Rapid genotyping of HLA-B27 among Korean population by real-time PCR melting curve analysis.

BACKGROUND

Real-time PCR and melting curve analysis is the relatively recent method for HLA-B27 genotyping, which has advantages of being simple and rapid.

METHODS

The accuracy of melting curve analysis for HLA-B27 was assessed in 153 clinical samples and 52 DNA samples from International Histocompatibility Workshop (IHW) cell lines, with sequence-based typing (SBT) as the reference method. We predicted melting reaction for various HLA-B27 subtypes using simulation software.

RESULTS

For clinical samples, 53 HLA-B27-positive and 100 negative results by melting curve analysis were confirmed by completely concordant SBT results. The B27:05 allele was found in 50 patients, and the B27:04 allele in 3 patients. Among 62 known alleles, 21 alleles had differences in the target sequence, including 10 alleles having mismatches in the primer binding site. In these alleles, differences in melting points (T(m)) were predicted to be ≤ 1.2°C. The predicted results were obtained when IHW samples were tested, which revealed slight lower T(m) for B27:06 and negative results for B27:07.

CONCLUSIONS

Genotyping of HLA-B27 by melting curve analysis was fast and reliable for routine laboratory testing for frequent alleles. In silico melting simulations provided useful information about the utility and limitation of this method for diverse HLA-B27 alleles.