Development of HLA-B*57:01 Genotyping Real-Time PCR with Optimized Hydrolysis Probe Design

J Mol Diagn. 2017 Sep;19(5):742-754. doi: 10.1016/j.jmoldx.2017.05.002. Epub 2017 Jul 18.


HLA-B*57:01 genotyping before abacavir (ABC) administration is a standard of care to avoid ABC-driven hypersensitivity reactions. Several HLA-B*57:01 tests have been developed, each with advantages and disadvantages. Some have limited accuracy, require special instrumentation, and/or are labor intensive and expensive. We developed a novel hydrolysis probe-based real-time PCR method of HLA-B*57:01 genotyping. Primer and probes were designed based on published sequence variations in exon 3 of HLA-B that distinguish HLA-B*57:01 from ABC-insensitive alleles such as HLA-B*57:03 and HLA-B*58:01. We designed PCR primers to amplify HLA-B*57:01 along with closely related alleles, such as HLA-B*57:03, directly from genomic DNA. Most ABC-insensitive alleles, including HLA-B*58:01, would not produce any products in the PCR reaction. Our hydrolysis probes enable differentiation of HLA-B*57:01 from the other amplified, but ABC-insensitive, alleles. In addition to using real-time PCR, we used restriction enzymes to generate differential digestion patterns that led to the development of an HLA-B*57:01 PCR-restriction fragment length polymorphism marker. When used to genotype a set of 75 selected clinical samples, our real-time PCR assay demonstrated 100% accuracy in distinguishing between the HLA-B*57:01-positive and -negative alleles when results were compared to those of sequence-specific oligonucleotide probe typing and reference laboratory testing. Our newly developed test will allow clinical laboratories with real-time PCR capabilities to perform HLA-B*57:01 genotyping in a timely and economical manner.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Base Sequence
  • DNA Probes*
  • Gene Frequency
  • Genotype
  • Genotyping Techniques*
  • HLA-B Antigens / chemistry
  • HLA-B Antigens / genetics*
  • Histocompatibility Testing / methods*
  • Histocompatibility Testing / standards
  • Humans
  • Hydrolysis
  • Real-Time Polymerase Chain Reaction / methods*
  • Real-Time Polymerase Chain Reaction / standards
  • Reproducibility of Results
  • Sensitivity and Specificity
  • Sequence Analysis, DNA


  • DNA Probes
  • HLA-B Antigens
  • HLA-B57 antigen