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QIAGEN
mericon
STEC Workflow Collaborative Study Protocol
May 2016
DRAFT
3
1.0
Introduction
The goal of this collaborative study is to estimate the reproducibility of the QIAGEN
mericon
® E. coli
O157 Screen Plus and
mericon
® E. coli STEC O-type Pathogen Detection workflow and to compare the
performance of the workflow to the US FSIS MLG 5.09 reference method for detection of
E. coli
O157:H7
in ground beef
.
Performance will be measured by POD statistical analyses, repeatability, and
reproducibility as described in the current AOAC microbiology guidelines. The OMA claim will be limited
to ground beef and beef trim.Raw ground beef (325 g test portions) will be inoculated with
E.
coli
O157:H7and compared to FSIS MLG 5.09 in a paired study. Randomized blind coded test portions of
the high, low and uninoculated batches will be sent to collaborators who will perform
the
mericon
workflowand the referencemethod. Collaborators will have the option to send samples to
QLabs for cultural confirmation. Collaborators will report raw data and QLabs will perform the statistical
analyses.
1.1
Description of the
mericon
STEC workflow
The
mericon
E. coli O157 Screen Plus and E. coli STEC O-Type methodsaremultiplex PCR
assaysthat amplify specific DNA fragments from pathogenic
E. coli
in food and include an
internal control. The internal control provides data regarding the presence of inhibitors in the
tested samples and the overall quality of the PCR run.
These assays were developed for use on the Rotor-Gene Q. The
mericon
E. coli O157 Screen Plus
kit and the
mericon
E. coli STEC O-type kit are not compatible with other real time cyclers. The
PCR Master Mix utilizes HotStarTaq
Plus
DNA Polymerase, patented multiplex PCR technology
such as Factor MP, and fast cycling technology including Q-Bond®. The assays include an
internal control and are used for detecting
E. coli
O157 and STECs, respectively, in raw ground
beef, rawbeef trim and spinach after 10 ± 1hours enrichment.
The methods usemodified Tryptone Soy Brothplus casamino acids (mTSB + CAA) for enrichment
of test portions. A 325-g food sample (ground beef or beef trim) is added to 975 mL of pre-
warmed mTSB + CAA. All samples are homogenized by stomaching at 230 rpm (2 min ± 10 s for
ground beef and beef trim) and incubated at 42 ± 1°C for 10 ± 1 h. There are two DNA
preparation procedures that can be utilized – the manual
mericon
DNA Bacteria Kit and the
automated QIAsymphony
mericon
BacteriaKit. For the manual
mericon
method, a 1-mL aliquot
of enrichment is centrifuged and the pellet is resuspended in Lysis Buffer and heat-treated.
After a second centrifugation, a 10-μL aliquot of the supernatant is used in the PCR reaction.
For the QIAsymphony method, a 0.5-mL aliquot of enrichment is loaded onto the instrument.
The instrument processes 0.4 mL by heat lysis and bead-based DNA extraction and purification.
A 10-μL aliquot of the resultant purified DNA is automatically loaded into a PCR reaction on a
Rotor-Disc-72.
PCR cycling parameters are consistent across all
mericon
pathogen detection assays. The
internal control must be uninhibited for the run to be valid. If the run is valid, the test sample
results are interpreted as positive or negative depending on whether the fluorescence exceeds a
OMAMAN-36 B : Collaborative Study Protocol
For ERP Use Only
January 2017
AOAC R search Institute
Expert Review Panel Use Only