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QIAGEN
mericon
STEC Workflow Collaborative Study Protocol
May 2016
DRAFT
20
Ground beef analysis (see Flowchart in Appendix 8.3)
1.
Prewarm modified Tryptic Soy Broth with casamino acids (mTSB+CAA) to 42°C.
2.
Place each pre-weighed325 g test portion into a filtered stomacher bag.
3.
Add 975 mL pre-warmed mTSB+CAA to each bag.
4.
Homogenize with a paddle blender at 230 rpm for 2.0 min ± 10 sec.
5.
Incubate all bags at 42 ± 1°C.
5.1
At sites with multiple collaborators, the enrichment setup should be staggered by at
least two hours between collaborators in order to stagger usage of the shared
RotorGene Q.
6.
Prepare sample and serial decimal dilutions for aerobic plate count (APC) according to BAM Chapter
3. Plate the appropriate dilutions in duplicate on plate count agar using the pour plate technique.
Incubate plates inverted for 48 ± 2 h at 35± 1°C. Count plates and report APCs as described.
7.
At 10 ± 1 h, mix the enrichment bags by hand and transfer and process two aliquots as follows for
the
mericon
method. The same analyst shall perform the manual and automated procedures. After
removing aliquots, return the enrichment bags to the incubator. Refer to the OMA draft method for
method details.
7.1
Pipette 1.0 mL of enrichment into a 2 mL microtube for processing by the manual
mericon
DNA Bacteria Kit and manual assay setup.
7.2
Pipette 500 μL of enrichment into a 2 mL microtube for processing with the automated
QIASymphony
mericon
Bacteria Kit and automated QIAsymphony assay setup.
7.3
Test the resulting DNA samples by the
mericon
E. coli O157 Screen Plus assay following
the method workflow.Record the C
T
values from each channel on the Data Report Form.
7.4
Determine and record the “Next Action” for each enrichment. Refer to the draft OMA
mericon
method for details. Three “Next Action” results are possible.
7.4.1
Test those DNA preps yielding “Additional testing required” in the screening
assay by the
mericon
E. coli STEC O-type assay as described in the method
workflow.
7.4.2
Those DNA preps yielding “No further action” in the screening assay will be
considered negative by the
mericon
method and no further testing will be
performed.
7.4.3
Those DNA preps yielding “Dilute sample and repeat test” are inhibited and
should be diluted 1/10 with RNase-free water and retested in the
mericon
E. coli
O157 Screen Plus assay.
7.5
For those DNA preps from 7.4.1 (Additional testing required), perform the manual
and/or automated assay setup as appropriate and analyze in the
mericon
E. coli O157
Screen Plus Assay following the method workflow. Record the C
T
values from each
channel on the Data Report Form.
7.6
Determine and record the “Next Action” for each enrichment. Refer to the draft OMA
mericon
method for details. There are again three results possible.
OMAMAN-36 B : Collaborative Study Protocol
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only