Low-cost, rapid quantitative Isothermal Assay for HIV RNA using ZNA

使用 ZNA 对 HIV RNA 进行低成本、快速定量等温测定

• 批准号: 8256266
• 负责人: Huimin Kong
• 金额: $ 30万
• 依托单位: BIOHELIX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8256266
• 关键词: Adherence; Affinity; Anti-Retroviral Agents; Back; Binding; Biological Assay; Blood; Chemistry; Clinical; Country; DNA; DNA-Directed DNA Polymerase; Detection; Dose; Economically Deprived Population; Economics; Enzymes; Equipment; Europe; Failure; Fluorescence; Goals; Human; Human Resources; Immune; Label; Laboratory Personnel; Licensing; Logistics; Measurement; Methods; Minor; Molecular; Molecular Conformation; Monitor; Multi-Institutional Clinical Trial; National Institute of Allergy and Infectious Disease; Nucleic Acid Probes; Nucleic Acids; Oligonucleotides; Patients; Performance; Pharmaceutical Preparations; Phase; Plasma; Polymerase Chain Reaction; Problem Solving; Property; RNA; Reporting; Research; Resources; Rest; Reverse Transcription; Risk; Sales; Sampling; Sensitivity and Specificity; Signal Transduction; Small Business Innovation Research Grant; Sodium Chloride; Specimen; Speed; Spermine; Spottings; Staging; System; Taq Polymerase; Technology; Temperature; Testing; Therapeutic; Time; Training; United States; Variant; Viral Load result; Viral load measurement; Virus; Work; base; cost; cost effective; design; health care delivery; helicase; inhibitor/antagonist; instrument; instrumentation; internal control; locked nucleic acid; melting; mortality; novel; phase 2 study; response; standard of care; viral RNA

DESCRIPTION (provided by applicant): In this Phase I project, we propose to investigate a novel primer chemistry and probe detection system called the Zip nucleic acids (ZNA"). ZNAs are oligonucleotides conjugated to a number of cationic spermine moieties that enhance the effective concentration of primers and probes near nucleic acid targets. This property has been reported to enhance the speed and sensitivity of RT-PCR (Moreau et al. 2009). ZNAs are compatible with Taqman detection formats (Paris et al. 2010). We expect this detection technology will further accelerate our RNA detection assays as well as increase our accuracy. We will compare the performance of ZNA to LNA DNA dual labeled (Tong et al. 2008, Li et al. 2010) and CPT probes. By the end of the study we will have completed the analytical study stage required for the commercial release of an IsoAmp HIV-1 quantitative assay. Our phase I research plan includes 4 aims: 1. Design and test ZNA primers targeting all subtypes of HIV-1, 2. Design and test with both ZNA taqman and ZNA cycling probes for the HIV assay, 3. Develop a simple work flow for extraction of RNA from dry blood spots (DBS) and dry plasma spots (DPS), and 4. Test the sensitivity and specificity of assays using the ZNA technology in combination with IsoAmp in a panel of HIV-1 isolates. At the conclusion of Phase I, we will be ready to identify the best probe technology to develop a commercial IsoAmp HIV-1 quantitative assay for commercial distribution in the US and abroad. We will also have a clear indication of the type of sample extraction method that best suits HDA viral load testing. In Phase II, we would develop a pre-IDE for a multi-site clinical study plan to seek FDA approval for sale in the US. We would also explore commercial release in the rest of the World. PUBLIC HEALTH RELEVANCE: Human immune-deficiency virus (HIV) viral load testing is the standard of care for monitoring anti-retroviral therapy in the United States and Europe. HIV quantitative tests rely of high throughput systems that use the polymerase chain reaction (PCR) and all suffer one major limitation: a need for expensive instrumentation, and skilled personnel to operate the equipment (Fiscus et al. 2006). In the developing World, low-cost CD4+ monitoring tests (Rodriguez et al. 2005) are used because of economic drivers, despite the fact that CD4+ monitoring lags a rise in viral RNA load in cases of therapeutic failure (Vaidya et al. 2010). BioHelix has developed an isothermal nucleic acid amplification chemistry called helicase dependent amplification (HDA) that can solve this problem. This technology has 4 advantages over PCR methods of viral load testing: 1) it relies on a low-cost instrument (1/10 the cost of PCR machines), 2) it can amplify RNA faster than DNA and can match the fastest PCR assays (Goldmeyer et al. 2007), 3) it is more tolerant of base variations in primers and probes than PCR, and 4) it is more tolerant to amplification inhibitors found in clinical samples than PCR. In this Phase I project, we will explore the potential application of Zip nucleic acids (ZNA) to enhance the performance of our HIV assays (Tang et al. 2010). By the end of the study we will have completed the analytical study stage required for the commercial release of an IsoAmp HIV-1 quantitative assay. In Phase II, we would develop a pre-IDE for a multi-site clinical study plan to seek FDA approval for sale in the US.

描述（由申请人提供）：在这个第一阶段项目中，我们建议研究一种称为 Zip 核酸 (ZNA") 的新型引物化学和探针检测系​​统。ZNA 是与许多阳离子精胺部分缀合的寡核苷酸，可增强核酸靶标附近引物和探针的有效浓度。据报道，这一特性可提高 RT-PCR 的速度和灵敏度（Moreau 等人） 等人。 2009）。 ZNA 与 Taqman 检测格式兼容（Paris 等人，2010）。 我们预计这项检测技术将进一步加速我们的 RNA 检测分析并提高我们的准确性。 我们将比较 ZNA 与 LNA DNA 双标记探针（Tong 等人，2008 年；Li 等人，2010 年）和 CPT 探针的性能。 在研究结束时，我们将完成商业发布所需的分析研究阶段 IsoAmp HIV-1 定量测定。 我们的第一阶段研究计划包括 4 个目标：1. 设计和测试针对 HIV-1 所有亚型的 ZNA 引物，2. 设计和测试用于 HIV 测定的 ZNA taqman 和 ZNA 循环探针，3. 开发一个简单的工作流程，用于从干血斑 (DBS) 和干血浆点 (DPS) 中提取 RNA，4. 测试测定的灵敏度和特异性 在一组 HIV-1 分离株中使用 ZNA 技术与 IsoAmp 相结合。 在第一阶段结束时，我们将准备好确定最佳探针技术来开发商业 IsoAmp HIV-1 定量检测，以便在美国和国外进行商业分销。 我们还将明确指出最适合 HDA 病毒载量检测的样本提取方法类型。 在第二阶段，我们将为多中心临床研究计划开发一个预IDE 寻求 FDA 批准在美国销售。 我们还将探索在世界其他地区的商业发布。 公共卫生相关性：人类免疫缺陷病毒 (HIV) 病毒载量检测是美国和欧洲监测抗逆转录病毒治疗的护理标准。 HIV 定量检测依赖于使用聚合酶链式反应 (PCR) 的高通量系统，并且所有系统都存在一个主要限制：需要昂贵的仪器和熟练的人员来操作设备（Fiscus 等，2006）。 在发展中国家，出于经济驱动因素，人们使用低成本的 CD4+ 监测测试（Rodriguez 等，2005），尽管在治疗失败的情况下，CD4+ 监测滞后于病毒 RNA 载量的上升（Vaidya 等，2010）。 BioHelix 开发了一种称为解旋酶依赖性扩增 (HDA) 的等温核酸扩增化学方法，可以解决这个问题。 与病毒载量检测的 PCR 方法相比，该技术有 4 个优点：1) 它依赖于低成本仪器（PCR 机器成本的 1/10），2) 它可以比 DNA 更快地扩增 RNA，并且可以匹配最快的 PCR 检测（Goldmeyer et al. 2007），3) 它比 PCR 更能容忍引物和探针的碱基变异，4) 更能容忍扩增 临床样本中发现的抑制剂多于 PCR。 在这个第一阶段项目中，我们将探索 Zip 核酸 (ZNA) 的潜在应用，以提高我们的 HIV 检测性能（Tang 等人，2010）。 到研究结束时，我们将完成商业发布 IsoAmp HIV-1 定量检测所需的分析研究阶段。 在第二阶段，我们将为多中心临床研究计划开发一个 pre-IDE，以寻求 FDA 批准在美国销售。