Development of an HIV reverse transcriptase aptamer-based detection assay

基于 HIV 逆转录酶适体的检测方法的开发

• 批准号: 8846946
• 负责人: JEFFREY J DESTEFANO
• 金额: $ 7.6万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-12-01 至 2016-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8846946
• 关键词: Affinity; Antibodies; Antibody Formation; Antigens; Antiviral Agents; Back; Base Sequence; Binding; Biochemical; Biological Assay; Biosensor; Capsid Proteins; Categories; Coupled; Data; Detection; Development; Diagnostic; Enzyme-Linked Immunosorbent Assay; Equipment; Evolution; Fluorescent Antibody Technique; Gel; HIV; HIV Antibodies; HIV Infections; Health; Human; Individual; Infection; Laboratories; Laboratory Scientists; Ligands; Methods; Microscope; Modification; Molecular Biology; Monitor; Nucleic Acids; Nucleic acid sequencing; Nucleotides; Pathogen detection; Poly A; Preclinical Drug Evaluation; Proteins; RNA; RNA-Directed DNA Polymerase; Radioactivity; Scientist; Scintillation Counting; Signal Transduction; Source; Specificity; Technology; Testing; Therapeutic; Time; Virion; Virus; Virus Diseases; Virus Replication; Western Blotting; Work; aptamer; base; cost; cost effective; drug resistant virus; falls; follow-up; gel electrophoresis; model development; nanoparticle; next generation; oligo (dT); public health relevance; rapid detection; receptor; research study; screening; tool

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.

描述（由申请人提供）：自1984年发现该病毒以来，HIV的检测和定量一直是科学家和临床医生的长期问题。目前，有几种测定可用于在现场或实验室中筛查HIV感染。它们通常分为几类：检测HIV RNA的基于PCR的测定，检测p24衣壳蛋白的ELISA测定，以及检测HIV逆转录酶（RT）的测定。也有快速检测筛选试验，检测HIV抗体，通常用作现场试验的初始筛选试验，因为它们高度敏感，只需几分钟即可完成。RT测定虽然在大多数情况下是成本有效的，但往往具有低灵敏度，这也是p24测定的缺点。基于PCR的测定具有高灵敏度，但通常昂贵且耗时，并且需要专门的设备。这些测定也像其他PCR测定一样受到污染。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中使用少量病毒进行的检测方法，甚至可以检测到可能由药物筛选或突变分析导致的非常低水平的感染。在本申请中，我们提出了使用我们在实验室开发的基于适体的技术的这种测定。我们的实验室已经开发出核酸适体，可以非常紧密地与HIV RT结合，并且能够检测到少至50-100个病毒颗粒。我们已经使用这些适体来开始开发基于适体的RT检测测定（阿尔塔），其是灵敏的、定量的和廉价的。我们设想这种技术作为下一代的艾滋病毒RT为基础的检测方法，并作为一个模型，为其他适配体为基础的病原体检测方法的发展。