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QIAGEN
mericon
STEC Workflow Collaborative Study Protocol
May 2016
DRAFT
10
(
c
)
DNA Amplification
.-(1) Store PCR loading block at 2-5 °C. (2) Use aseptic techniques. (3) Change
pipette tips between samples. (4) Change pipette tips for the PCR master mix and the DNA samples. (5)
Use gloves and protective laboratory wear. (6) Do not touch any PCR equipment or supplies without
wearing gloves. (7) In case of contamination, laboratory benches and pipettes can be decontaminated
with a 1/10 dilution of commercial bleach solution.
(
d
)
Enrichment medium.-
Pre-warm mTSB + CAA to 42 ± 1°C.
G.
Test Portion Enrichment
(
a
)
Raw ground beef and raw beef trim.-
Add a 325 g test portion to 975 mL mTSB + CAApre-warmed
to 42 ± 1°C. Homogenize in a paddle blender for 2.0 min ± 10 sec and incubate at 42 ± 1°C for 10 ± 1 h.
H.
DNA Extraction and Assay Setup
(
a
)
Manual mericonDNA Bacteria Kit method
.-(1) Gently mix enriched test portions to disperse
organisms throughout the enrichment broth. (2) Transfer 1.0 mL of enrichment culture to a sterile 2 mL
screw cap tube and centrifuge for 5 minutes at 13,000 x
g
. (3) Discard the supernatant with a pipettor,
taking care not to disrupt the pellet. (4) Add 200 μL Fast Lysis Buffer to the bacterial pellet, tightly cap
the tube, and resuspend the pellet by brief, vigorous vortex mixing. (5) Place the microcentrifuge tube
into a heat block at 100 ± 1°C for 10 min ± 10 sec. (6) Remove the tube and allow it to cool to 15-24°C for
2 min ± 10 sec. (7) Centrifuge the tube at 13,000 x
g
for 5 min. (8) Transfer 100 μL of the supernatant to
a fresh 1.5 mL microcentrifuge tube. This is the extracted DNA. (9) Dilute an aliquot of the extracted
DNA 1/10 with RNase-free water.
(
b
)
Manual Assay Setup.-
(1) Place the desired number of PCR 72-well strip tubes into the adapters of
the cooling block for the Rotor-Gene Q. (2) Reconstitute
mericon
Assay by adding 130 μL Multiplex PCR
Master Mix (tube(s) with blue lid) to each vial of
mericon
Assay (yellow lid). (3) Add 10 μL
reconstituted
mericon
Assay to each strip tube. (4) Setup sample reactions by adding 10 μL sample DNA
to each appropriate strip tube. (5) Setup control reaction by adding 10 μLRNase-free water to the
negative PCR control strip tube. (6) Proceed to Analysis.
(
c
)
Automated QIAsymphony Bacteria Kit method.-
(1) Gently mix enriched test portions to disperse
organisms throughout the enrichment broth. (2) Transfer 500 μL of each enrichment culture into sterile
2 mL microtubes. (3) Close all the drawers and hoods of the QIAsymphony SP/AS instrument. (4) Switch
on the instrument and wait until the “Sample Preparation” screen appears and the initialization
procedure as finished. (5) Log in to the instrument. (6) Ensure the “Waste” drawer is prepared properly
and perform an inventory scan of the “Waste” drawer, including the tip chute and liquid waste. Replace
the tip disposal bag if necessary. (7) Load the required elution rack into the “Eluate” drawer and perform
an inventory scan of the “Eluate” drawer. (8) Load the required reagent cartridge(s) and consumables
into the “Reagents and Consumables” drawer. (9) Press the “R+C” button in the touchscreen to open the
screen that shows the consumables status (“Consumables/8-RodCovers/Tubes/Filter-tips/Reagent
Cartridges”). Press the “Scan Bottle” button to scan the bar code of the TopElute bottle with the
handheld bar code scanner. Press the “OK” button. (10) Perform an inventory scan of the “Reagents and
Consumables” drawer. (11) Place the samples into the appropriate tube carrier and load them into the
OMAMAN-36 B : Collaborative Study Protocol
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only