Description

1. Intended Use
The HLA-B27 AIMATIS Assay is a fast and accurate real-time PCR test for the detection of the HLA-B27 allele, a specific variant of the human leukocyte antigen B (HLA-B) gene that is strongly associated with seronegative spondyloarthropathies. The kit is used to confirm a suspected diagnosis of ankylosing spondylitis, reactive arthritis, juvenile rheumatoid arthritis or anterior uveitis. The qualitative assay discriminates the presence or absence of HLA-B27 in a human genomic DNA extract and detects the majority of disease-relevant HLA-B27 subtypes.

2. Introduction
HLA-B molecules are cell surface proteins that play an important role in immunity. The frequency of the HLA-B27 variant is about 8% in the
general Caucasian population, whereas it is found in over 90% of patients suffering from ankylosing spondylitis (AS). The disease,
predominantly striking men between the age of 20 and 40 years, is characterized by inflammation of the sacroiliac joint and progressing
stiffness of the spine. Due to the high correlation with the disease, HLA-B27 genotyping is suitable for the differential diagnosis of AS. Other
subsets of spondyloarthritis are also associated with HLA-B27, although to a lesser degree.
3. Kit Contents
AIMATIS 2x Master Mix 1 vial 1000 / 320 μl HLA-B27 Assay
Mix 1 vial 550 / 550 μl HLA-B27
Positive Control 1 vial 75 / 75 μl
HLA-B27 Negative Control 1 vial 75 / 75 μl
The kit contains reagents for 100 / 32 reactions in a final volume of 20 μl
each.
The AIMATIS 2x Master Mix comprises HotStart Taq DNA polymerase and dNTPs in an optimized buffer system.
The HLA-B27 Assay Mix consists of gene-specific primers and dual-labeled hydrolysis probes for HLA-B27 and a control gene. A positive and
a negative control for HLA-B27 are supplied with the kit.

4. Storage and Stability
HLA-B27 AIMATIS Assay is shipped on cooling blocks. On arrival, store the kit at -30 to -15°C. Alternatively, store at 2 to 8°C for short-term
use within one month. The kit withstands up to 20 freeze/thaw cycles with no loss of activity. Avoid prolonged exposure to intense light. If
stored correctly, the kit will retain full activity until the expiration date indicated on the label.

5. Product Description
5.1. Principle of the Test
The test is based on the fluorogenic 5’ nuclease assay, also known as TaqMan® assay. Each reaction contains gene-specific primer pairs
which amplify a 202 bp fragment of the HLA-B27 gene and a control gene, the latter serving as PCR control. Further components are two
dual-labeled, gene-specific hydrolysis probes which hybridize to the target sequence of the corresponding fragment. The proximity of the 5’-
fluorescent reporter and 3’-quencher dye on intact probes prevents the reporter from fluorescing. During the extension phase of PCR the 5’ –
3’ exonuclease activity of the Taq DNA polymerase cleaves the 5’-fluorescent reporter from the hybridized probe. The physical separation of
the fluorophore from the quencher dye generates a fluorescent signal in real-time, which is proportional to the accumulated PCR product.
In samples positive for HLA-B27 both, the FAM-labeled HLA-B27 probe as well as the HEX-labeled PCR control probe bind to the
appropriate gene fragment. A strong fluorescence signal is detected in the FAM channel (520nm) and in the HEX channel (556nm). In samples
negative for HLA-B27 only the HEX-labeled PCR control probe hybridizes to the complementary strand of the control gene fragment. A strong
fluorescence signal is detected in the HEX channel and no or only a baseline signal in the FAM channel.