hygiena KIT 2300 42 E.coli O157 Detection Kit User Manual

hygiena KIT 2300 42 E.coli O157 Detection Kit

Product Information

The E.coli O157 Detection Kit is a qualitative detection kit used for the detection of Escherichia coli serogroup O157, which includes serotype Escherichia coli O157:H7. The kit is approved with license number 100601 and comes with a manual version of 5,
dated February 2022. The kit contains all the necessary components required for the detection process, including Master Mix, Enzyme Solution, Internal Control, Control Template positive control, and Negative Control.

Kit Contents

detection process
Negative Control (transparent cap)| Component required for the detection process

Applicability

The E.coli O157 Detection Kit can be used in a variety of settings, including clinical laboratories, food safety laboratories, and environmental testing laboratories. The kit is designed for use in the qualitative detection of Escherichia coli serogroup O157, including serotype Escherichia coli O157:H7.

Storage and Stability

The kit should be stored at 2-8°C and is stable until the expiration date listed on the kit. Once opened, the kit should be used within the expiration date.

Product Usage Instructions

Required Materials
Most of the required equipment and reagents are available through HygienaTM. Please contact us for further information.

Precautions and Preparations

Before starting the detection process, it is important to follow the necessary precautions and preparations. This includes ensuring that all required equipment and reagents are available, preparing the necessary buffers, and following all safety protocols.

Enrichment and DNA Extraction

Enrichment and DNA extraction are important steps in the detection process. Certified methods should be followed for enrichment and DNA extraction.

Procedure

The detection process involves a workflow, program setup, and data interpretation. These steps should be followed carefully to ensure accurate results.

Workflow

The workflow involves several steps, including sample preparation, DNA extraction, PCR amplification, and detection of amplified products.

Program Setup
The program setup involves setting up the PCR reaction and running the program according to the specifications provided in the manual.

Data Interpretation

Data interpretation involves analyzing the results of the detection process and determining whether Escherichia coli serogroup O157 is present in the sample.

Troubleshooting

If any issues arise during the detection process, it is important to follow the troubleshooting steps provided in the manual.

Support

If further support is needed, please contact the manufacturer for assistance.

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 -25 °C to -15 °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.

Applicability
The kit described in this Instruction Manual has been developed for real-time PCR instruments with a FAM and a VIC/HEX detection channel. The performance of the kit was tested with the following real-time PCR instruments: LightCycler® 480 (Roche Diagnostics), Applied BiosystemsTM 7500 Fast (Thermo Scientific), Mx3005P® (Agilent), and others.
The foodproof® E. coli O157 Master Mix (vial 1, yellow cap) is sequence- specific for a highly conserved sequence within the O antigen gene cluster of Escherichia coli of the serogroup O157. Inclusivity has been tested with 60 strains with known serogroup O157 whereas all of them could be detected (100% inclusivity). The exclusivity has been determined using 73 E. coli of other serogroup than O157 and 47 Non-E. coli strains.
A relative detection limit of 1 to 10 cells per 25 g sample can be achieved with all kinds of foods. The foodproof® E. coli O157 De Egg powder, coconut, sausages, smoked fish, ice cream, watermelon, sliced cabbage, food dye, pasta, dough, wet and dry pet food: enrichment in lactose broth for 24 h +/- 2 h at 35 °C, sub-cultivation in 1/10 BHI, 3 h at 37 °C tection Kit detects down to 103 – 104 cfu/ml in enrichment cultures, depending on the sample preparation kit used: foodproof® ShortPrep II Kit.

Kit Contents
A schematic representation of the foodproof® E. coli O157 Detection Kit with all its components.

| Component| Details
---|---|---

1

| ****

Master Mix (yellow cap)

| 3 x 600 µl, ready-to-use primer and hydrolysis probe mix specific for parameter DNA and the parameter-specific Internal Control (IC).

Store at -25 °C to -15 °C.

Avoid repeated freezing and thawing! Protect from light!

2

| ****

Enzyme Solution (red cap)

| 3 x 32 µl, contains Taq DNA Polymerase and Uracil-DNA Glycosylase (heat labile) for prevention of carry-over contamination.

Store at -25 °C to -15 °C.

3

| ****

Internal Control (white cap)

| 3 x 32 µl, contains a stabilized solution of plasmid DNA and a yellow dye for better visualization. For use as an internal amplification control.

Store at -25 °C to -15 °C.

Optional: After first thawing store at 2 °C to 8 °C for up to one month.

4

| ****

Control Template (purple cap)

| 1 x 50 µl, contains a stabilized solution of DNA. For use as a PCR run positive control.

Store at -25 °C to -15 °C.

Optional: After first thawing store at 2 °C to 8 °C for up to one month.

5

| ****

Negative Control (transparent cap)

| 1 x 1 ml, contains PCR-grade water. For use as a PCR run negative control. Store at -25 °C to -15 °C.

Optional: After first thawing store at 2 °C to 8 °C for up to one month.

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.

Material

• Nuclease-free, aerosol-resistant pipette filter tips
• Real-time PCR compatible strips or plates with optical cap or foil
• Sterile reaction tubes for preparing PCR mixes and dilutions
• PCR strip or plate centrifuge

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.
• 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).o

Keep the PCR mix away from light.

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® E. coli O157 Detection Kit is intended for the rapid detection of E. coli O157 DNA isolated from enrichment cultures of all kinds of foods and primary production stage (PPS) samples that are potentially contaminated with E. coli O157. The detection kit must not be used in diagnostic procedures.
Pre-enrichment broth and temperature according to DIN EN ISO 16654:2001 or BAM (Chapter 4a) or USDA for 18 to 24 h. Other suitable, validated enrichment procedures can also be used.
Recommended DNA extraction kits:

• KIT 2301 75 / 76 – StarPrep One Kit / large (suitable for most matrices)
• KIT 2301 71 – ShortPrep II (for a very quick extraction)

Certified Methods
The foodproof® E. coli O157 Detection Kit was validated according to the AOAC RI Performance Tested Methodssm program (license number 100601) for detection of E. coli strains of serogroup O157.
This kit is based on the foodproof® E. coli O157 Detection Kit – Hybridization Probes (LightCycler® 1.x, 2.0) which has been AOAC-RI and NordVal validated.
The validation extension includes:

• 25 g plus 225 ml mBPWp at 37 ± 0.5 °C for 24 h. After 5 h, ACV supplements are added, and the samples are incubated static for 18 h. Matrix tested: Egg salad, apple juice. Reference method was according FDA-BAM/USDA.
• 25 g plus 585 ml mTSB + N at 42 ± 0.5 °C for 20 h. Matrix tested: Large Bockwurst. Reference method was according USDA/FSIS MLG 5.04 (2008).

DNA isolation was done according to the package insert of the foodproof® ShortPrep II Kit.

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

Workflow

Thaw the solutions, mix by flicking the tubes four to five times, and briefly spin vials in a microcentrifuge before opening

1. PREPARE PCR MIX
   Add 18 µl of Master Mix (yellow cap), 1 µl Enzyme Solution (red cap) and 1 µl Internal Control (white cap) for each reaction to a suitable tube
   (n samples + 2 controls + at least one additional reaction to cover pipetting loss).
   Mix carefully but thoroughly by pipetting up and down.

2. ADD PCR MIX
   Pipette 20 µl of prepared PCR mix into each strip or plate well.

3. ADD SAMPLES AND CONTROLS
   Pipette 5 µl of samples, negative control (colorless cap) or Control Template (purple cap) into respective wells.

4. SEAL
   Seal strips/plate accurately.

5. CENTRIFUGE
   Briefl y spin strips/plate in a suitable centrifuge.

6. START REAL-TIME PCR RUN
   Cycle samples as described in the program setup (2.4.2).

Program Setup

Program your real-time PCR instrument before setting up the PCR reactions. Select the following channels:

FAM (E.coli O157), and VIC (Internal Control).

As an alternative to VIC, HEX can be used.

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 TAMRA as quencher and no passive reference dye.
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.

Troubleshooting

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.
Reagents are not homogeneously mixed.| Mix reagents thoroughly before pipetting.
Fluorescence intensity varies.| 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.

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 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 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

1. 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.

2. 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 labeled at the 5´-end with a reporter fluorophore and at the 3´-end with a quencher.

3. 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.

4. 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 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®, 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 number: R 302 10.

Change Index
Version 5, February 2022:
Rebranding, new document layout and updated content.

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

References

• Support | Hygiena

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