hygiena KIT230020 (LP) E coli and Shigella Detection User Guide

: September 16, 2024
: Hygiena

Table of Contents

hygiena KIT230020 (LP) E coli and Shigella Detection
Product Specifications
Product Usage Instructions
FAQ
PROGRAM SETUP
DATA INTERPRETATION
PREPARATION OF THE PCR MIX
More Information
References
Read User Manual Online (PDF format)
Download This Manual (PDF format)

hygiena KIT230020 (LP) E coli and Shigella Detection

Product Specifications

Product: E. coli and Shigella Detection LyoKit
Product No: KIT230020 (LP), KIT230021 (RP), KIT230329 (DP)
PCR kit for the qualitative detection of Escherichia coli and Shigella spp. DNA using real-time PCR instruments
Manufactured in: Germany
For in vitro use only

Product Usage Instructions

Program Setup:

Program your real-time PCR instrument before setting up the PCR reactions.
Select the following channels: FAM (Escherichia coli and Shigella spp.) and HEX (Internal Control).
Set the program cycle as per the provided details.

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 based on the provided table.

Preparation of the PCR Mix:

Take appropriate precautions to prevent contamination, e.g., by using filter tips and wearing gloves.
Place strips in a rack and ensure lyophilized pellets are at the bottom of all tubes.
Add samples and controls, seal the tubes with provided 8-cap strips, and mix thoroughly.
Centrifuge the strips briefly before starting the real-time PCR run as described.

Start Real-time PCR Run:

Cycle samples according to the program setup.
Place tubes in a vertical, balanced order into the cycler for efficient processing.

FAQ

Q: How should I interpret the results?
- A: Always compare your sample results to positive and negative controls. Refer to the provided table for result interpretation based on the FAM and HEX channels.
Q: What precautions should I take during preparation?
- A: To prevent contamination, use filter tips, wear gloves, and ensure proper sealing of tubes after adding samples and controls.

Product No: KIT230020 (LP), KIT230021 (RP), KIT230329 (DP)

PCR kit for the qualitative detection of Escherichia coli and Shigella spp. DNA using real-time PCR instruments.

PROGRAM SETUP

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

FAM (Escherichia coli and Shigella spp.) and HEX (Internal Control).

Pre-incubation: 1 cycle

Step 1: 37 °C for 4 min
Step 2: 95 °C for 5 min

Amplification: 35-50 cycles

Step 1: 95 °C for 5 sec
*Step 2:** 60 °C for 60 sec
Fluorescence detection
** 35 cycles for confirmation of single colonies

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.
For the Dualo 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 (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.

FAM	HEX	Result Interpretation
+	+ or –	Positive for Escherichia coli and/or Shigella spp.
–	+	Negative for Escherichia coli and Shigella spp.
–	–	Invalid

Note: The table is valid for protocols with 35 cycles only. If more than 35 cycles are used, Cq values > 35 should be considered as negative results.

PREPARATION OF THE PCR MIX

Take appropriate precautions to prevent contamination, e.g., by using fi lter 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 lyophilizedpellets to the bottom of all tubes.
DECAP
- Immediately before fi lling, 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 H2O 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 fi rst and last column.