hygiena KIT230020 (LP) foodproof E. Coli And Shigella Detection LyoKit User Guide

hygiena KIT230020 (LP) foolproof E. Coli And Shigella Detection LyoKit

Product Information

Specifications

• Product Name: E. coli and Shigella Detection LyoKit
• Revision: A, November 2023
• Product Numbers: KIT230020 (LP), KIT230021 (RP), KIT230329 (DP)
• Application: PCR kit for the qualitative detection of Escherichia coli and Shigella spp. DNA using real-time PCR instruments

Product Usage Instructions

Program Setup

Program your real-time PCR instrument with the following settings:

• Select FAM channel for Escherichia coli and Shigella spp.
• Select the HEX channel for Internal Control.
• Set the cycle parameters as specified in the manual.

Data Interpretation

Verify results using positive and negative controls before interpreting sample results. Compare samples to controls and interpret based on the table provided in the manual.

Preparation of the PCR Mix

1. Place strips in a PCR tube rack after removing them from the aluminum bag.
2. Decap tubes just before filling them with samples.
3. Add samples and controls to the tubes.
4. Seal the tubes with the provided caps.
5. Mix thoroughly to resuspend pellets.
6. Centrifuge briefly to ensure proper mixing.
7. Start the real-time PCR run following the specified cycle.

FAQ

Q: Can I reuse the PCR tube strips?

• A: No, it is recommended to use new PCR tube strips for each PCR run to avoid contamination.

Q: How should I store the LyoKit after use?

• A: Store the LyoKit in a cool and dry place, away from direct sunlight and moisture to maintain its integrity.
• Product No. KIT230020 (LP), KIT230021 (RP), KIT230329 (DP)
• PCR kit for the qualitative detection of Escherichia coli and Shigella spp. DNA using real-time PCR instruments.

PROGRAM SETUP

• Program your real-time PCR instrument before setting up the PCR reactions.

Select the following channels:

• FAM (Escherichia coli and Shigella spp.) and HEX (Internal Control).
• 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 35 cycles for confirmation of single colonies
• For some real-time PCR instruments, the probe quencher as well as the use of a passive reference dye must be specified.
• This kit contains probes with a non-fluorescent “dark” quencher and no passive reference dye.
• For the Dualo 32® R2 real-time PCR instrument, please open the software, click on ‘New’, and select the respective template file.
• Template files can be added by clicking on ‘Add’ in the ‘Select template file’ window.

DATA INTERPRETATION

• Verify the results of positive (Control Template) and negative (H2O) controls, before interpreting the sample results.
• Always compare samples to positive and negative controls.
• Review data from each channel and interpret results as described in the table.

Note: The table is valid for protocols with 35 cycles only. If 50 cycles are used, Cq values > 35 should be considered as negative results.

PREPARATION OF THE PCR MIX

Take appropriate precautions to prevent contamination, e.g., by using filter tips and wearing gloves.

1. PLACE STRIPS IN RACK
   • Take the needed number of PCR tube strips out of an aluminum bag.
   • Important: close the bag tightly afterward. 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.
2. DECAP
   • Immediately before filling, carefully open strips and discard caps.
   • Do not leave it open longer than necessary.
3. 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.
4. SEAL
   • Carefully seal the tubes with the provided 8-cap strips.
5. MIX
   • Resuspend the pellet after sealing by mixing thoroughly.
   • Alternatively, resuspend the pellet by pipetting up and down multiple times in Step 3.
6. CENTRIFUGE
   • Briefly spin strips, e.g., 5 seconds at 500 – 1,000 x g, in a suitable centrifuge.
7. START REAL-TIME PCR RUN
   • Cycle samples as described above.
   • Place tubes in a vertical, balanced order into the cycler, e.g., two strips can be placed in the first and last column.

• foodproof®
• E.coli and Shigella Detection LyoKit
• KIT230020 /021 /329 Kit for 96 reactions Store kit at 2 to 8 °C
• For food testing purposes FOR IN VITRO USE ONLY Made in Germany
• Hygiena®
• Camarillo, CA 93012 USA
• diagnostics.support@hygiena.com
• www.hygiena.com

Documents / Resources

| hygiena KIT230020 (LP) foodproof E. Coli And Shigella Detection LyoKit [pdf] User Guide
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References

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