HLA-B27 real time PCR Kit

REAGENTS PROVIDED
Each kit contains enough reagents to perform 96 tests. Each kit
also contains a package insert.
PACKAGE INSERT
For real time PCR
Ref.
A1
A2
A3
HLA-B27 real time
PCR Kit
96 Determinations
(Do not use for determining the tissue type!)
Cap Color
blue
red
green
ASSAY PROCEDURE
Required materials provided:
• PCR reagents
• Package insert
INTENDED USE
The AnDiaTec® HLA-B27 real time PCR Kit is designed for the analysis
of the HLA-B27 genetic region by real time detection of the B27 allele.
The presence of this allele gives an indication of a genetic predisposition
for autoimmune diseases as rheumatoid arthritis or ankylosing
spondylitis. DNA containing sample materials such as buccal swabs
or whole blood can be used for testing.
Required materials not provided:
1. Real time PCR instrument (e.g. Applied Biosystems, Stratagene,
Qiagen/Corbett Research, Bio-Rad, Roche LC480)
2. Optical microtiter plates or optical reaction tubes
3. Optical adhesive covers
4. DNA extraction kit
5. Pipets (0.5 µL - 1000 µL)
6. Sterile filtertips for micropipets
7. Sterile tubes
8. Table centrifuge
SUMMARY
Human Leukocyte Antigens (HLA) are crucial for the immune system
to discriminate between „self“ and „non-self“. The HLA-B27 allele
located at the HLA-B locus is highly associated with inflammatory
rheumatic diseases, especially ankylosing spondylitis (AS, Bechterew‘s
disease). Up to 90% of AS-patients are HLA-B27 positive compared
to 8% of healthy caucasians. AS is a chronic inflammatory disease
which mainly affects joints in the spine and the sacroiliac joint in the
pelvis. Severe cases of AS possibly lead to fusion and acampsia of
the spine. Other diseases with a high prevalence of the HLA-B27 allele
are Reiter‘s syndrome, anterior uveitis and inflammatory bowel disease.
An early determination of an HLA-B27 positive genotype helps to
confirm first signs of rheumatic diseases and to start subsequently
with specific therapy.
WARNINGS AND PRECAUTIONS
• This assay needs to be carried out by skilled personnel!
• Clinical samples should be regarded as potentially infectious
materials.
• This assay needs to be performed according to GLP (Good
Laboratory Practice).
AMPLIFICATION
The PCR technology is utmost sensitive. Thus, amplification of a single
molecule generates millions of identical copies. These copies may
evade through aerosols and sit on surfaces.
In order to avoid contamination of samples with previously amplified
DNA, it is important to physically strictly divide sample and reagent
preparation units from sample amplification units. Pipets, vials and
other working materials should not circulate among working units!
• Do not use the kit after its expiry date
• Set up (if possible) two separate working areas:
1. Isolation of the DNA
2. Amplification/detection of amplification products
• Always use sterile pipet tips with filter
• Wear separate coats and gloves in each area
• Routinely decontaminate your pipets and the laboratory benches
with ethanol-free decontaminant
• Avoid aerosols
PRINCIPLE OF THE TEST
The AnDiaTec® HLA-B27 real time PCR Kit contains specific primers,
probes and additional reagents for the screening of the HLA-B27 allele
in clinical samples.
The kit provides a ready-to-use Amplification Mix. The possibly present
HLA-B27 allele is amplified by means of Polymerase Chain Reaction
(PCR) and detected in real time by the optical unit in the FAM channel
of the PCR machine. Subsequently, the results are analyzed and
interpreted for the presence of the HLA-B27 allele.
It is not allowed to use this test for determining the tissue type.
The Amplification Mix also contains an Internal Control, which is
simultaneously amplified. The amplification of this Internal Control is
measured in the VIC/HEX/JOE channel and does not affect the HLAB27 allele specific amplification. In case of an HLA-B27 allele negative sample, the proper amplification of this Internal Control guarantees
a successful DNA extraction and PCR performance.
937100
Presentation
1 vial, 1,5 mL
1 vial, 50 µL
1 vial, 200 µL
STORAGE AND HANDLING
• All reagents (A1 to A3) should be stored at -20°C.
• All reagents can be used until the expiry date printed on the labels.
• Do not freeze and thaw the reagents several times (>2). In case
of sporadic use, aliquot the reagents.
• Cool all reagents during the working steps.
• Avoid exposure of A1 to light.
Cat. No.: 937100
For in vitro diagnostic use
Type of Reagent
HLA-B27 Mix
Positive Control
Negative Control
1
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PROCEDURE
3. INTERPRETATION OF THE RESULTS
HLA-B27 allele specific amplification is detected by FAM fluorescence
and the Internal Control by VIC/HEX/JOE fluorescence using following
settings:
The complete procedure is separated into three steps.
1. Sample preparation and DNA extraction
2. Amplification and detection
3. Interpretation of the results using the software of the real time PCR
instrument
1. DNA EXTRACTION
1.1 Extract genomic DNA from buccal swabs, blood samples or other
samples by use of a commercial DNA extraction kit according to
the manufacturer‘s instructions.
Detection
Reporter Dye
Quencher
HLA-B27 allele
Internal Control
FAM
VIC/HEX/JOE
NFQ
NFQ
Reference Dye
1.2 If the real time PCR is not performed immediately, store the
extracted DNA at -20°C.
none
3.1 The Positive Control shows a HLA-B27 genotype, as well as
amplification of the Internal Control.
The Negative Control included in the kit must not show any signal.
2. HLA-B27 real time PCR Protocol
Please note: The amplification signal of the Internal Control in
the VIC/HEX/JOE channel has to be detected in each DNA sample.
However, only in case of an allele-specific amplification, the Internal
Control may be reduced or even inhibited due to competition.
If no signal can be detected in any channel, a diagnostic statement
can not be made. The sample has to be retested.
Please read carefully the manufacturer‘s instructions before starting
the procedure! Each assay should include a Negative and a Positive
Control.
Always use filtertips for pipetting. Keep reagents cool during all
working steps!
3.2 Following results can arise:
• FAM fluorescence is detected:
The AnDiaTec® HLA-B27 real time PCR Kit contains a ready-to-use
amplification mix: A1 HLA-B27 Mix
The result ist positive.
The sample has an HLA-B27 positive genotype.
2.1 Prior to using the ready-to-use HLA-B27 Mix, please vortex.
Pipet 15 µL of the Amplification Mix in an extra well of a 96 well
optical microtiter plate or in an extra optical tube. The number of
wells or tubes used is calculated by the number of samples plus
the Negative and Positive Control.
The occurence of VIC/HEX/JOE fluorescence is inessential as
high concentrations of HLA-B27 DNA may reduce or even inhibit
the amplification of the Internal Control.
2.2 Add 5 µL extracted DNA sample to the prepared Amplification
Mix. Do the same with the Positive Control (A2) and the Negative
Control (A3). Pipet the Negative Control first. To avoid
contamination, it is advisable to cover the wells containing the
Negative Control with an adhesive seal while pipetting the Positive
Control and sample DNA. Remove this adhesive seal after having
prepared all wells.
•
No FAM bur VIC/HEX/JOE fluorescence is detected:
The result is negative.
The sample has no HLA-B27 positive genotype.
The detected signal of the Internal Control excludes the possibility
of an inhibition of the PCR.
2.3 Cover the 96 well optical microtiter plate with an optical adhesive
cover. Spin down briefly.
•
2.4 Run the real time PCR using the following temperature protocol:
A diagnostic statement cannot be made.
Neither FAM nor VIC/HEX/JOE fluorescence is detected:
An inhibition of the PCR occured.
95°C for 2 min
45 cycles of:
95°C for 10 sec
58°C for 30 sec measure at the end of this step!
72°C for 30 sec
Quidel Germany GmbH
AnDiaTec division
Leibnizstr.11
D-70806 Kornwestheim
937100
2
Tel.: ++49 (0)7154 806 23 0
Fax: ++49 (0)7154 806 23 24
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