hygiena KIT230119 LP Legionella Quantification LyoKit User Guide

microproof®
Legionella Quantification LyoKit
Ready Reference Guide
Revision A, November 2023

Product No. KIT230119 (LP), KIT230120 (RP)
PCR kit for the quantitative detection of Legionella DNA using real-time PCR instruments. 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 (Legionella pneumophila), HEX (Legionella spp.), ROX (Legionella pneumophila serogroup 1) and Cy5 (Internal Control). 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
Step 3 : 72 °C for 60 sec

• Fluorescence detection
  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 dyes.
  For colony confirmation, a shortened PCR protocol is available. Please refer to the manual.

PREPARATION OF STANDARD CURVE

Use Quantification Standard A, B, C and D to prepare a standard curve (see table below).
Briefly vortex and centrifuge Quantification Standards before use.
For each Quantification Standard, pipet 25 µL into the designated wells in duplicates.
A typical experiment consists of 9 wells needed for standards (duplicates) and a negative control, plus n wells (n = number of samples).

Quantification
Standard| Cap Color| Concentration to Be Entered as Standard (GU/reactions)
---|---|---
FAM Channel| HEX Channel| ROX Channel
A| purple| 25,000| 25,000| 25,000
B| red| 2,500| 2,500| 2,500
C| yellow| 250| 250| 250
D| white| 25| 25| 25

PREPARATION OF THE PCR MIX

Take appropriate precautions to prevent contamination, e.g., by using fi lter tips and wearing gloves.
For data interpretation and calculation, refer to the complete product manual.

1. PLACE STRIPS IN RACK
   Take needed number of PCR tube strips out of aluminum bag.
   Important: Seal 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 each tube.

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

3. ADD SAMPLES AND CONTROLS
   Pipet 25 µL of samples, standards and Negative Control (colorless cap) into respective wells. If using less volume, add PCR-grade H2O to reach 25 µL.

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

5. MIX
   Resuspend pellet by mixing thoroughly.
   Alternatively resuspend pellet by pipetting up and down multiple times in step

7. CENTRIFUGE
   Briefl y spin strips, e.g., 5 seconds at 500 – 1,000 x g, in a suitable centrifuge.

8. 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 columns.

KIT230119 / 20
Kit for 96 reactions
Store kit at 2 to 8 °C
FOR IN VITRO USE ONLY
Made in Germany
Camarillo, CA 93012 USA
diagnostics.support@hygiena.com
www.hygiena.com

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