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QIAGEN
mericon
STEC Workflow Collaborative Study Protocol
May 2016
DRAFT
9
(
d
)
Microtubes, 2.0 mL, with caps.-
MLS.
(
e
)
Modified tryptone soy broth with casamino acids (mTSB + CAA)
.-prepared according to FSIS MLG
1.08, or equivalent commercial formula.
(
f
)
Water.-
Nuclease-free PCR grade, or equivalent, MLS.
E.
Safety Precautions
(
a
)
mericon E. coli O157 Screen Plus Kit, mericon E. coli STEC O-Type
Kit,
and mericon DNA Bacteria
Kit.-
All chemicals should be considered potentially hazardous. When working with chemicals, always
wear a suitable lab coat and disposable gloves. Product usage should follow good laboratory practices.
(
b
)
QIAsymphonymericonBacteria Kit.-
The buffers in the reagent cartridge contain guanidine salts,
which can form highly reactive compounds when combined with bleach. If liquid containing these buffers is
spilled, clean with a suitable laboratory detergent and water. If the spilled liquid contains potentially
infectious agents, clean the affected area first with laboratory detergent and water, and then with 1% (v/v)
sodium hypochlorite solution. Reagents in this kit are highly flammable and are harmful by inhalation,
skin contact and swallowing.
(
c
)
Real-Time PCR System
.-Improper use of the Rotor-Gene Q may cause personal injuries or damage
to the instrument. The Rotor-Gene Q must only be operated by qualified personnel who have been
appropriately trained. Servicing of the Rotor-Gene Q must only be performed by QIAGEN Field Service
Specialists.
(
d
)
Enrichment.-
Shiga-toxin producing
E. coli
is a Biosafety Level 2 organism. Biological samples such
as enrichments have the potential to transmit infectious diseases. Follow all applicable local,
state/provincial, and/or national regulations on disposal of biological wastes. Wear appropriate
protective equipment, which includes but is not limited to protective eyewear, face shield, clothing/lab
coat, and gloves. All work should be conducted in properly equipped facilities utilizing the appropriate
safety equipment (for example, physical containment devices). Individuals should be trained in
accordance with applicable regulatory and company/institution requirements before working with
potentially infectious materials. All enrichment broths should be sterilized following any culture-based
confirmatory steps.
F.
General Preparation
(
a
)
General.-
(1) Use aseptic techniques. (2) Use Filter bags during enrichment to minimize
particulates. (3) Clean the workstations with a disinfectant of choice (e.g., sodium hypochlorite solution,
phenol solution, Quaternary ammonium solution, etc.) before and after use.
Note: Do not use sodium
hypochlorite for cleaning the QIAsymphony.
(4) Separate work areas for the following: media
preparation, sample preparation, DNA lysis, and DNA amplification and detection. (5) Do not reuse kit
disposables. (6) Change pipette tips between samples. (7) Wear personal protective equipment.
(
b
)
DNA Lysis.-
(1) Pre-warm heat block to 100 ± 1°C. (2) Change pipette tips between sample
transfers. (3) Do not disrupt the pellet when discarding the supernatant. (4) Be sure to allow the samples
to cool to room temperature (24 ± 2°C) after the heat lysis procedure.
OMAMAN-36 B : Collaborative Study Protocol
For ERP Use Only
January 2017
AOAC Research Institute
Expert Review Panel Use Only