High accuracy of HLA-B*27 genotyping by allele-specific real-time polymerase chain reaction in a heterogeneous population compared to flow cytometry and single nucleotide polymorphism detection assays

Pathology. 2020 Feb;52(2):256-261. doi: 10.1016/j.pathol.2019.09.013. Epub 2020 Jan 3.


HLA-B27 is a risk marker for ankylosing spondylitis and other associated seronegative spondyloarthropathies. We compared three methods of HLA-B*27 typing in a New South Wales (NSW) population: flow cytometry, rs4349859 single nucleotide polymorphism (SNP) detection assay, and allele-specific real-time polymerase chain reaction (RT-PCR) analysis of exons 2 and 3. Over a 5-month period, 543 samples underwent flow cytometric testing and RT-PCR high-resolution melt analysis of rs4349859 SNP and of exon 2 (5' fragment) and exon 3. In the third method, positive samples were further analysed with fluorescent resonance emission transfer (FRET) RT-PCR of exon 2 fragments, 2a and 2b. HLA-B*27 and other genotypes were confirmed by Sanger sequencing of a 600 base pair fragment of exons 2 and 3. In our cohort, the rs4349859 SNP method had 78.6% sensitivity and 98.7% specificity. Screening with exon 2 (5' fragment) and exon 3 RT-PCR provided 100% sensitivity. Further testing with exon 2a and 2b FRET RT-PCR produced 100% specificity. This cascade approach with allele-specific RT-PCR assays was able to differentiate all samples into HLA-B*27 subtypes. HLA-B*27 genotyping with allele-specific RT-PCR assays, to screen for and confirm HLA-B27 positive samples, was more sensitive and specific than flow cytometry and rs4349859 SNP assays. It is a potentially cost-effective method for differentiating HLA-B27 subtypes. Our cascade genetic testing approach is suitable for replacing the current flow cytometric HLA-B27 assay for the heterogeneous NSW population.

Keywords: HLA-B27; ankylosing spondylitis; melting; real-time PCR.

Publication types

  • Comparative Study

MeSH terms

  • Alleles
  • Flow Cytometry
  • Genotype
  • Genotyping Techniques*
  • HLA-B27 Antigen / genetics*
  • Humans
  • New South Wales
  • Polymorphism, Single Nucleotide
  • Real-Time Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Spondylarthropathies / diagnosis*
  • Spondylarthropathies / genetics


  • HLA-B27 Antigen