Low cost molecular assay for HIV

低成本 HIV 分子检测

• 批准号: 8054221
• 负责人: BERTRAND LEMIEUX
• 金额: $ 85.82万
• 依托单位: BIOHELIX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-15 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8054221
• 关键词: Antibodies; Automation; Bathing; Binding; Biological; Biological Assay; Biotin; Blood; Buffers; Characteristics; Clinical; Clinical Research; Communicable Diseases; Complex; Contracts; DNA; Data; Databases; Detection; Developing Countries; Devices; Diagnostic; Digoxigenin; Drug Formulations; Educational process of instructing; Engineering; Enzymes; Epidemic; Equilibrium; Fluorescein-5-isothiocyanate; HIV; HIV vaccine; HIV-1; Haptens; Health; Hospitals; Hour; Human; Human immunodeficiency virus test; Incubated; Institutes; Institutional Review Boards; Journals; Laboratories; Lateral; Latex; Latex Particles; Life; Marketing; Methods; Microspheres; Molecular; Multi-Institutional Clinical Trial; Nucleic Acids; Oral; Paper; Participant; Patient Recruitments; Patients; Performance; Persons; Phase; Plasma; Polymerase Chain Reaction; Positioning Attribute; Process; Publications; RNA; RNA-Directed DNA Polymerase; Reaction; Reagent; Recording of previous events; Reporting; Resources; Sales; Sampling; Screening procedure; Sensitivity and Specificity; Site; Solutions; South Africa; Specimen; Spottings; Streptavidin; System; Technology; Testing; Time; Tube; United States; United States Food and Drug Administration; Universities; Vaccination; Virus; Virus Diseases; Walking; Water; Western Blotting; aged; base; cost; design; follow-up; helicase; high risk; high school; improved; instrument; instrumentation; internal control; manufacturing facility; migration; prevent; programs; validation studies; viral RNA

DESCRIPTION (provided by applicant): We propose to develop a rapid, low cost, threshold assay for the detection of HIV infection of HIV vaccination trial participants. The assay we propose to develop will use our proprietary helicase dependent amplification (HDA) platform as well as a low cost device specifically designed to perform molecular tests without contaminating the laboratory with amplification products. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. It is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). We propose to use a lateral flow device to detect amplification products. Such devices have already proven their utility in separating nucleic acid amplification products, and are widely used in several "moderate complexity" and CLIA-waived tests listed in the FDA device database. Detection of amplicons will be accomplished by using latex particles conjugated to antibodies that bind to the probes used to detect the HDA reaction products. The assay uses a sandwich format to detect haptens (biotin, FITC and Digoxigenin) incorporated into one of the HDA primers and into each of the detection probes. The lateral flow strip used for these assays has two capture zones, and thus allows for the detection of one analyte (HIV) as well as a competitive internal control. Assays that fail to give a band in the lateral flow device window are scored as invalid, while assays with a band in the control line alone are scored as true negative. Assays with bands at both the control and test line position are scored as positives at the threshold value (typically 200 copies of the analyte). Assays with a strong band in the test line but no control line are scored as strong positives (typically over 5000 copies of analyte). Preliminary studies suggest a limit of detection of 100 copies/mL plasma when nucleic acids extracted from plasma samples with a sequence-specific capture method are used as templates for RT-HDA. We propose to further test the specificity and sensitivity of the HIV test with clinical specimens obtained from 5 high-risk patient recruitment sites. Assays will be performed at BioHelix, and at Vanderbilt University. Kits will be supplied to Dr. Yi-Wei Tang (Vanderbilt University) so his laboratory can evaluate the assays. In addition, we propose to perform a validation study at the National Institute for Communicable Diseases (NICD) of South Africa to gain clearance to sell kits in South Africa. Finally, we may also contract with Assuragen to engineer a clone of Armored RNA for our competitive internal control. By the end of Phase II, we will have preliminary data for submission of an IDE to the FDA to kick-off a clinical study to seek pre market approval (PMA) for sale of the assay system for human diagnostics in the US.

描述（由申请人提供）：我们建议开发快速，低成本，阈值分析，以检测HIV感染HIV疫苗接种试验参与者。我们建议开发的测定方法将使用我们专有的解旋酶依赖性扩增（HDA）平台以及专门设计用于执行分子测试的低成本设备，而无需用放大产品污染实验室。 HDA与聚合酶链反应（PCR）相似，因为它使用两个引物来指数增大核酸。它与PCR不同，因为它完全是等温的（因此不需要昂贵的热循环器）。我们建议使用横向流动装置检测放大产品。这种设备已经证明了它们在分离核酸扩增产物中的实用性，并且在FDA设备数据库中列出的几种“中等复杂性”和CLIA捕获的测试中广泛使用。通过使用与用于检测HDA反应产物的探针结合的抗体结合的乳胶颗粒来实现扩增子的检测。该测定法使用三明治格式来检测掺入HDA引物之一的触觉（生物素，FITC和二高素），并进入每个检测探针中。这些测定法的侧向流带有两个捕获区，因此可以检测一个分析物（HIV）以及一个竞争性的内部控制。无法在横向流量设备窗口中提供频带的测定法被评分为无效，而单独使用控制线中的频带的测定将其评分为真为负。在对照线位置和测试线位置的频带的测定在阈值值（通常为分析物的200份）时评分为阳性。测试线中具有强频带的测定法，但没有控制线的评分为强阳性（通常超过5000份分析物）。初步研究表明，当从具有序列特异性捕获方法的血浆样品中提取的核酸被用作RT-HDA的模板时，检测100份/mL血浆的限制。我们建议通过从5个高危患者招募部位获得的临床标本进一步测试HIV检验的特异性和灵敏度。测定将在Biohelix和Vanderbilt大学进行。套件将提供给Yi-Wei Tang博士（Vanderbilt University），以便他的实验室可以评估测定法。此外，我们建议在南非国家传染病研究所（NICD）进行验证研究，以获得在南非出售工具包的许可。最后，我们还可以与Assuragen合同，以设计竞争性内部控制的装甲RNA的克隆。到第二阶段结束时，我们将拥有向FDA提交IDE的初步数据，以启动临床研究，以寻求市场批准（PMA）以销售美国人类诊断的测定系统。