HLA‐DR typing by PCR amplification with sequence‐specific primers (PCR‐SSP) in 2 hours: An alternative to serological DR typing in clinical practice including donor‐recipient matching in cadaveric transplantation

Abstract

In most PCR-based tissue typing techniques the PCR amplification is followed by a post-amplification specificity step. In typing by PCR amplification with sequence-specific primers (PCR-SSP), typing specificity is part of the amplification step, which makes the technique almost as fast as serological tissue typing. In the present study primers were designed for DR "low-resolution" typing by PCR-SSP, i.e. identifying polymorphism corresponding to the serologically defined series DR1-DRw18. This resolution was achieved by performing 19 PCR reactions per individual, 17 for assigning DR1-DRw18 and 2 for the DRw52 and DRw53 superspecificities. Thirty cell lines and 121 individuals were typed by the DR "low-resolution" PCR-SSP technique, TaqI DRB-DQA-DQB RFLP analysis and serology. The concordance between PCR-SSP typing and RFLP analysis was 100%. The reproducibility was 100% in 40 samples typed on two separate occasions. No false-positive or false-negative typing results were obtained. All homozygous and heterozygous combinations of DR1-DRw18 could be distinguished. Amplification patterns segregated according to dominant Mendelian inheritance. DNA preparation, PCR amplification and post-amplification processing, including gel detection, documentation and interpretation, were performed in 2 hours. In conclusion, PCR-SSP is an accurate typing technique with high sensitivity, specificity and reproducibility. The method is rapid and inexpensive. DR "low-resolution" typing by the PCR-SSP technique is ideally suited for analyzing small numbers of samples simultaneously and is an alternative to serological DR typing in routine clinical practice including donor-recipient matching in cadaveric transplantations.

The DERAA HLA–DR alleles in patients with early polyarthritis: Protection against severe disease and lack of association with rheumatoid arthritis autoantibodies

“…Genomic DNA was extracted from 2.5 ml of EDTA-treated whole blood (Wizard DNA kit; Promega, Madison, WI) and then stored at 4°C. Genomic typing was performed using sequence-specific primer PCR techniques specific for HLA class II molecules (24,25). Primer sets for low-resolution typing of HLA-DR and HLA-DQ antigens and for high-resolution typing of DRB1*01, DRB1*04, DRB1*11, DRB1*13, and DRB1*14 were obtained from Pel-Freez Clinical Systems (Brown Deer, WI).…”

Association of the PD‐1.3A allele of the PDCD1 gene in patients with rheumatoid arthritis negative for rheumatoid factor and the shared epitope

“…An analysis of HLA-DRB1 genotypes was performed using the sequence-specific primer-polymerase chain reaction method (DR Low Resolution kit; Olerup SSP, Saltsjöbaden, Sweden) as previously described (14,15). Interpretation of the bands was done according to the manufacturer's instructions.…”

HLA-DRB1*04 and DRB1*10 are associated with resistance and susceptibility, respectively, in Brazilian and Vietnamese leprosy patients

“…27 The DRB1 alleles were identified using sequence-specific primers (SSP) for PCR amplification as described previously. 28 …”