title: Real-time PCR using fluorescent resonance emission transfer probes for HLA-B typing.
abstract: HLA genotyping by polymerase chain reaction (PCR) has some inherent labor-intensive and effort-demanding limitations. To overcome them, we have developed a real-time PCR with hybridization probes approach able to obtain a medium-low resolution HLA-B genotyping with fewer tubes and probes and with a shorter time requirement. Our strategy used 18 simultaneous reactions amplifying HLA-B alleles and an internal control. Monitorization of both amplifications in each tube is performed by the simultaneous application of two fluorescent resonance emission transfer probes: the first probe, different for each tube, is specific for the HLA-B locus and the second probe detects the control gene. A medium-low resolution (300 HLA-B allelic groups) typing is obtained for each sample by analyzing the melting curve patterns. Because some alleles may be determined without using the complete set of reactions, we present an alternative strategy: a first round of seven reactions and, according to the result, a second (or third) round of PCRs to solve the ambiguities. This method was validated in pretyped clinical samples and the results were completely concordant. Moreover, fewer ambiguous results were obtained. In summary, we present a new, faster, and more accurate method than currently used PCR techniques to type HLA-B alleles.
authors.affiliation: Laboratory of Immunobiology Research and Applications to Diagnosis, Banc de Sang i Teixits, Barcelona, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Faner
authors.forename: Natàlia
authors.initials: N
authors.lastname: Casamitjana
authors.forename: Jordi
authors.initials: J
authors.lastname: Coll
authors.forename: Pepi
authors.initials: P
authors.lastname: Caro
authors.forename: Ricardo
authors.initials: R
authors.lastname: Pujol-Borrell
authors.forename: Eduard
authors.initials: E
authors.lastname: Palou
authors.forename: Manel
authors.initials: M
authors.lastname: Juan
mesh_terms: D004247:DNA / Q000097:blood; D015343:DNA Probes, HLA / Q000737:chemistry / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D005838:Genotype; D015235:HLA-B Antigens / Q000235:genetics*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
chemical_list: D015343:DNA Probes, HLA; D005456:Fluorescent Dyes; D015235:HLA-B Antigens; D004247:DNA