Genotyping of the CCR5 Chemokine Receptor by Isothermal NASBA Amplification and Differential Probe Hybridization
| Autor: | Joseph W. Romano, Roxanne N. Shurtliff, Savita Pahwa, Mark H. Kaplan, S. Tetali, Xue Ping Wang, Eun Mi Lee, Christine C. Ginocchio |
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| Rok vydání: | 1999 |
| Předmět: |
Microbiology (medical)
Genotype Receptors CCR5 Clinical Biochemistry Immunology Gene Dosage Biology Sensitivity and Specificity Gene dosage Article Random Allocation Nucleic acid thermodynamics Humans Immunology and Allergy Lymphocytes RNA Messenger Genotyping Alleles Oligonucleotide Hybridization probe virus diseases Nucleic Acid Hybridization Nucleic acid amplification technique Virology Molecular biology NASBA Mutation DNA Probes Nucleic Acid Amplification Techniques |
| Zdroj: | Clinical Diagnostic Laboratory Immunology. 6:959-965 |
| ISSN: | 1098-6588 1071-412X |
| DOI: | 10.1128/cdli.6.6.959-965.1999 |
| Popis: | The human CCR5 chemokine receptor functions as a coreceptor with CD4 for infection by macrophage-tropic isolates of human immunodeficiency virus type 1 (HIV-1). A mutated CCR5 allele which encodes a protein that does not function as a coreceptor for HIV-1 has been identified. Thus, expression of the wild-type and/or mutation allele is relevant to determining the infectibility of patient peripheral blood mononuclear cells (PBMC) and affects disease progression in vivo. We developed a qualitative CCR5 genotyping assay using NASBA, an isothermal nucleic acid amplification technology. The method involves three enzymes and two oligonucleotides and targets the CCR5 mRNA, which is expressed in PBMC at a copy number higher than 2, the number of copies of DNA present encoding the gene. The single oligonucleotide set amplifies both alleles, and genotyping is achieved by separate hybridizations of wild-type- and mutation-specific probes directly to the single-stranded RNA amplification product. Assay sensitivity and specificity were demonstrated with RNAs produced in vitro from plasmid clones bearing the DNA encoding each allele. No detectable cross-reactivity between wild-type and mutation probes was found, and 50 copies of each allele were readily detectable. Analysis of patient samples found that 20% were heterozygous and 1% were homozygous for the CCR5 mutation. Thus, NASBA is a sensitive and specific means of rapidly determining CCR5 genotype and provides several technical advantages over alternative assay systems. |
| Databáze: | OpenAIRE |
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