Development of a new HLA-DRB real-time PCR typing method.
Polymerase chain reaction (PCR)-based human leukocyte antigen (HLA) typing methods currently used in most histocompatibility laboratories, such as PCR-sequence-specific primers (PCR-SSP) and PCR-sequence-specific oligonucleotide probes (PCR-SSO), are time-consuming and are at risk of contamination during the post-PCR process. The aim of this study was to develop a real-time PCR (rtPCR)-based HLA-DRB1 and -DRB3/4/5 low-medium resolution typing method to avoid these problems. This new method combined the use of specific primers and probes for HLA-DRB alleles. One pair of DRB gene primers and two DRB-specific probes (FAM and VIC) were used per reaction in each of a set of 16 PCR reaction tubes. To provide an internal positive control, each tube also contained a pair of primers and a TET probe for glyceraldehyde phosphate dehydrogenase. This allowed a very significant reduction in the number of reactions and the processing time, whereas typing resolution increased. After successful testing on 100 samples, the technique was validated in 200 clinical samples that had previously been typed for HLA-DRB using a standard PCR-based method. Identical results were obtained with all samples. This new method also reduced ambiguous results and was faster and less cumbersome than currently used PCR-SSP or PCR-SSO techniques.
Casamitjana
Natàlia
N
Laboratori d'Immunobiologia per a la Recerca i Aplicacions Diagnòstiques, Serveis Sanitaris de Referència--Centre de Transfusió i Banc de Teixits, Barcelona, Spain.
Faner
Rosa
R
Santamaria
Albert
A
Colobran
Roger
R
Ribera
Anna
A
Pujol-Borrell
Ricardo
R
Juan
Manel
M
Palou
Eduard
E
D000483:Alleles; D017931:DNA Primers; D006684:HLA-DR Antigens / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D006650:Histocompatibility Testing / Q000379:methods; D006801:Humans; D016377:Organ Transplantation; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase
D017931:DNA Primers; D006684:HLA-DR Antigens; D019914:Taq Polymerase