hygiena KIT230117 (LP) Vibrio Detection LyoKit User Guide

: June 16, 2024
: Hygiena

Table of Contents

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

hygiena KIT230117 (LP) Vibrio Detection LyoKit

PROGRAM SETUP

Product No. KIT230117 (LP), KIT230118 (RP)
PCR kit for the qualitative detection of Vibrio parahaemolyticus, V. vulnificus and V. cholerae DNA and their respective toxin genes using real- time PCR instruments.
Before starting, it is strongly recommended to read the entire product manual available on our website.

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

FAM (V. parahaemolyticus, tdh), HEX (V. vulnificus, trh1 and trh2), ROX (V. cholerae, ctx) and Cy5 (Internal Control).

Program Setup

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

Amplifi cation: 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
Step 3**: ramp up to 65 °C

Fluorescence detection
Fluorescence detection during 37 – 65 °C ramp with 2 – 4 measurements/°C

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.
Color Compensation is necessary for users of the LightCycler® 480 System: Color Compensation Set 3 (Product No. KIT230005) has to be used for LC480 I instrument; Color Compenation Set 5 (Product No.
KIT230011) has to be used for LC480 II instrument.

DATA INTERPRETATION

Verify 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. For melting curve interpretation of toxin genes (tdh, trh1, trh2 and ctx), refer to the product manual.

FAM	HEX	ROX	Cy5	Result Interpretation
+	+	+	+ or –	Positive for V. parahaemolyticus, V. vulnificus and V.

cholerae
–| +| +| + or –| Positive for V. vulnificus and V. cholerae
+| –| +| + or –| Positive for V. parahaemolyticus and V. cholerae
+| +| –| + or –| Positive for V. parahaemolyticus and V. vulnificus
–| +| –| + or –| Positive for V. vulnificus
+| –| –| + or –| Positive for V. parahaemolyticus
–| –| +| + or –| Positive for V. cholerae
–| –| –| +| Negative for V. parahaemolyticus, V. vulnificus and V. cholerae
–| –| –| –| Invalid

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.

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
Immediately before filling, carefully open strips and discard caps.
Do not leave open longer than necessary.
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 H2 O to reach 25 µL.
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
Briefly 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 first and last column.