hygiena KIT230129 RoboPrep Fusion User Guide

: May 15, 2024
: Hygiena

Table of Contents

PROGRAM SETUP
DATA INTERPRETATION
PREPARATION OF THE PCR MIX
References
Read User Manual Online (PDF format)
Download This Manual (PDF format)

**hygiena KIT230129 RoboPrep Fusion User Guide

**

Product No. KIT230129 (LP), KIT230130 (RP), KIT230369 (DP)

PCR kit for the qualitative detection of the Listeria sense strict species (L. monocytogenes, L. steelier, L. Ivanovic, L. welshimeri, L. innocuity and L. martini) including the simultaneous identification of Listeria monocytogenes. 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: u FAM (Listeria monocytogenes), HEX (Listeria sense strict) and ROX (Internal Control).
PROGRAM SETUP

Pre-incubation: 1 cycle
Step 1: 37 °C for 4 min
Step 2: 95 °C for 5 min

Amplify cation: 50 cycles
Step 1 : 95 °C for 5 sec
Step 2*: 60 °C for 60 sec

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

For the Varimax real-time PCR instrument, please use transparent low profile tubes (KIT230369, DP).

For the Dual 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 results of positive (Control Template) and negative controls (H2 O), 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	Result Interpretation
+	+	+ or –	Positive for L. monocytogenes
–	+	+ or –	Positive for Listeria sense strict
–	–	+	Negative for L. monocytogenes, Listeria sense strict
–	–	–	Invalid

PREPARATION OF THE PCR MIX

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

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.
DECAP
Carefully open strips immediately before fi ling and discard caps. Do not leave open longer than necessary
ADD SAMPLES AND CONTROLS
Pipette 5 to 25 µL of samples, 25 µL negative control (colorless cap) or Control Template (purple cap) into respective wells.
Varying sample volumes are due to different enrichment broths and DNA extraction procedures. If using less volume, add PCR-grade H2 O to reach a total volume of 25 µ
SEAL
Carefully seal the tubes with the provided 8-cap strips.
MIX
Resuspend pellet after sealing by mixing thoroughly. Alternatively, resuspend pellet by pipetting up and down multiple times in Step 3.
CENTRIFUGE
Briefl y spin strips, e.g., 5 seconds at 500 – 1,000 x g, in a suitable centrifuge.
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 fi rst and last column