hygiena KIT230077 STEC Screening LyoKit Instruction Manual
- June 22, 2024
- Hygiena
Table of Contents
hygiena KIT230077 STEC Screening LyoKit
Specifications
- Product Name: STEC Screening LyoKit
- Product No.: KIT230077 /78
- License Number: 102004
- Approval: Revision A, May 2024
Product Information
Number of Reactions
The kit is designed for 96 reactions with a final reaction volume of 25 μL
each. Up to 94 samples plus positive and negative control can be analyzed per
run.
Storage and Stability
Store all components at 2 to 8 °C. They are guaranteed to be stable through
the expiration date printed on the label. Opening of the kit does not shorten
the expiration date. The PCR strips must be stored in the provided aluminum
bag with silica gel pads. Protect from light and moisture.
LyoKit Tube Profiles
The LyoKit is available in two different tube profiles: white low profile
tubes, 0.1 mL (LP) and clear regular profile tubes, 0.2 mL (RP). The majority
of real-time PCR cyclers use low profile tubes, 0.1 mL (LP). However, the
Dualo 32® R2 and a few other cyclers require clear low profile tubes, 0.1 mL
(DP) or clear regular profile tubes, 0.2 mL (RP). Please refer to the cycler
and tube compatibility chart for more information.
Applicability
The foodproof STEC Screening LyoKit is intended for the rapid detection of
Shiga toxinproducing E. coli DNA isolated from enrichment cultures prepared by
valid methods with relevant kinds of samples that are potentially contaminated
with these microorganisms, e.g., meat or sprouts. The kit described in these
Product Instructions has been developed for real-time PCR instruments with a
FAM, a HEX, a ROX and a Cy5 detection channel. The performance of the kit was
tested with the following real-time PCR instruments: BAX® System Q7,
LightCycler® 480, LightCycler® 96 (Roche Diagnostics), AriaMx, Mx3005P®
(Agilent Technologies), CFX96TM (Bio-Rad), PikoReal24 and Applied BiosystemsTM
7500 Fast (Thermo Scientific). Detection of Shiga toxin-producing Escherichia
coli with the foodproof STEC Screening LyoKit is in accordance with ISO/TS
13136. The kit must not be used in diagnostic procedures.
Kit Contents
A schematic representation of the foodproof STEC Screening LyoKit with all
its components.
- LP: KIT230077
- RP: KIT230078
Tube profile and instrument compatibility chart is available online
Different tube profiles are available: white low-profile tubes (LP) and clear regular-profile tubes (RP).
Product Usage Instructions
Required Material
Gather enrichment cultures prepared by valid methods, potential sample
sources, and necessary lab equipment. Most of the required equipment and
reagents are available through Hygiena®. Please contact us for further
information. Use a real-time PCR cycler suitable for detection of respective
probes as well as for using low or regular profile strip tubes. In case the
strip tubes don’t fit for the instrument, the samples should be transferred to
appropriate PCR vessels after resuspension of the lyophilized PCR mix.
- Nuclease-free, aerosol-resistant pipette filter tips.
- PCR strip / plate centrifuges
- Without vortex: Mini microcentrifuge for 4 x 8-strips
- With vortex: Multispin MSC-6000 for 4 x 8-strips
- With vortex: CVP-2 for 12 x 8-strips and plates
- DNA Extraction Kits
- foodproof StarPrep Three Kit (KIT230187) or
- foodproof Magnetic Preparation Kit I (KIT230180)
Precautions and Preparations
The kit provides all reagents required for the PCR. However, in order to
achieve reliable results, the entire assay procedure must be performed under
nuclease-free conditions.
Follow the instructions below to avoid nucleases, carry-over or cross- contamination:
- Keep the kit components separate from other reagents in the laboratory.
- Use nuclease-free labware (e.g., pipettes, pipette tips, reaction vials).
- Wear gloves when performing the assay.
- To avoid cross-contamination of samples and reagents, use fresh aerosol barrier pipette tips.
- To avoid carry-over contamination, transfer the required solutions for one experiment into a fresh tube, rather than directly pipetting from stock solutions.
- Physically separate the workplaces for DNA preparation, PCR setup and PCR to minimize the risk of carry-over contamination. Use a PCR hood for all pipetting steps.
- Sample Material: Use any sample material suitable for PCR in terms of purity, concentration and absence of inhibitors.
- DNA Extraction: We provide sample preparation kits suitable for all kind of food and other samples.
- Positive Control: Always run a positive control with the samples. Use the provided control DNA (Control Template) or a positive sample preparation control.
- Negative Control: Always run a negative control with the samples. To prepare a negative control, replace the template DNA with PCR-grade water. Include a negative control during sample preparation to monitor reaction purity and cross-contamination. This extraction control can be used as an additional negative control reaction.
- Confi rmation: If required, positive results may be confi rmed by appropriate methods (e.g., reference method).
- Waste Disposal: All contaminated and potentially infectious material, like enrichment cultures or food samples, should be autoclaved before disposal and eliminated according to local rules and regulations. For more information, e.g., proper disposal of unused chemicals, please refer to the appropriate safety data sheet (SDS).
Keep the PCR mix away from light and moisture. For more information, please refer to the appropriate safety data sheet (SDS). The SDS is available online at www.hygiena.com/sds.
Enrichment and DNA Extraction
Follow certified methods for enrichment and DNA extraction from the samples.
The foodproof STEC Screening LyoKit is intended for the rapid detection of
Shiga toxinproducing E. coli DNA isolated from enrichment cultures. For DNA
extraction, please use the kits mentioned in 2.1 Required Material.
Certified Methods:
Refer to the provided documentation for approved enrichment and DNA extraction
protocols. The foodproof STEC Screening LyoKit was validated according to the
AOAC RI Performance Tested MethodsSM program (License No. 102004) for the
category raw meat for screening and for confirmation of Shiga toxin- (stx1 &
stx2) and intimin- (eae) positive E. coli STEC strains. The validation
includes 375 g test portions enriched in Modified Tryptone Soy Broth (mTSB)
(1:4) at 42 ± 1 °C for 12 to 24 h and 25 g test portions enriched in mTSB
(1:10) for 8 to 24 h. DNA isolation was done according to the package insert
of the foodproof StarPrep® Three Kit – Extraction Procedure A. For 25 g meat
samples with 8 to 20 h enrichment time and for 375 g meat samples with 12 to
20 h enrichment time, 500 μL of the enrichment culture were used for DNA
extraction. For enrichment times between 20 h and 24 h, 100 μL of the
enrichment culture were used for DNA extraction. The foodproof STEC Screening
LyoKit was validated in combination with the foodproof STEC Identification
LyoKit and positive samples were confirmed via the method described in Annex A
in the package insert of the foodproof STEC Identification LyoKit and via the
reference method described in the USDA/FSIS-MLG 5C.00.
Procedure
Follow the workflow, program setup, and data interpretation steps as outlined
in the user manual. This protocol describes how to perform the analysis of DNA
extracts by real-time PCR.
Workflow:
A step-by-step process for sample preparation, PCR setup, and result
interpretation.
-
PLACE STRIPS IN THE RACK
Take the needed number of PCR tube strips out of aluminium Important: close bag tightly afterwards. Place strips in a suitable -
PCR tube rack.
If needed, gently tap the tubes to move the lyophilized pellets to the bottom of all tubes. -
DECAP
Open strips carefully immediately before filling and discard caps.
Important: Recap immediately after hydration. Do not leave it open longer than necessary. -
ADD SAMPLES AND CONTROLS
Pipette 25 μL of samples, negative control (colourless cap) or Control Template (purple cap) into respective wells. If using less volume, add PCR- grade H2O to reach 25 μL. To reduce the risk of cross-contamination, prepare only one strip at a time. -
SEAL
Seal the tubes with the provided 8-cap strips tightly. -
MIX
Resuspend the pellet after sealing by mixing thoroughly. Alternatively, resuspend the pellet by pipetting up and down multiple times in step 3. -
CENTRIFUGE
Briefly spin strips, e.g., 5 sec at 500 – 1,000 x g, in a suitable centrifuge. -
START REAL-TIME PCR RUN
Cycle samples as described in the program setup (2.4.2). Place tubes in a vertical, balanced order into the cycler, e.g., two strips can be placed in the fi rst and last column.
Program Setup:
Configure the PCR machine according to the kit instructions for optimal
performance. Program your real-time PCR instrument before setting up the PCR
reactions. Select the following channels:
- FAM (stx1), HEX (stx2), ROX (eae), and Cy5 (Internal Control).
For some real-time PCR instruments, the probe quencher as well as the usage of a passive reference dye has to be specified. This kit contains probes with a non-fluorescent “dark” quencher and no passive reference dye. A Color Compensation is necessary for users of the LightCycler 480 System: Color Compensation Set 3 (Product No. KIT230005). For users of the Agilent Mx3005P instrument: Click “Instrument” and “Filter Set Gain Settings” to open the Filter Set Gain Settings dialog box in which the gain settings may be viewed and modified: for FAM the Filter Set Gain Setting has to be modified to “x1”.
Data Interpretation:
Analyze the results based on the provided guidelines to determine the presence
of target genes. Verify results of positive (Control Template) and negative
controls (H2O), before interpreting sample results. Always compare samples to
positive and negative controls. Review data from each channel and interpret
results as described in the table.
Troubleshooting
Consult the troubleshooting section in the manual for common issues and solutions.
Support
Contact customer support for any questions or assistance. If you have
questions or experience any problems with our products, please contact us:
Our aim is to provide you with a solution as quickly and effectively as possible. We would also like you to contact us if you have any suggestions for improving the product or in case you would like to use our product for a different application. We highly value your feedback.
ADDITIONAL INFORMATION
Testing Principle
The foodproof kit provides all necessary reagents and a control template
for reliable interpretations of results. To ensure the maximum reliability of
the kit and to prevent misinterpretation of negative results due to inhibition
of the amplification, an Internal Control (IC) is included. A hydrolysis probe
was designed to bind specifically to the IC, allowing detection in the
respective channel, whereas the target DNA is detected in another channel. In
case of a negative result due to amplification inhibition of the sample DNA of
interest, the amplification of the IC is suppressed as well, whereas a
negative result for the sample DNA of interest and amplification of the IC
clearly indicates the absence of parameter in the sample. The real-time PCR
kit minimizes contamination risk and contains all reagents (except for
template DNA) needed for the detection of target DNA. Primers and probes
provide specific detection of target DNA in food and environmental samples,
including primary production stage samples. The described performance of the
kit is guaranteed for use only on the real-time PCR instruments listed in
Section 1.2: Applicability. For other instruments, please contact us.
Step-by-Step Procedure
- Using the kit’s sequence-specific primers in a polymerase chain reaction (PCR), the PCR instrument and the supplied reagents amplify fragments of specific sequences for target DNA.
- The PCR instrument detects these amplified fragments in real-time through fluorescence generated by cleavage of the hybridized probe due to the 5′ nuclease activity of the Taq DNA polymerase. The probe is labelled at the 5′ end with a reporter fluorophore and at the 3′ end with a quencher.
- During the annealing/elongation phase of each PCR cycle, the probe hybridizes to an internal sequence of the amplicon and is cleaved by the 5′ nuclease activity of the Taq DNA polymerase. This cleavage of the probe separates the reporter dye from the quencher dye, increasing the reporter dye signal.
- The PCR instrument measures the emitted fluorescence of the reporter dye.
Prevention of Carry-Over Contamination
The heat-labile Uracil-DNA N-Glycosylase (UNG) is suitable for preventing
carry-over contamination between PCRs. This technique relies on the
incorporation of deoxyuridine triphosphate (dUTP) during all amplification
reactions, and the pretreatment of all successive PCR mixtures with the heat-
labile UNG. The UNG cleaves DNA at any site where a deoxyuridine residue has
been incorporated. The resulting abasic sites are hydrolyzed due to the high
temperatures during the initial denaturation step, and can no longer serve as
PCR templates. The heat-labile UNG is inactivated during the initial
denaturation step. Native DNA (e.g., the isolated target genomic DNA) does not
contain uracil and is therefore not degraded by this procedure. Since dTTP is
replaced with dUTP and UNG is included in this kit, decontamination can be
achieved with the provided reagents.
Trademarks
foodproof® and LyoKit® are trademarks of Hygiena Diagnostics GmbH. Hygiena®
and BAX® are registered trademarks of Hygiena. Other brand or product names
are trademarks of their respective holders.
License
License Notice
The purchase price of this product includes limited, nontransferable rights
under US Patent No. 7,687,247 owned by Life Technologies Corporation to use
only this amount of the product to practice the claims in said patent solely
for activities of the purchaser for bioburden testing, environmental testing,
food testing, or testing for genetically modified organisms (GMO) in
accordance with the instructions for use accompanying this product. No other
rights are conveyed, including no right to use this product for in vitro
diagnostic, therapeutic, or prophylactic purposes. Further information on
purchasing licenses under the above patent may be obtained by contacting the
Licensing Department, Life Technologies Corporation, 5791 Van Allen Way,
Carlsbad, CA 92008. Email:
outlicensing@lifetech.com.
Reference Number
The reference number and original Hygiena Diagnostics GmbH article numbers: R
602 11-1 (KIT230077) and R 602 11-2 (KIT230078).
Change Index
-
Version 1, September 2013:
First version of the package insert. -
Version 2, March 2017:
License Notice changed. Introduction of vortex centrifuges into the PCR Setup Procedure. -
Version 3, October 2020:
Addition of AOAC certification information -
Version 4, February 2022:
Rebranding, new document layout and content, new product number. -
Revision A, December 2023:
Change of index format, minor editorial changes. R 602 11 20 -> INS- KIT230077-78-REVA
FAQ
Q: Can this kit be used for diagnostic purposes?
A: No, the kit is not intended for diagnostic procedures.
Hygiena
Camarillo, CA 93012
USA
diagnostics.support@hygiena.com
Manufactured byHygiena Diagnostics GmbH
GmbHHermannswerder 17
14473 Potsdam
Germany
www.hygiena.com
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