hygiena KIT 2300 77 STEC Screening LyoKit User Manual

: June 10, 2024
: Hygiena

hygiena KIT 2300 77 STEC Screening LyoKit

STEC Screening LyoKit

Product Information

The STEC Screening LyoKit is a qualitative detection kit used to detect the presence of Shiga toxin-producing Escherichia coli (STEC) by screening for genes of Shiga toxins (stx1 & stx2) and intimin (eae). The kit is approved with license number 102004 and comes with a manual version 4, February 2022. The kit is available in two order numbers: KIT 2300 77 and KIT 2300 78.

General Information

Number of Reactions: Not specified
Storage and Stability: Not specified
LyoKit Tube Profiles: white low profile tubes (LP) and clear regular profile tubes (RP)

Applicability

The STEC Screening LyoKit is applicable for the qualitative detection of STEC in various samples.

Kit Contents

Component	Details
Microplate	12 x 8-tube strips, prefilled with lyophilized ready-to-use PCR

mix. Available are different tube profiles: white low profile tubes
(LP), and clear regular profile tubes (RP).
12 x 8-cap strips| For use in real-time PCR after addition of samples.
2 x H2O PCR-grade (colorless cap)| Not specified.

Product Usage Instructions

Required Materials

STEC Screening LyoKit
Sample
PCR instrument and software
Pipettes and tips
Nuclease-free water
Microcentrifuge tubes
Sterile loops or pipette tips for inoculation
Incubator

Precautions and Preparations

Read the manual carefully before starting the procedure.
Wear gloves and a lab coat to prevent contamination.
Use nuclease-free water to prepare samples and reagents.
Follow aseptic techniques while handling samples and
reagents.
Ensure that the PCR instrument is calibrated and functioning
properly.
Store the kit components as instructed in the manual.

Enrichment and DNA Extraction

Certified Methods

The kit recommends using certified methods for sample enrichment and DNA extraction. These methods are not included in the kit and should be performed before starting the procedure.

Procedure

Workflow

Enrichment and DNA extraction of the sample using certified methods.
Prepare the PCR reaction mix by reconstituting the lyophilized mix in the microplate tubes with nuclease-free water.
Add the extracted DNA to the PCR reaction mix and mix well.
Transfer the PCR reactions to the 12 x 8-cap strips.
Perform real-time PCR on the strips using a PCR instrument and software.
Interpret the results using the data interpretation guidelines provided in the manual.

Program Setup

The program setup for real-time PCR may vary depending on the instrument and software used. Follow the manufacturer’s instructions for setting up the program.

Data Interpretation

Interpret the results according to the guidelines provided in the manual. Positive and negative controls should be included in each run for validation purposes.

Troubleshooting

If any issues arise during the procedure, refer to the troubleshooting section in the manual for guidance.

Support

If further assistance is required, contact technical support as provided in the manual.

Additional Information

Testing Principle

The STEC Screening LyoKit detects genes of Shiga toxins (stx1 & stx2) and intimin (eae) using real-time PCR.

Trademarks
No trademarks are mentioned in the manual.

Reference Number
The kit is approved with license number 102004.

Change Index
The change index is not specified in the manual.

OVERVIEW

General 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 °C 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. Protect from light and moisture.

LyoKit Tube Profiles
The LyoKit is available in three different tube profiles: white low profile tubes (LP), clear regular profile tubes (RP), and clear low profile tubes (DP).
The majority of real-time PCR cyclers use low profile tubes (LP). For the Dualo 32® R2 and a few other cyclers, please use clear low profile tubes (DP). For a detailed overview, please have a look at our compatibility chart.

Applicability
The foodproof® STEC Screening LyoKit – 5’Nuclease – is intended for the rapid detection of Shiga toxin-producing E. coli DNA isolated from enrichment cultures prepared by valid methods and inoculated with all relevant kinds of foods and primary production stage (PPS) samples that are potentially contaminated with Shiga toxin-producing E. coli.
The kit described in this Instruction Manual 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: LightCycler® 480, LightCycler® 96 (Roche Diagnostics), AriaMx, Mx3005P (Agilent Technologies), CFX96 (Bio-Rad) 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.

Kit Contents
A schematic representation of the foodproof® STEC Screening LyoKit with all its components.

LP: KIT 2300 77
RP: KIT 2300 78

	Component	Details

1

| ****

Microplate

| 12 x 8-tube strips, prefilled with lyophilized ready-to-use PCR mix.

Available are different tube profiles: white low profile tubes (LP), and clear regular profile tubes (RP).*

2| 12 x 8-cap strips| For use in real-time PCR after addition of samples.
3| 2 x H2O PCR-grade (colorless cap)| 1 ml nuclease-free, for use as a PCR run negative control.

4

| Control Template (purple cap)| 250 µl, contains a stabilized solution of DNA for use as a PCR run positive control.

Tube profile and instrument compatibility chart is available online: www.bc- diagnostics.com/foodproof-compatibility-chart

INSTRUCTIONS

Required Material

Most of the required equipment and reagents are available through HygienaTM. 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.

Material

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

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 nuclease, 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 samples and primary production stage 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.
Confirmation: If required, positive results may be confirmed 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.bc-diagnostics.com.

Enrichment and DNA extraction

The foodproof® STEC Screening LyoKit is intended for the rapid detection of Shiga toxin-producing E. coli DNA isolated from enrichment cultures prepared by valid methods and inoculated with all relevant kinds of foods and primary production stage (PPS) samples.

Certified Methods
The foodproof® STEC Screening LyoKit was validated according to the AOAC RI Performance Tested MethodsSM program (license number 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

This protocol describes how to perform the analysis of DNA extracts by real- time PCR.

Workflow

PLACE STRIPS IN RACK
Take needed number of PCR tube strips out of aluminum bag.
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 direct before fi lling and discard caps.
Important: do not leave open longer than necessary.
ADD SAMPLES AND CONTROLS Pipette 25 µl of samples, negative control (colorless 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 pellet after sealing by mixing thoroughly.
Alternatively, resuspend pellet by pipetting up and down multiple times in step 3.
CENTRIFUGE
Briefl y 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

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).

hygiena-KIT-2300 77-STEC-Screening-LyoKit-7

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 (Order No. KIT 2300 05).
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
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.

FAM	HEX	ROX	Cy5	Result Interpretation
+	+	+	+ or –	Positive for stx1 , stx2 and eae
–	+	+	+ or –	Positive for stx2 and eae
+	–	+	+ or –	Positive for stx1 and eae
+	+	–	+ or –	Positive for stx1 and stx2
–	+	–	+ or –	Positive for stx2
+	–	–	+ or –	Positive for stx1
–	–	+	+ or –	Positive for eae
–	–	–	+	Negative for STEC
–	–	–	–	Invalid

Troubleshooting

Problem	Possible Cause	Recommendation

Squashed or crooked tubes, or open / dislodged tube lids after run, or the cycler does not open or close properly.| Wrong tube format.| Choose the correct tube format for your cycler. Tube profile and instrument

compatibility chart is available online: www. bc- diagnostics.com/compatibility-chart

If necessary, the samples can be transferred to appropriate PCR vessels after resuspension of the lyophilized PCR mix.

Wrong placement of tubes.| Place tubes into the cycler in a vertical and balanced order, as described in the instructions for the PCR instrument.
No signal increase is observed, even with positive controls.| Incorrect detection channel has been chosen.| Set channel settings for respective dyes accordingly.
Pipetting errors.| Check for correct reaction setup and repeat the PCR run.

Always run a positive control along with your samples.

No data acquisition programmed.| Check the cycle programs.
A sample shows no signals, including the internal control. Positive and negative control have proper signals.| Inhibitory effects of the sample material (e.g., caused by insufficient purification).| Use the recommended DNA extraction kit.

Dilute samples or pipette a lower amount of sample DNA (e.g., 20 µl PCR-grade water and 5 µl sample instead of 25 µl sample).

Negative control samples are positive.| Carry-over contamination.| Exchange all critical solutions and reagents for DNA/RNA extraction.

Repeat the complete experiment with fresh batches of all reagents.

Always handle samples, kit components and consumables in accordance with commonly accepted practices to prevent carry-over contamination.

Add positive controls after sample and negative control reaction vessels have been sealed.

Fluorescence intensity is too low.| Inappropriate storage of kit components.| Store lyophilized PCR mix at 2 °C to 8 °C, protected from light and moisture.
Low initial amount of target DNA.| If possible, increase the amount of sample DNA. Depending on the chosen DNA isolation method, inhibitory effects may occur.
Strong decrease of fluorescence baseline.| Resuspension of lyophilized PCR mix not complete.| Always resuspend lyophilized PCR mix thoroughly.

Use the recommended vortex centrifuge with the correct settings.

---|---|---
Fluorescence intensity varies or changes abruptly during the run.| Insufficient centrifugation of the PCR strips, e.g., resuspended PCR mix is still in the upper part of the vessel or bubbles trapped in the mix.| Always centrifuge PCR strips.

Use the centrifuge models and settings recommended in this manual.

Avoid the introduction of air bubbles during pipetting.

Outer surface of the vessel or the seal is dirty (e.g., by direct skin contact).| Always wear gloves when handling the vessels and seal.

Do not mark vessels on the outside of the tubes or directly on top of the reaction mix.

Pellets are difficult to dissolve.| The lyophilized PCR mix started to rehydrate.| Store the lyophilized PCR mix always in the aluminum bag with the silica gel pads. Make sure that the lids are tightly closed.

Remove strips from the aluminum bag only shortly before PCR setup.

Open strip shortly before filling.

Support

If you have questions or experience any problems with our products, please contact us:

www.hygiena.com/technical-support-request

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 appl feedback.

ADDITIONAL INFORMATION

Testing Principle

The foodproof® kit provides all necessary reagents and a control template for reliable interpretations of results. To ensure 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 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 inhibition of the amplification by 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 above.

Step-by-Step Procedure

Using the kit‘s sequence-specifi c primers in a polymerase chain reaction (PCR), the PCR instrument and the supplied reagents amplify fragments of specifi c sequences for target DNA.
The PCR instrument detects these amplified fragments in realtime through
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 PCR’s. This technique relies on the incorporation of deoxyuridine triphosphate (dUTP) during all amplifi cation 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®, microproof®, vetproof®, ShortPrep®, RoboPrep®, and LyoKit® are trademarks of BIOTECON Diagnostics GmbH.
HygienaTM is a registered trademark of Hygiena.
Other brand or product names are trademarks of their respective holders.

Reference Number
The reference number and original BIOTECON Diagnostics article numbers:
R 602 11 -1, and R 602 11 -2.

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 order number.

Hygiena LLC
CA 93012 Camarillo
USA
www.hygiena.com/technical-support-request
Manufactured by
BIOTECON
Diagnostics GmbH
Hermannswerder 17
14473 Potsdam
Germany
[email protected]
www.bc-diagnostics.com

foodproof®
STEC
Screening LyoKit
Order No.
LP: KIT 2300 77
RP: KIT 2300 78
Kit for 96 reactions (lyophilized) for a maximum of 94 samples
Store kit at 2 °C to 8 °C
For testing of food
and environmental samples
Approval: