945100_enterotaqman_manual

STORAGE AND HANDLING
All reagents (A1 to A8) should be stored at -20°C. All reagents can be
used until the expiration date printed on the labels. Avoid multiple freezing
and thawing cycles of reagents (< 2). If used sporadically, prepare
aliquots of the reagents. Cool all reagents during the working steps.
Helicobacter pylori
Avoid exposure of the Primer and Probe Mix (A2) to light.
real time PCR Kit
ASSAY PROCEDURE
Required material provided:
(Clarithromycin resistance)
Cat. No.: 900100
Required material not provided:
For in vitro diagnostic use 96 reactions
1. LightCycler instrument (Roche)2. LightCycler capillaries INTENDED USE
The AnDiaTec® Helicobacter pylori real time PCR Kit (Clarithromycin resistance) is a screening assay for the detection of Helicobacter pylori, 5. Pipets (0.5µl - 1 ml) with sterile filter tips and its clarithromycin resistance status in the capillary system of the One of the major factors for chronic gastritis and peptic ulcerations of WARNINGS AND PRECAUTIONS
the stomach is the colonisation of the stomach by the gram-negativemicroorganism Helicobacter pylori. The therapy comprises of the • This assay needs to be carried out by skilled personel! application of certain antibiotics, of which one of the most important is • Clinical samples should be regarded as potentially infectious clarithromycin. The resistance of Helicobacter pylori against clarithromycin is the major cause of failure in eradication therapies. It • This assay needs to be run according to GLP (Good Laboratory was shown that mutations in the 23S rRNA gene (A-G change at nucleotide position 2143 or 2144) is responsible for the resistance ofHelicobacter pylori against clarithromycin. The success rate for the AMPLIFICATION
clarithromycin treatment of Helicobacter pylori in strains with single The PCR technology is utmost sensitive. Thus, amplification of a single mutation is only 40%, but 85% in the wild type strains.
molecule generates millions of identical copies. These copies mayevade through aerosols and sit on surfaces.
Stomach biopsies or stool samples can be used for testing with theAnDiaTec® Helicobacter pylori real time PCR Kit (Clarithromycin In order to avoid contamination of samples with DNA which was previously amplified, it is important to physically strictly divide sample PRINCIPLE OF THE TEST
and reagent preparation units from sample amplification units. Pipets,vials and other working materials should not circulate among working The AnDiaTec® Helicobacter pylori real time PCR Kit (Clarithromycin resistance) contains specific primers and additional material for thedetection of the Helicobacter pylori 23S rRNA gene. The base present • Do not use the kit after its expiration date at nucleotide position 2143 and 2144 (mutation or wildtype) is • Set up (if possible) two seperate working areas: characterized by melting curve analysis determining the binding affinity of the probe to the DNA template. The identification of different 2. Amplification/ detection of amplification products genotypes in each single sample is conducted exclusively by real time • Always use sterile pipet tips with filter • Wear separate coats and gloves in each area The AnDiaTec® kit does also differentiate a silent mutation on position • Routinely decontaminate your pipets and the laboratory benches 2142 which does not cause Clarithromycin resistance.
REAGENTS PROVIDED
• The LightCycler-PCR process is owned by Hoffmann-La Roche Ltd.
Each kit contains enough reagents to perform 96 tests. Each kitalso contains a package insert.
PROCEDURE
For real time PCR
The complete procedure is separated in four steps:1. Sample preparation and DNA extraction Type of reagents
Presentation
Cap Color
2. Preparation of the ready-to-use Enzyme Mix.
Enzyme Buffer (mit dNTP) 3 vials, 60 µL each blue 3. Amplification and combined detection of the DNA templates followed 4. Interpretation of the genotypes using the LightCycler software and 1. SAMPLE PREPARATION AND DNA EXTRACTION
4. ANALYSIS OF GENOTYPES UND INTERPRETATION OF
1.1 Stomach biopsies: Transfer the sample into a sterile microtube and add 40µL of Proteinase K solution (A8), 40µL Lysis Buffer (A7), and 320µL DNAse-free water. Incubate for 6 h at 37°C in thethermoblock.Extract genomic DNA by use of a commercial DNA 4.2 Switch on the Color Compensation Filter prior to PCR (required because of the simultaneous use of 2 differently labelled probes).
Stool samples: Extract genomic DNA by use of a commercial DNA 4.3The result of the quantification is shown in channel F2/F1 and isolation kit directly from the sample.
1.2 If the LightCycler PCR is not performed immediately, store extracted Both positive controls must show amplification in F2/F1 and F3/F1 the negative control must not show any amplification.
2. PREPARATION OF THE READY-TO-USE ENZYME MIX
• A signal is detected in channel F2/F1 and F3/F1:
The result is positve: The sample contains Helicobacter
2.1 Centrifuge one vial Enzyme Mix (A1a) and one vial Enzyme Buffer(A1b) briefly at 13000 rpm.
pylori DNA.
• No signal is detected in channel F2/F1and F3/F1: 2.2 Transfer 60µL of Enzyme Buffer (A1b) into the Enzyme Mix (A1a).
The result is negative: The sample contains no
2.3 Mix carefully by pipetting up and down (do not vortex!). This is
Helicobacter pylori DNA
4.4 The melting curve analysis should be performed with the 3. Helicobacter pylori real time PCR Protocol
following settings: • Digital filter: enabled Please read carefully the manufacterer‘s instructions before starting 4.5 The assessment of the measured values and the respective Tm the procedure! The Master Mix volume for the respective number of Analysis is done at the end of the PCR with the „LightCycler Manual samples and controls should be pipetted as follows.
Tm Estimation Report“, thereby the resulting Tm values differentiate 3.1. The Enzyme Mix volume per reaction and sample should be between wild type and mutant genotypes.
multiplied with the number of samples to be performed, including controls A4, A5 and A6 (N). For reasons of unprecise pipetting, addan extra (virtual) sample. Proceed in the same manner with all additional reagents.Cool all reagents during all work steps!
Compare the melting curves of the samples with the melting curvesof both controls (wildtype and mutant): Mix gently (do not vortex!) the following reagents in a sterile vial:
the present H. pylori is Clarithromycin sensitive (wildtype).
Enzyme Mix (ready-to-use) (A1a+A1b), Primer and Probe Mix (A2)and MgCl (A3). This mixture is the Master Mix. Spin down briefly in 3.2 Pipet 18µL of Master Mix using micropipets with sterile filter tips the present H. pylori is Clarithromycin resistant (mutant).
in each of the LightCycler capillaries. 2µL of the DNA samples,positive and negative controls (A4, A5 and A6) are then added to each of these capillaries. It is recommended to pipet the negative control first to avoid contamination. Immediately lock the capillaries the present H. pylori consists of a sensitive and a resistant
Spin down briefly (in a LightCycler capillary centrifuge).
strain (double infection).
Perform the following LightCycler PCR Protocol:
95°C for 10 Min.
and Tm = 60,5°C (+/- 1°C) for F3/F1
the present H. pylori is Clarithromycin sensitive (silent
95°C for 0 sec.
55°C for 10 sec. ramping time: 20°C/sec. -aqu. mode SINGLE
4.6 Samples with ambigous melting curves (flat curve) have to be 72°C for 15 sec.
repeated. Use the melting curve amplitudes of the positive controlsas reference.
95°C for 0 sec.
50°C for 5 sec.
AnDiaTec GmbH & Co. KG
80°C for 0 sec.
ramping time: 0,1°C/sec. -aqu. mode CONT 40°C for 30 sec.

Source: http://www.andiatec.com/de/_produkte/900100.pdf