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Genotyping of HLA-B27 by Real-Time PCR without Hybridization Probes

¹ Department of Clinical Chemistry, Medisch Spectrum Twente, Hospital Group, Enschede, PO Box 50000, NL-7500 KA, The Netherlands
^a author for correspondence: fax 31-53-487-3075, e-mail labmst@euronet.nl

The HLA-B27 allele, present in 9% of the Caucasian population, is strongly associated with several rheumatic diseases, e.g., ankylosing spondylitis, Reiter syndrome, and acute anterior uveitis (1). Various methods have been developed for the identification of the HLA-B27 allele. Initially we used flow cytometry for serological HLA-B27 typing. Our molecular biological assay (2), based on allele-specific PCR (3), relies on specific primer recognition of a sequence in the third exon that is unique to the B27 allele. The forward primer is specific for most B alleles, whereas the sequence of the reverse primer is unique to the B27 alleles and is complementary to all of them except for subtype B2707 because of the AT at its 3' end. This method unambiguously identified all B27-positive samples except for the rare B2707 subtype (2). Flow cytometry, in contrast, produced ambiguous results in 3% of samples because of antibody cross-reactivity (2). In the last 5 years, we have analyzed >2000 samples with unequivocal results and negligible complications, but the necessary post-PCR identification steps such as gel electrophoresis and confirmation by allele-specific hybridization analysis make the procedure time-consuming and prone to contamination.

Recently, a new generation of high-speed thermal cyclers has been developed based on real-time PCR analysis. These instruments, for example, the LightCycler^TM (Roche Molecular Biochemicals), fluorometrically monitor real-time formation of PCR products during thermal cycling with SYBR Green I. This double-stranded DNA-selective fluorescent dye provides a convenient, rapid way to detect and quantify any PCR product, regardless of sequence. During each phase of DNA synthesis, the SYBR Green I dye, which is already included in the reaction mixture, binds to the amplified PCR products, which subsequently can be detected by their fluorescence. The specificity and sensitivity of the reaction can be . . . [Full Text of this Article]

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References

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