DETECTION OF HLA-B27 ALLELES BY GROUP-SPECIFIC AMPLIFICATION AND TIME-RESOLVED FLUOROMETRY

This newly developed HLA-B27 assay combines a polymerase chain reactio n (PCR) from blood spot samples with solution hybridisation in microti tration plate and with time-resolved fluorometry (TRF) as the detectio n system. In a multiplex amplification reaction, the 144 base pair reg ion of HLA-BU alleles is amplified with allele-specific primers simult aneously with the region of beta-actin gene as an internal control. Am plified products are collected onto streptavidin (SA)-coated microtitr ation wells, denatured and hybridised with a europium (Eu)-labelled HL A-B27 specific probe and a samarium (Sm)-labelled beta-actin specific probe. Finally, Eu and Sm fluorescence is enhanced and detected in a t ime-resolved fluorometer. The typing results obtained with 110 blood s pot samples showed an exact match with serological class I HLA-typing. When this technique was further evaluated, 348 blood spot samples wer e clearly categorised into two populations, HLA-B27 positives and nega tives. This new PCR-TRF method permits the automation of HLA-B27 assay s and saves time and labour in routine diagnostics. (C) 1998 Elsevier Science B.V. All rights reserved.