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QIAGEN
mericon
STEC Workflow Collaborative Study Protocol
May 2016
DRAFT
5
4.3.4.5
For those DNA preps from 4.3.4.4.1 (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.
4.3.4.6
Determine and record the “Next Action” for each enrichment. Refer to
the draft OMA
mericon
method for details. There are again three results
possible.
4.3.4.6.1
Those DNA preps yielding “Additional testing required” are
positive for STEC, either
E. coli
O157:H7 (orange channel
positive) or
E. coli
serogroups O26, O45, O103, O111, O121,
or O145 (Green channel positive).
4.3.4.6.2
Those DNA preps yielding “No further action” are negative
for STEC, including
E. coli
O157:H7, O26, O45, O103, O111,
O121, and O145.
4.3.4.6.3
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 STEC O-Type assay.
4.3.4.7
Regardless of
mericon
results, proceed to step 4.3.5 for confirmation of
all enrichments using the MLG 5.09 reference method for
E. coli
O157:H7.
4.3.5
At 15-24 h, mix the enrichment bags by hand and remove an aliquot for
processing according to MLG 5.09 as summarized below.Refer to MLG 5.09 for
details. [Alternatively, if pre-arranged, remove a 20-mL aliquot of each
enrichment and place into appropriate screw-cap tubes for shipment to QLabs
for confirmation.Follow instructions provided by QLabs for shipment. Make a
note on the Data Report Form of the date when enrichment aliquots were
shipped.]
4.3.5.1
Performa screening assay specified in MLG 5A.04. Record which
screening assay was used and the results.
4.3.5.2
Regardless of the screening assay results, perform anti-O157 IMS on an
aliquot from each enrichment bag.
4.3.5.3
After performing IMS, plate acid-treated and untreated IMS beads
onmRBA. Test resultant typical colonies by latex agglutinationfor O157
and transfer up to 5 positive colonies to SBA.
4.3.5.4
Test isolated colonies on SBA by latex agglutination for O157 and H7,
confirmfor toxin production byBAX STEC Screen Assay or Premier EHEC
EIA, and determine biochemical ID by VITEK 2 GN or API20E.
4.3.5.5
Record all results on the Data Report Form.
4.4
Most Probable Number (MPN) Analysis
OMAMAN-36 B : Collaborative Study Protocol
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only