A1 Journal article – refereed
Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes




List of Authors: Lehmusvuori A, Kiviniemi M, Ilonen J, Soukka T
Publisher: Elsevier
Publication year: 2014
Journal: Analytical Biochemistry
Journal name in source: Anal Biochem
Journal acronym: Analytical biochemistry
Volume number: 465
Number of pages: 6

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.



Last updated on 2019-20-07 at 17:12