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.
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.
Center for BioTechnology, Karolinska Institute, NOVUM, Huddinge, Sweden.
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Olerup
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Zetterquist
D000483:Alleles; D001483:Base Sequence; D002102:Cadaver; D005802:Genes, MHC Class II; D006684:HLA-DR Antigens / Q000235:genetics*; D006650:Histocompatibility Testing / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D015203:Reproducibility of Results; D016415:Sequence Alignment; D017385:Sequence Homology; D013997:Time Factors; D014019:Tissue Donors; D014181:Transplantation Immunology
D006684:HLA-DR Antigens; D015345:Oligonucleotide Probes