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首页> 外文期刊>Clinical and diagnostic laboratory immunology >Genotyping of the CCR5 Chemokine Receptor by Isothermal NASBA Amplification and Differential Probe Hybridization
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Genotyping of the CCR5 Chemokine Receptor by Isothermal NASBA Amplification and Differential Probe Hybridization

机译：等温NASBA扩增和差异探针杂交对CCR5趋化因子受体的基因分型。

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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.
机译：人CCR5趋化因子受体充当CD4的共受体，以被人类免疫缺陷病毒1型（HIV-1）的巨噬细胞嗜性分离株感染。已经鉴定出突变的CCR5等位基因，该等位基因编码的蛋白质不具有HIV-1的共受体功能。因此，野生型和/或突变等位基因的表达与确定患者外周血单个核细胞（PBMC）的可感染性有关，并影响体内疾病的进展。我们使用等温核酸扩增技术NASBA开发了定性CCR5基因分型测定法。该方法涉及三种酶和两种寡核苷酸，并靶向CCR5 mRNA，该蛋白在PBMC中表达的拷贝数高于2，即编码该基因的DNA拷贝数。单个寡核苷酸组可同时扩增两个等位基因，并通过将野生型和突变特异性探针直接与单链RNA扩增产物直接杂交来实现基因分型。用带有编码每个等位基因的DNA的质粒克隆体外产生的RNA证明了测定的敏感性和特异性。在野生型和突变探针之间未发现可检测到的交叉反应性，每个等位基因的50个拷贝很容易被检测到。对患者样品的分析发现，CCR5突变的杂合子为20％，纯合子为1％。因此，NASBA是快速确定CCR5基因型的灵敏且特异的手段，与替代测定系统相比，具有多种技术优势。

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