Detecting Early Emerging Drug-Resistant HIV Populations

描述(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)感染总是会导致耐药突变的选择和积累，这些突变是由于HIV聚合酶的内在低保真度而产生的。这些不断增长的变异的早期检测可能具有临床意义。在确定临床重要性之前，需要有在大量密切相关的准病毒物种中可靠地检测微小基因变化的技术。我们开发了一种新的、闭合管、单血管RT-PCR方法来检测和定量野生型和突变病毒序列的混合种群。我们的方法，称为基因编码，使用两个单独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的遗传物质的混合种群。掺入猝灭剂标记的核苷酸会导致反应荧光的降低，与聚合酶链式反应产物的积累成比例。在聚合酶链式反应的扩展阶段进行荧光测量，以确定多个通道中的周期阈值(Ct)。未知样品的百分比组成可以通过测量两个通道的Ct之间的差异(定义为DeltaCt)并将该DeltaCt绘制到先前建立的标准曲线上来确定。标准曲线是通过绘制一个目标与另一个目标的百分比或比率与一组已知标准样品的增量Ct的关系来生成的。混合种群测试系统分析野生型与突变型RNA的百分比。数据显示，在标准混合物中，deltaCt发生了很大的变化，动态范围从100%到0.01%。在一个总共含有1.5×10/7目标分子的反应中，我们可以检测到每10,000个野生型RNA拷贝中就有一个突变拷贝。该方法还可以确定分析物的百分比组成。我们将优化我们的系统，以检测和量化17个抗逆转录病毒耐药性艾滋病毒突变。这些突变导致对三种通常被禁止的艾滋病治疗药物产生抗药性：逆转录酶(RT)核苷抑制剂、非核苷RT抑制剂和蛋白酶抑制剂。这项技术的发展可以包括更多的艾滋病毒变种，这可能会带来一种易于运行和广泛使用的研究和临床诊断方法。在未来，科学家可能会更容易地检测和评估抗药性艾滋病毒变种，就像它们出现在患者或实验样本中一样。

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.