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3M MDA2 E. coli O157:H7 Collaborative Study
May 2016
OMA-2016-May-XXXX
DRAFT DOCUMENT
8
a)
Invert room temperature (20-25
o
C) lysis tubes to mix. Proceed to next step within 4 hours.
1
A 20 µL aliquot of each enriched sample is transferred to separate lysis tubes using a new
2
pipette tip after each sample transfer. Place uncovered samples in the 3M Molecular
3
Detection Heat Block for 15 ± 2 minutes at 100 ± 1
o
C. During heating, the lysis samples will
4
change from pink (cool) to yellow (hot). Following the heat lysis transfer samples place
5
samples in the 3M Molecular Detection Chill Block Insert for 10 ± 1 minutes. The 3M
6
Molecular Chill Block Insert, used at ambient temperature without the 3M Molecular
7
Detection Chill Block Tray, should sit directly on the laboratory bench. When cool, the lysis
8
solution will revert to a pink color.
9
b)
Add 20 µL of each lysed sample and control to separate reagent tubes, and mix by pipetting
10
up and down five times. Analyze a matrix control tube with the samples for each matrix to
11
verify that no interference with the assay was caused by the matrix. Use sterile 3M BPW ISO
12
for the Negative Control (NC). Transfer a 20 µL aliquot to the NC and the Reagent Control
13
(RC) tubes.
14
c)
Add a 20 µL aliquot of a randomly picked sample to the matrix control tube, mixed, and
15
recapped. Using the 3M
™
software, follow prompts to identify samples and controls. Load
16
all samples into the Speed Loader Tray (SLT), place into the Molecular Detection System, and
17
initiate the 3M
™
MDA2 -
E. coli
O157
(including H7) assay. Results are obtained within 60
18
minutes.
19
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j)
Interpretation and Test Result Report
21
22
An algorithm interprets the light output curve resulting from the detection of the nucleic acid
23
amplification. Results are analyzed automatically by the software and are color-coded based on
24
the result. A Positive or Negative result is determined by analysis of a number of unique curve
25
parameters. Presumptive positive results are reported in real-time while Negative and Inspect
26
results will be displayed after the run is completed.
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NOTE: Even a negative sample will not give a zero reading as the system and 3M MDA2 –
E. coli
29
O157 (including H7) amplification reagents have a “background” relative light unit (RLU) reading.
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In the rare event of any unusual light output, the algorithm labels this as “Inspect.” 3M
32
recommends the user to repeat the assay for any Inspect samples. If the result continues to be
33
Inspect, proceed to confirmation test using your preferred method or as specified by local
34
regulations.
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36
k)
Confirmation
37
38
All samples of primary enrichments were confirmed by following the appropriate reference
39
method confirmation, beginning with transfer from the primary enrichment to secondary
40
enrichment broth (if applicable), followed by subsequent plating and confirmation of isolates
41
using appropriate biochemical and serological methods.
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4)
Reporting Raw Data
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45
a)
Report data using the data report form in Appendix 8.2.
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47
b)
Upon completion the laboratory will fax or email the completed data form to the Study Director.
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OMAMAN-35 B: Collaborative Study Protocol
ERP USE ONLY
January 2017
AOAC Research Institute
Expert Review Panel Use Only