Hygiena foodproof® E. coli O157 Detection Kit

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

This Instruction Manual describes a real-time PCR kit for instruments with FAM and VIC/HEX detection channels. Performance was tested with instruments including LightCycler® 480 (Roche Diagnostics), Applied Biosystems™ 7500 Fast (Thermo Scientific), and Mx3005P® (Agilent).

The foodproof® E. coli O157 Master Mix is sequence-specific for a conserved sequence within the O antigen gene cluster of Escherichia coli serogroup O157. Inclusivity testing covered 60 strains of serogroup O157 (100% detection). Exclusivity was determined using 73 E. coli strains from other serogroups and 47 Non-E. coli strains.

A relative detection limit of 1 to 10 cells per 25 g sample can be achieved across various food types. For specific food matrices like egg salad or apple juice, detection is down to 10³ - 10⁴ cfu/ml in enrichment cultures after specific incubation steps, depending on the sample preparation kit used (e.g., foodproof® ShortPrep II Kit).

Kit Contents

[Diagram: A schematic representation of the foodproof® E. coli O157 Detection Kit components, showing five numbered vials in a blue box.]

• Component 1 (Master Mix, yellow cap): 3 x 600 µl, ready-to-use primer and hydrolysis probe mix specific for target DNA and Internal Control (IC). Store at -25 °C to -15 °C. Avoid repeated freezing/thawing and protect from light.
• Component 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.
• Component 3 (Internal Control, white cap): 3 x 32 µl, contains a stabilized solution of plasmid DNA and a yellow dye for visualization. Used 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.
• Component 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.
• Component 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 required equipment and reagents are available through Hygiena™. Please contact them for further information.

Essential Equipment:

• A real-time PCR cycler suitable for detection of respective probes.
• 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

To achieve reliable results, the entire assay procedure must be performed under nuclease-free conditions. Follow these instructions to avoid nuclease, carry-over, or cross-contamination:

• Keep kit components separate from other reagents.
• Use nuclease-free labware (pipettes, pipette tips, reaction vials).
• Wear gloves during the assay.
• Use fresh aerosol barrier pipette tips to avoid cross-contamination.
• Transfer solutions to a fresh tube for each experiment to avoid carry-over contamination, rather than pipetting directly from stock solutions.
• Physically separate workplaces for DNA preparation, PCR setup, and PCR to minimize contamination risk. Use a PCR hood for all pipetting steps.
• Sample Material: Use material suitable for PCR in terms of purity, concentration, and absence of inhibitors.
• DNA Extraction: Hygiena provides sample preparation kits suitable for food and primary production stage samples.
• Positive Control: Always run a positive control with samples, using the provided Control Template or a positive sample preparation control.
• Negative Control: Always run a negative control with samples. Prepare by replacing template DNA with PCR-grade water. Include an extraction control during sample preparation for monitoring reaction purity and cross-contamination.
• Confirmation: Positive results may be confirmed by appropriate methods (e.g., reference method) if required.
• Waste Disposal: Autoclave all contaminated and infectious material (enrichment cultures, food samples) before disposal according to local rules. Refer to the safety data sheet (SDS) for disposal of unused chemicals.

Important: Keep the PCR mix away from light.

The SDS is available online at www.bc-diagnostics.com.

Enrichment and DNA extraction

The foodproof® E. coli O157 Detection Kit is for rapid detection of E. coli O157 DNA from enrichment cultures of food and primary production stage (PPS) samples. It is not for diagnostic procedures.

Pre-enrichment should follow DIN EN ISO 16654:2001, BAM (Chapter 4a), or USDA standards for 18 to 24 h. Other validated enrichment procedures are also acceptable.

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 Methods™ program (license number 100601) for detecting 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), validated by AOAC-RI and NordVal.

Validation extension included:

• Matrix: Egg salad, apple juice. Procedure: 25 g sample + 225 ml mBPWp at 37 ± 0.5 °C for 24 h, with ACV supplements added after 5 h, followed by static incubation for 18 h. Reference method: FDA-BAM/USDA.
• Matrix: Large Bockwurst. Procedure: 25 g sample + 585 ml mTSB + N at 42 ± 0.5 °C for 20 h. Reference method: USDA/FSIS MLG 5.04 (2008).

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

Procedure

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

Workflow

Thaw solutions, mix by flicking tubes 4-5 times, and briefly spin vials in a microcentrifuge before opening.

1. Prepare PCR Mix: Add 18 µl Master Mix (yellow cap), 1 µl Enzyme Solution (red cap), and 1 µl Internal Control (white cap) per reaction to a suitable tube (n samples + 2 controls + 1 extra reaction for pipetting loss). Mix carefully by pipetting.
   [Diagram: Shows tubes with volumes 18 µl (Master Mix), 1 µl (Enzyme Solution), and 1 µl (Internal Control) being combined.]
2. Add PCR Mix: Pipette 20 µl of prepared PCR mix into each strip or plate well.
   [Diagram: Shows 20 µl of PCR mix being pipetted into wells.]
3. Add Samples and Controls: Pipette 5 µl of samples, negative control (colorless cap), or Control Template (purple cap) into respective wells.
   [Diagram: Shows 5 µl of sample or control being pipetted into wells.]
4. Seal: Seal strips/plate accurately.
   [Diagram: Shows a plate being sealed.]
5. Centrifuge: Briefly spin strips/plate in a suitable centrifuge.
   [Diagram: Shows PCR strips being spun in a centrifuge.]
6. Start Real-Time PCR Run: Cycle samples as described in the program setup (2.4.2).
   [Diagram: Shows a real-time PCR instrument running a test.]

Program Setup

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

• FAM (E. coli O157) and VIC (Internal Control). HEX can be used as an alternative to VIC.

PCR Program:

• Pre-incubation (1 cycle): Step 1: 37 °C for 4 min; Step 2: 95 °C for 5 min.
• Amplification (50 cycles): Step 1: 95 °C for 5 sec; Step 2: 60 °C for 60 sec (Fluorescence detection).

For some instruments, the probe quencher and passive reference dye must be specified. This kit uses TAMRA as quencher and no passive reference dye.

Agilent Mx3005P users: In "Instrument" > "Filter Set Gain Settings", set FAM gain to "x1".

Data Interpretation

Verify results of positive (Control Template) and negative controls (H₂O) before interpreting sample results. Always compare samples to controls. Review data from each channel and interpret as follows:

Troubleshooting

Support

For questions or problems with Hygiena products, please contact them:

www.hygiena.com/technical-support-request

Hygiena aims to provide quick and effective solutions. Feedback for product improvement or alternative applications is highly valued.

Additional Information

Testing Principle

The foodproof® kit includes all necessary reagents and a control template for reliable result interpretation. An Internal Control (IC) is included to ensure maximum reliability and prevent misinterpretation of negative results due to amplification inhibition. A hydrolysis probe binds specifically to the IC for detection in one channel, while target DNA is detected in another. If amplification is inhibited by sample DNA, the IC amplification is also suppressed. Conversely, if sample DNA is absent and the IC amplifies, it indicates the absence of the target parameter. The real-time PCR kit minimizes contamination risk and provides all necessary reagents (except template DNA) for target DNA detection. Primers and probes ensure specific detection of target DNA in food and environmental samples, including primary production stage samples. The kit's performance is guaranteed only on the listed real-time PCR instruments.

Step-by-Step Procedure Explanation:

1. Using the kit's sequence-specific primers, the PCR instrument and reagents amplify target DNA fragments.
2. The instrument detects amplified fragments in real time via fluorescence generated by cleavage of hybridized probes by Taq DNA polymerase's 5'-nuclease activity. Probes are labeled with a reporter fluorophore (5'-end) and a quencher (3'-end).
3. During annealing/elongation, the probe hybridizes to the amplicon and is cleaved, separating the reporter and quencher dyes, thus increasing the reporter signal.
4. The PCR instrument measures the emitted fluorescence.

Prevention of Carry-Over Contamination

The heat-labile Uracil-DNA N-Glycosylase (UNG) prevents carry-over contamination. This involves incorporating deoxyuridine triphosphate (dUTP) in all amplification reactions and pre-treating PCR mixtures with UNG. UNG cleaves DNA at deoxyuridine residues. The resulting abasic sites are hydrolyzed at high temperatures during initial denaturation, preventing them from serving as PCR templates. UNG is inactivated during initial denaturation. Native DNA, lacking uracil, is not degraded. By replacing dTTP with dUTP and including UNG, this kit facilitates decontamination.

Trademarks

foodproof®, microproof®, vetproof®, ShortPrep®, RoboPrep®, and LyoKit® are trademarks of BIOTECON Diagnostics GmbH. Hygiena™ 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.