Development of an HIV reverse transcriptase aptamer-based detection assay

描述(申请人提供)：自1984年发现艾滋病毒以来，艾滋病毒的检测和定量一直是科学家和临床医生面临的一个长期问题。目前，有几种方法可用于现场或实验室中筛查艾滋病毒感染。它们通常分为几类：基于聚合酶链式反应的检测HIV RNA的检测，检测p24衣壳蛋白的ELISA检测，以及检测HIV逆转录酶(RT)的检测。还有检测艾滋病毒抗体的快速检测筛查试验，通常用作现场检测的初始筛查试验，因为它们高度敏感，只需几分钟就能完成。虽然RT检测在大多数情况下具有成本效益，但往往灵敏度较低，这也是p24检测的一个缺点。基于聚合酶链式反应的分析具有很高的灵敏度，但通常成本高、耗时长，并且需要专门的设备。这些检测也像其他的聚合酶链式反应检测一样容易受到污染。艾滋病毒领域将受益于一种灵敏、定量、快速和简单的廉价检测方法。一种可以在实验室环境下使用少量病毒进行的检测，可以检测到可能由药物筛选或突变分析引起的极低水平的感染。在本申请中，我们提出了一种使用我们实验室开发的基于适体的技术的检测方法。我们实验室已经开发出与HIV RT结合非常紧密的核酸适配子，能够检测到低至50-100个病毒颗粒。我们已经利用这些适配子开始建立一种灵敏、定量、廉价的基于适配子的RT检测方法(ARTA)。我们设想这项技术将成为下一代基于HIV RT的检测方法，并作为开发其他基于适体的病原体检测方法的模型。

DESCRIPTION (provided by applicant): Detection and quantitation of HIV has been a longstanding issue for scientists and clinicians since the discovery of the virus in 1984. Currentl there are several assays available for screening for HIV infection in the field or the laboratory. They typically fall into a few categories: PCR-based assays that detect HIV RNA, ELISA assays that detect p24 capsid protein, and assays that detect HIV reverse transcriptase (RT). There are also rapid detection screening assays that detect antibodies to HIV and are typically used as the initial screening tests in field assays because of they are highly sensitive and take just minutes to complete. The RT assays though cost effective in most cases, tend to have low sensitivity which is also a drawback of p24 assays. PCR-based assays have high sensitivity but are typically costly and time consuming and require specialized equipment. These assays are also subject to contamination like other PCR assays. The HIV field would benefit from an inexpensive assay that is sensitive, quantitative, fast and simple. An assay that can be performed in a laboratory setting with small quantities of virus and could detect even very low levels of infection that might result from drug screens or mutational analysis. In this application we propose such an assay using aptamer-based technology that we have developed in our lab. Our lab has developed nucleic acid aptamers that can bind to HIV RT with very tightly and are capable of detecting as few as 50-100 virus particles. We have used these aptamers to begin to develop an Aptamer-based RT detection Assay (ARTA) that is sensitive, quantitative, and inexpensive. We envision this technology as the next generation of HIV RT-based detection assays and as a model for development of other aptamer-based pathogen detection assays.