title: Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes.
abstract: Genotyping in closed tube is commonly performed using polymerase chain reaction (PCR) amplification and allele-specific oligonucleotide probes using fluorescence resonance energy transfer (FRET). Here we introduce a homogeneous human leukocyte antigen (HLA)-DQA1∗05 end-point PCR assay based on switchable lanthanide luminescence probe technology and a simple dried blood sample preparation. The switchable probe technology is based on two non-luminescent oligonucleotide probes: one carrying a non-luminescent lanthanide chelate and the other carrying a light-absorbing antenna ligand. Hybridization of the probes in adjacent positions to the target DNA leads to the formation of a highly luminescent lanthanide chelate complex by self-assembly of the reporter molecules. Performance of the HLA-DQA1∗05 assay was evaluated by testing blood samples collected on sample collection cards and was prepared by lysing the punched samples (3-mm discs) using alkaline reaction conditions and high temperature. Testing of 147 blood samples yielded 100% correlation to the heterogeneous DELFIA technology-based reference assay. Genotyping requires carefully designed probe sequences able to discriminate matched and mismatched target sequences by hybridization. Furthermore, definite genotype discrimination was achieved because inherently non-luminescent switchable probes together with time-resolved measurement mode led to very low background signal level and, therefore, very high signal differences averaging 54-fold between DQA1∗05 and other alleles.
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, 20520 Turku, Finland. Electronic address: ari.lehmusvuori@utu.fi.
authors.forename: Ari
authors.initials: A
authors.lastname: Lehmusvuori
authors.affiliation: Immunogenetics Laboratory and MediCity Research Laboratory, University of Turku, 20520 Turku, Finland.
authors.forename: Minna
authors.initials: M
authors.lastname: Kiviniemi
authors.affiliation: Immunogenetics Laboratory and MediCity Research Laboratory, University of Turku, 20520 Turku, Finland; Department of Clinical Microbiology, University of Eastern Finland, FI-70211 Kuopio, Finland.
authors.forename: Jorma
authors.initials: J
authors.lastname: Ilonen
authors.affiliation: Department of Biochemistry/Biotechnology, University of Turku, 20520 Turku, Finland.
authors.forename: Tero
authors.initials: T
authors.lastname: Soukka
mesh_terms: D015342:DNA Probes / Q000737:chemistry* / Q000235:genetics; D005063:Europium / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D060005:Genotyping Techniques / Q000379:methods*; D059848:HLA-DQ alpha-Chains / Q000235:genetics*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*
chemical_list: D015342:DNA Probes; D059848:HLA-DQ alpha-Chains; C069050:HLA-DQA1 antigen; D005063:Europium
keywords: Genotyping; Lanthanide chelate; PCR; Switchable sensor