AOAC-RI ERP Book Micro Jan 19 2017

OMAMAN-35 B: Collaborative Study Protocol ERP USE ONLY January 2017

3M MDA2 E. coli O157:H7 Collaborative Study

May 2016

OMA-2016-May-XXXX

a) Invert room temperature (20-25 o C) lysis tubes to mix. Proceed to next step within 4 hours. A 20 µL aliquot of each enriched sample is transferred to separate lysis tubes using a new pipette tip after each sample transfer. Place uncovered samples in the 3M Molecular Detection Heat Block for 15 ± 2 minutes at 100 ± 1 o C. During heating, the lysis samples will change from pink (cool) to yellow (hot). Following the heat lysis transfer samples place samples in the 3M Molecular Detection Chill Block Insert for 10 ± 1 minutes. The 3M Molecular Chill Block Insert, used at ambient temperature without the 3M Molecular Detection Chill Block Tray, should sit directly on the laboratory bench. When cool, the lysis b) Add 20 µL of each lysed sample and control to separate reagent tubes, and mix by pipetting up and down five times. Analyze a matrix control tube with the samples for each matrix to verify that no interference with the assay was caused by the matrix. Use sterile 3M BPW ISO for the Negative Control (NC). Transfer a 20 µL aliquot to the NC and the Reagent Control c) Add a 20 µL aliquot of a randomly picked sample to the matrix control tube, mixed, and recapped. Using the 3M ™ software, follow prompts to identify samples and controls. Load all samples into the Speed Loader Tray (SLT), place into the Molecular Detection System, and initiate the 3M ™ MDA2 - E. coli O157 (including H7) assay. Results are obtained within 60 solution will revert to a pink color. (RC) tubes.

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minutes.

j)

Interpretation and Test Result Report

An algorithm interprets the light output curve resulting from the detection of the nucleic acid amplification. Results are analyzed automatically by the software and are color-coded based on the result. A Positive or Negative result is determined by analysis of a number of unique curve parameters. Presumptive positive results are reported in real-time while Negative and Inspect

results will be displayed after the run is completed.

NOTE: Even a negative sample will not give a zero reading as the system and 3M MDA2 – E. coli O157 (including H7) amplification reagents have a “background” relative light unit (RLU) reading.

In the rare event of any unusual light output, the algorithm labels this as “Inspect.” 3M recommends the user to repeat the assay for any Inspect samples. If the result continues to be Inspect, proceed to confirmation test using your preferred method or as specified by local

AOAC Research Institute Expert Review Panel Use Only

regulations.

k) Confirmation

All samples of primary enrichments were confirmed by following the appropriate reference method confirmation, beginning with transfer from the primary enrichment to secondary enrichment broth (if applicable), followed by subsequent plating and confirmation of isolates

using appropriate biochemical and serological methods.

4) Reporting Raw Data

a) Report data using the data report form in Appendix 8.2.

b) Upon completion the laboratory will fax or email the completed data form to the Study Director.

DRAFT DOCUMENT

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