Rapid DNA typing of HLA-B27 allele by real-time PCR using lightcycler technology.

Abstract

For clinical diagnostic routine we developed a fast DNA typing of HLA-B27 by PCR and real-time detection using LightCycler technology. The method combines the sensitivity and specificity of PCR with the swiftness of the LightCycler system. The amplification step was performed with a primer set coding for a region in the third exon common to B*2701 to B*2705. The PCR cycles were monitored continuously using the SYBR Green I dye. Beta-globin was used as an internal control. An analysis of 32 samples with one PCR run was completed within 40 minutes. After amplification a melting curve analysis permitted the accurate identification of the PCR amplicons. The mean melting temperatures (Tm) were 90.5 degrees C and 87.3 degrees C, which are characteristic for HLA-B27 and beta-globin, respectively. A comparison of 300 samples which were typed for HLA-B27 with a conventional sequence-specific polymerase chain reaction (SSP-PCR) and with the new method demonstrated a perfect correlation (specificity 100%). In summary, the method described is fast, reliable, cost-effective and well adapted for routine laboratory testing.