Detection of HLA-B27 alleles by group-specific amplification and time-resolved fluorometry

This newly developed HLA-B27 assay combines a polymerase chain reaction (PCR) from blood spot samples with solution hybridisation in microtitration plate and with time-resolved fluorometry (TRF) as the detection system. In a multiplex amplification reaction, the 144 base pair region of HLA-B27 alleles is amplified with allele-specific primers simultaneously with the region of beta-actin gene as an internal control. Amplified products are collected onto streptavidin (SA)-coated microtitration wells, denatured and hybridised with a europium (Eu)-labelled HLA-B27 specific probe and a samarium (Sm)-labelled beta-actin specific probe. Finally, Eu and Sm fluorescence is enhanced and detected in a time-resolved fluorometer. The typing results obtained with 110 blood spot samples showed an exact match with serological class I HLA-typing. When this technique was further evaluated, 348 blood spot samples were clearly categorised into two populations, HLA-B27 positives and negatives. This new PCR-TRF method permits the automation of HLA-B27 assays and saves time and labour in routine diagnostics.