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Detection of HLA-B27 alleles by group-specific amplification and time-resolved fluorometry. 1998

L Välimaa, and M Sjöroos, and M Luhtala, and P Toivanen, and T Lövgren, and J Ilonen
Department of Biotechnology, University of Turku, Finland.

This newly developed HLA-B27 assay combines a polymerase chain reaction (PCR) from blood spot samples with solution hybridisation in microtitration plate and with time-resolved fluorometry (TRF) as the detection system. In a multiplex amplification reaction, the 144 base pair region of HLA-B27 alleles is amplified with allele-specific primers simultaneously with the region of beta-actin gene as an internal control. Amplified products are collected onto streptavidin (SA)-coated microtitration wells, denatured and hybridised with a europium (Eu)-labelled HLA-B27 specific probe and a samarium (Sm)-labelled beta-actin specific probe. Finally, Eu and Sm fluorescence is enhanced and detected in a time-resolved fluorometer. The typing results obtained with 110 blood spot samples showed an exact match with serological class I HLA-typing. When this technique was further evaluated, 348 blood spot samples were clearly categorised into two populations, HLA-B27 positives and negatives. This new PCR-TRF method permits the automation of HLA-B27 assays and saves time and labour in routine diagnostics.

UI MeSH Term Description Entries
D009693 Nucleic Acid Hybridization Widely used technique which exploits the ability of complementary sequences in single-stranded DNAs or RNAs to pair with each other to form a double helix. Hybridization can take place between two complimentary DNA sequences, between a single-stranded DNA and a complementary RNA, or between two RNA sequences. The technique is used to detect and isolate specific sequences, measure homology, or define other characteristics of one or both strands. (Kendrew, Encyclopedia of Molecular Biology, 1994, p503) Genomic Hybridization,Acid Hybridization, Nucleic,Acid Hybridizations, Nucleic,Genomic Hybridizations,Hybridization, Genomic,Hybridization, Nucleic Acid,Hybridizations, Genomic,Hybridizations, Nucleic Acid,Nucleic Acid Hybridizations
D005188 False Negative Reactions Negative test results in subjects who possess the attribute for which the test is conducted. The labeling of diseased persons as healthy when screening in the detection of disease. (Last, A Dictionary of Epidemiology, 2d ed) False Negative Reaction,Reaction, False Negative,Reactions, False Negative
D005470 Fluorometry An analytical method for detecting and measuring FLUORESCENCE in compounds or targets such as cells, proteins, or nucleotides, or targets previously labeled with FLUORESCENCE AGENTS. Fluorimetry,Fluorometric Analysis,Analysis, Fluorometric
D006493 Heparin A highly acidic mucopolysaccharide formed of equal parts of sulfated D-glucosamine and D-glucuronic acid with sulfaminic bridges. The molecular weight ranges from six to twenty thousand. Heparin occurs in and is obtained from liver, lung, mast cells, etc., of vertebrates. Its function is unknown, but it is used to prevent blood clotting in vivo and vitro, in the form of many different salts. Heparinic Acid,alpha-Heparin,Heparin Sodium,Liquaemin,Sodium Heparin,Unfractionated Heparin,Heparin, Sodium,Heparin, Unfractionated,alpha Heparin
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000483 Alleles Variant forms of the same gene, occupying the same locus on homologous CHROMOSOMES, and governing the variants in production of the same gene product. Allelomorphs,Allele,Allelomorph
D012680 Sensitivity and Specificity Binary classification measures to assess test results. Sensitivity or recall rate is the proportion of true positives. Specificity is the probability of correctly determining the absence of a condition. (From Last, Dictionary of Epidemiology, 2d ed) Specificity,Sensitivity,Specificity and Sensitivity
D013997 Time Factors Elements of limited time intervals, contributing to particular results or situations. Time Series,Factor, Time,Time Factor
D015345 Oligonucleotide Probes Synthetic or natural oligonucleotides used in hybridization studies in order to identify and study specific nucleic acid fragments, e.g., DNA segments near or within a specific gene locus or gene. The probe hybridizes with a specific mRNA, if present. Conventional techniques used for testing for the hybridization product include dot blot assays, Southern blot assays, and DNA:RNA hybrid-specific antibody tests. Conventional labels for the probe include the radioisotope labels 32P and 125I and the chemical label biotin. Oligodeoxyribonucleotide Probes,Oligonucleotide Probe,Oligoribonucleotide Probes,Probe, Oligonucleotide,Probes, Oligodeoxyribonucleotide,Probes, Oligonucleotide,Probes, Oligoribonucleotide
D015796 HLA-B27 Antigen A specific HLA-B surface antigen subtype. Members of this subtype contain alpha chains that are encoded by the HLA-B*27 allele family. HLA Class I Histocompatibility Antigen, B-27 alpha Chain,HLA-B27,Antigen, HLA-B27,HLA B27 Antigen,HLA Class I Histocompatibility Antigen, B 27 alpha Chain

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