Detecting Early Emerging Drug-Resistant HIV Populations

描述（由申请人提供）：用抗逆转录病毒药物治疗人类免疫缺陷病毒1型（HIV）感染总是导致耐药突变体的选择和积累，这是由于HIV聚合酶本质上的低保真度引起的。早期发现这些生长变异可能具有临床意义。在确定临床重要性的水平之前，需要有可靠地检测大量密切相关的病毒准物种中微小遗传变化的技术。我们开发了一种新颖的闭管单管RT-PCR方法来检测和定量野生型和突变型病毒序列的混合种群。我们的方法称为GENE-CODE，使用两个独立的荧光团标记的正向引物和一个共同的反向引物来分析密切相关遗传物质的混合群体。加入淬灭剂标记的核苷酸导致反应荧光与PCR产物的积累成比例地减少。荧光测量在PCR的延伸阶段进行，以确定多通道的周期阈值（Ct）。未知样品的组成百分比可以通过测量两个通道的Ct（定义为deltaCt）之间的差来确定，并将该deltaCt绘制到先前建立的标准曲线上。标准曲线是通过绘制一组已知标准样本的一个目标与另一个目标与deltaCt的百分比或比率来生成的。混合群体测试系统分析野生型和突变型RNA的百分比。数据显示，在标准混合物中，deltaCt在100%至0.01%耐药突变序列的动态范围内变化很大。在含有1.5 x 10/7总靶分子的反应中，我们可以检测到每10,000个野生型RNA拷贝中有一个突变拷贝。该方法还可测定分析物的组成百分比。我们将优化我们的系统，以检测和量化17种抗逆转录病毒抗性HIV突变。这些突变赋予了对三种常用的艾滋病治疗药物的耐药性：逆转录酶（RT）核苷抑制剂、非核苷RT抑制剂和蛋白酶抑制剂。这项技术的发展包括更多的艾滋病毒变体，可能会导致一种简单易用和广泛使用的研究和临床诊断方法。在未来，科学家可以更容易地检测和评估耐药的艾滋病病毒变体，就像它们在患者或实验样本中出现一样。

DESCRIPTION (provided by applicant): Treatment of human immunodeficiency virus type 1 (HIV) infection with antiretroviral agents invariably leads to selection and accumulation of drug-resistant mutants that arise due to the intrinsically low fidelity of HIV polymerase. Early detection of these growing variants may be clinically relevant. Before the level of clinical importance can be ascertained, technologies that reliably detect small genetic changes in a large sea of closely related viral quasi-species need to be available. We have developed a novel, closed-tube, single vessel RT-PCR method to detect and quantitate mixed populations of wild-type and mutant viral sequences. Our method, called GENE-CODE, uses two separate fluorophore-labeled forward primers and one common reverse primer to analyze mixed populations of closely related genetic materials. Incorporation of a quencher-labeled nucleotide results in a decrease in reaction fluorescence in proportion to the accumulation of PCR product. Fluorescence measurements are performed during extension phase of the PCR to determine the cycle threshold (Ct) in multiple channels. The percent composition of an unknown sample can be determined by measuring the difference between the Ct of the two channels (defined as deltaCt) and plotting that deltaCt onto a previously established standard curve. The standard curve is generated by plotting percent or ratio of one target to the other versus deltaCt for a set of known standard samples. The mixed population test system analyzes the percentage of wild-type to mutant RNA. The data show a large change in deltaCt with a dynamic range from 100% to 0.01% drug-resistant mutant sequence in a standard mixture. We can detect one copy of mutant per 10,000 wild-type RNA copies in a reaction containing 1.5 x 10/7 total target molecules. This method also determines percent make-up of the analyte. We will optimize our system to detect and quantitate 17 antiretroviral resistant HIV mutations. These mutations confer resistance to the three types of commonly proscribed AIDS therapeutics : nucleoside inhibitors of reverse transcriptase (RT), non-nucleoside RT inhibitors and protease inhibitors. Development of this technology to include a larger number of HIV variants could lead to a simple-to-run and widely-used method for research and clinical diagnostics. In the future, scientists may more easily detect and evaluate drug-resistant HIV variants just as they emerge within patient or experimental samples.