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All robustness parameters were evaluated using raw beef trim inoculated at a level
expected to yield fractional positive results (2 to 8 positive samples out of 10 replicates). A
bulk sample of beef trim was inoculated with
E.coli
O157, mixed thoroughly, and separated
into 25g test portions. An additional 300 g of uninoculated trim was added to create the 325
g test portion. For each operational parameter, 10 inoculated replicates were analyzed
along with 5 un-inoculated replicates. Lysed DNA samples were diluted 1:10 using
nuclease free water.
The first parameter, incubation time, was evaluated by analyzing replicates after 8, 9 and
10 hours+5 minof incubation. The second parameter, manual heat lysis time, was
evaluated by analyzing replicates heated to 100+1ºC in a dry bath for 8, 10 and 12 minutes
(+15 sec). The third parameter, Master Mix to sample ratio, was evaluated by adding
8:12,10:10 or 12:8 µL of PCR Master Mix to DNA sample volume. Both
mericon
Pathogen
Detection Assays were evaluated using nine different treatment combinations. The
treatment parameters are presented in Table 6.
Table 5: Robustness Treatment Conditions
Treatment
Combination
Enrichment
Incubation Time
(Hours)
Lysis Heat Time
(Minutes)
Assay Composition
(Assay Mix µL: DNA µL)
1
8
8
12 µL:8 µL
2
8
8
8 µL:12 µL
3
8
12
12 µL:8 µL
4
8
12
8 µL:12 µL
5
10
8
12 µL:8 µL
6
10
8
8 µL:12 µL
7
10
12
12 µL:8 µL
8
10
12
8 µL:12 µL
9
9
10
10 µL:10 µL
-All test portions were sampled from a single enrichment
Product Consistency (Lot to Lot) and Stability Evaluation
The Method Developer Study covering product consistency and stability was performed in
the method developer’s laboratory (QIAGEN R&D, Hilden Germany)
Product Consistency Study
This study examined three lots of each
mericon
kits for lot-to-lot variability and three Rotor-
Gene Q instruments for variability. Included were the
E.coli
Screen Plus and
E. coli
STEC
O-type PCR kits (3 lots of each primer/probe set and 3 lots of Master Mix), and the DNA
OMAMAN-36 E/ AOAC PTM Report
ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only