Rapid DNA Typing of HLA-B27 Allele By Real-Time PCR Using LightCycler Technology
Tiemann, C.; Vogel, A.; Dufaux, B.; Zimmer, M.; Joerg-Krone, R.; and Hans-Jochen Hagedorn
Clinical Laboratory 2001(3-4) Clin. L.ab. 2001;.47:131-134
2001
ISSN/ISBN: 1433-6510 PMID: 11294575 Document Number: 7472
For clinical diagnostic routine we developed a fast DNA typing of HLA-B27 by PCR and real-time detection using LightCycler TM technology. The method combines the sensitivity and specifïcity 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. ß-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 identifïcation of the PCR amplicons. The mean melting temperatures (Tm) were 90.5°C and 87.3°C, which are characteristic for HLA-B27 and ß-globin, respectively. A comparison of 300 samples which were typed for HLA-B27 with a conventional sequencespecific 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.
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