hygiena KIT230110 (LP) foodproof Clostridium botulinum Detection LyoKit User Guide

hygiena KIT230110 (LP) foodproof Clostridium botulinum Detection LyoKit

Specifications

• Product Name: Clostridium botulinum Detection LyoKit
• Revision: A, November 2023
• Product Numbers: KIT230110 (LP), KIT230111 (RP)
• Functionality: PCR kit for the qualitative detection of botulinum type A, B, E, and F neurotoxin/producing Clostridium species
• Compatibility: Real-time PCR instruments
• Manufactured: Germany

Product Usage Instructions

Program Setup

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

• FAM (botulinum neurotoxin types A and E)
• HEX (botulinum neurotoxin types B and F)
• ROX (Internal Control)

2. Follow the specified cycle settings provided in the manual.

Data Interpretation

1. Verify results of positive (Control Template) and negative (H2O) controls before interpreting sample results.
2. Compare sample results to positive and negative controls.
3. Review data from each channel and interpret results based on the provided table.

Preparation of the PCR Mix

1. Take appropriate precautions to prevent contamination by using filter tips and wearing gloves.
2. Place strips in a rack and ensure lyophilized pellets are at the bottom of all tubes.
3. Add samples and controls, then carefully seal the tubes with the provided caps.
4. Mix thoroughly by resuspending the pellet either by gentle tapping or pipetting.
5. Briefly centrifuge the strips to ensure proper mixing.
6. Start the real-time PCR run as described in the manual.

For Food Testing Purposes:

This product is for in vitro use only. Follow all instructions carefully for accurate results.

Made in Germany

Frequently Asked Questions (FAQ)

Q: Can this kit differentiate between botulinum neurotoxin types A and E, and B and F?

• A: Without a melting curve analysis, the botulinum neurotoxin types can still be detected, but differentiation between A and E or B and F is not possible.

Q: What precautions should I take during the preparation of the PCR mix?

• A: Take appropriate precautions to prevent contamination by using filter tips, wearing gloves, and ensuring proper sealing of tubes.
• Product No. KIT230110 (LP), KIT230111 (RP)
• PCR kit for the qualitative detection of botulinum type A, B, E, and F neurotoxin/producing Clostridium species (C. botulinum, C. baratii, and C. butyricum) using real-time PCR instruments.
• Without a melting curve analysis, the botulinum neurotoxin types (BoNT) can still be detected but not differentiate between A and E or B and F.
• Before starting, it is strongly recommended to read the entire product manual available on our website.

PROGRAM SETUP

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

Select the following channels:

• FAM (botulinum neurotoxin types A and E), HEX (botulinum neurotoxin types B and F), and ROX (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
• Melting   Curve: 1 cycle
  • Step 1: 95 °C for 50 sec
  • Step 2: 37 °C for 50 sec
• 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.
• A Color Compensation is necessary for users of the LightCycler® 480 System: Color Compensation Set 3 (Product No. KIT230005).

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.

Amplification Curve| FAM| HEX| ROX| Result Interpretation
---|---|---|---|---
+| –| + or –| Positive for BoNT A and/or E
–| +| + or –| Positive for BoNT B and/or F
+| +| + or –| Positive for BoNT A and/or E, Positive for BoNT B and/or F
–| –| + or –| Negative for BoNT A, B, E, F
–| –| –| Invalid
Melting Curve| FAM| HEX| Botulinum Neurotoxin Type (BoNT)
none| none| A
none| none| B
68 ± 2 °C| none| E
none| 55 ± 2 °C and 64.5 ± 2 °C| F
(64 ± 2 °C)| 55 ± 2 °C and 62.5 ± 2 °C| Control Template

PREPARATION OF THE PCR MIX

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

1. PLACE STRIPS IN THE 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® Clostridium botulinum Detection LyoKitKIT230110 /11
• 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 KIT230110 (LP) foodproof Clostridium botulinum Detection LyoKit [pdf] User Guide
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References

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