hygiena KIT230127 LP Magnetic Preparation Kit User Guide

hygiena KIT230127 LP Magnetic Preparation Kit

Specifications

• Product Name: Animal Detection 1 LyoKit
• Revision: A
• Date: November 2023
• Product Numbers: KIT230127 (LP), KIT230128 (RP)
• Application: PCR kit for the qualitative detection of porcine, bovine, and equine with differentiation of horse, donkey, and zebra

Program Setup

Before setting up the PCR reactions, program your real-time PCR instrument with the following channels:

• FAM (porcine)
• HEX (bovine)
• ROX (equine: horse, donkey, zebra)
• Cy5 (Internal Control)

Run the program with the following settings:

• 4 minutes – 1 cycle

• 5 minutes – 5 seconds | 60 seconds* – 35 cycles

• 50 seconds, 50 seconds, ramp** – 1 cycle

• Melting Curve: 1 cycle

• Fluorescence detection

** Ramp refers to the gradual increase or decrease of temperature over a period of time.

Data Interpretation

To interpret the results, follow these steps:

1. Verify results of positive (Control Template) and negative controls (H2O).
2. Always compare samples to positive and negative controls.
3. Review data from each channel.
4. Interpret the results according to the table provided.

Preparation of the PCR Mix

Follow these steps to prepare the PCR mix:

1. Take appropriate precautions to prevent contamination, such as using filter tips and wearing gloves.

2. Place the required number of PCR tube strips out of the aluminum bag. Close the bag tightly afterwards.

3. Place the strips in a suitable PCR tube rack. If necessary, gently tap the tubes to move the lyophilized pellets to the bottom of all tubes.

4. Immediately before filling, carefully open the strips and discard the caps. Do not leave them open longer than necessary.

5. Add samples and controls to the open tubes.

6. Carefully seal the tubes with the provided 8-cap strips.

7. Resuspend the pellet after sealing by mixing thoroughly.
   Alternatively, resuspend the pellet by pipetting up and down multiple times.

8. Briefly spin the strips, e.g., 5 seconds at 500 – 1,000 x g, in a suitable centrifuge.

9. Start the real-time PCR run according to the program setup.
   Place the tubes in a vertical, balanced order into the cycler.

Frequently Asked Questions

What is the purpose of this kit?
This kit is designed for the qualitative detection of porcine, bovine, and equine with differentiation of horse, donkey, and zebra.

Where can I find the complete product manual?
The complete product manual is available on our website. It is strongly recommended to read it before starting.

What precautions should I take to prevent contamination?
To prevent contamination, use filter tips and wear gloves while handling the PCR mix.

How should I interpret the results?
To interpret the results, compare the samples to the positive and negative controls. Review the data from each channel and refer to the provided table for result interpretation.

Revision A, November 2023

Product No. KIT230127 (LP), KIT230128 (RP)

PCR kit for the qualitative detection of porcine, bovine and equine with differentiation of horse, donkey and zebra. 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 (porcine), HEX (bovine), ROX (equine: horse, donkey, zebra) and Cy5 (Internal Control).

• Pre-incubation: 1 cycle Step 1: 37 °C for 4 min Step 2: 95 °C for 5 min
• Amplifi cation: 35 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 : 60 °C for 50 sec Step 3**: ramp up to 80 °C

For CFX96TM real time PCR cycler step 2 of melting curve is: 50 °C for 50 seconds. For ABI 7500 Fast, step 2 of melting curve is: 50 °C for 60 seconds, step 3: ramp up to 85 °C. 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 (Product No. KIT230005).

DATA INTERPRETATION

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

FAM| HEX| ROX| Cy5| Result Interpretation – Amplification
---|---|---|---|---
+| +| +| + or –| Positive for porcine, bovine and equine
–| +| +| + or –| Positive for bovine and equine
+| –| +| + or –| Positive for porcine and equine
+| +| –| + or –| Positive for porcine and bovine
–| +| –| + or –| Positive for bovine
+| –| –| + or –| Positive for porcine
–| –| +| + or –| Positive for equine
–| –| –| +| Negative for porcine, bovine and equine
–| –| –| –| Invalid

Channel| Result Interpretation – Melting Curve
ROX| Donkey: 64 – 67 °C| Zebra: 68 – 70.5 °C| Horse: 71 – 74 °C

The Control Template contains only donkey and horse. Therefore, two peaks will be displayed. Please use the Control Template as a reference. Melting curve peaks can vary about ± 1 °C depending on PCR instrument used.

foodproof® is a registered trademark of Hygiena. The above mentioned real-time PCR instruments are registered trademarks of their respective holders.

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 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.

2. DECAP
   Immediately before filling, carefully open strips and discard caps.
   Do not leave open longer than necessary.

3. ADD SAMPLES AND CONTROLS
   Pipette 25 µL of samples (from Sample Preparation Kit III), Negative Con-trol (colorless cap) or Control Template (purple cap) into respective wells.
   For samples prepared with StarPrep Five, use 22 µL PCR-grade water + 3 µL sample for a total volume of 25 µL.

4. SEAL
   Carefully seal the tubes with the provided 8-cap strips.

5. MIX
   Resuspend pellet after sealing by mixing thoroughly.
   Alternatively, resuspend 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®
Animal Detection 1 LyoKit

KIT230127 / 28
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 | [email protected] | www.hygiena.com

References

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