Hygiena foodproof® Spoilage Yeast Detection 2 LyoKit

PROGRAM SETUP

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

• FAM (Saccharomyces cerevisiae var. diastaticus)
• HEX (Wickerhamomyces anomalus)
• ROX (Kazachstania exigua and Schizosaccharomyces pombe)
• ATTO 490LS (Internal Control)

PCR Thermal Cycling Profile:

The PCR instrument should be programmed with the following steps:

• 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 10 sec, Step 2: 60 °C for 60 sec (with fluorescence detection)
• Melting Curve (1 cycle): Step 1: 95 °C for 50 sec, Step 2: 37 °C for 50 sec, Step 3: ramp up to 85 °C

For the Dualo 32® (for beverage testing) real-time PCR instrument, open the software, click 'New', and select the respective template file. Template files can be added via 'Add' in the 'Select template file' window.

DATA INTERPRETATION

Verify results of positive (Control Template) and negative controls (H&sub2;O), 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.

Melting Curve Analysis

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 required number of PCR tube strips from the aluminum bag. Important: close the 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, Negative Control (colorless cap) or Control Template (purple cap) into respective wells. If using less volume, add PCR-grade H&sub2;O to reach 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.