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QIAGEN
mericon
STEC Workflow Collaborative Study Protocol
May 2016
DRAFT
4
4.3.1
The candidate and reference methods use the same enrichment procedure for
ground beef, differing only in the time of enrichment. Therefore, each
collaborator will receive only one set of ground beef test portions and one
portion for aerobic plate count. Each collaborator will analyze test portions as
follows.
4.3.2
Addeach 325-g test portion to 975 mL pre-warmed (42°C) modified Tryptone
Soy Broth with casaminoacids (mTSB+CAA) in a filtered stomacher bag,
homogenizewith paddle blender for 2.0 ± 10 sec., and enrich at 42±1°C
(Appendix 8.3).
4.3.2.1
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.
4.3.3
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.
4.3.4
At 10±1 h, mix the enrichment bags by hand and remove two aliquots as
follows. 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.
4.3.4.1
Pipette 1.0 mL of enrichment into a 2 mL microtube for processing by
the manual
mericon
DNA Bacteria Kit and manual assay setup.
4.3.4.2
Pipette 500 μL of enrichment into a 2 mL microtube for processing with
the automated QIASymphony
mericon
Bacteria Kit and automated
QIAsymphony assay setup.
4.3.4.3
Test the resulting DNA preps 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.
4.3.4.4
Determine and record the “Next Action” for each enrichment. Refer to
the draft OMA
mericon
method for details. Three “Next Action” results
are possible.
4.3.4.4.1
For those DNA preps yielding “Additional testing required”,
proceed to the
mericon
E. coli STEC O-type assay.
4.3.4.4.2
Those DNA preps yielding “No further action” in the
screening assay will be considered negative and no further
testing using the
mericon
kit will be performed.
4.3.4.4.3
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.
OMAMAN-36 B : Collaborative Study Protocol
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only