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)：在这个第一阶段的项目中，我们建议研究一种名为Zip核酸(ZNA)的新型底物化学和探针检测系统。ZNAs是与许多阳离子精胺基团连接的寡核苷酸，可提高核酸靶标附近的引物和探针的有效浓度。据报道，这一特性可以提高RT-PCR的速度和灵敏度(Moreau等人)。2009年)。ZNAs与Taqman检测格式兼容(Paris等人2010)。我们希望这项检测技术将进一步加快我们的RNA检测分析，并提高我们的准确性。我们将比较ZNA和LNA DNA双重标记的性能(童等人)。2008年，Li等人。2010)和CPT探头。到研究结束时，我们将完成IsoAmp HIV-1定量检测商业化发布所需的分析研究阶段。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检测的Zna TaqMan和Zna循环探针，3.开发从干血点(DBS)和干浆点(DPS)中提取RNA的简单工作流程，以及4.在一组HIV-1分离株中使用ZNA技术结合IsoAmp来测试检测的敏感性和特异性。在第一阶段结束时，我们将准备确定最佳探针技术，以开发一种商业IsoAmp HIV-1定量检测方法，在美国和国外进行商业分销。我们还将明确指示最适合HDA病毒负载测试的样本提取方法类型。在第二阶段，我们将为一项多地点临床研究计划开发一种前IDE，以寻求FDA批准在美国销售。我们还将探索在世界其他地区进行商业发行。 公共卫生相关性：在美国和欧洲，人类免疫缺陷病毒(HIV)病毒负荷测试是监测抗逆转录病毒治疗的护理标准。艾滋病毒定量检测依赖于使用聚合酶链式反应(PCR)的高通量系统，而且都面临一个主要限制：需要昂贵的仪器和熟练的人员来操作设备(Fiscus等人)。2006)。在发展中国家，低成本的CD4+监测测试(Rodriguez et al.2005年)是因为经济驱动因素，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等人。2010)。Biohelix已经开发出一种等温核酸扩增化学，称为解旋酶依赖扩增(HDA)，可以解决这个问题。这项技术与检测病毒载量的PCR方法相比有4个优点：1)它依赖于低成本的仪器(1/10的PCR机成本)，2)它可以比DNA更快地扩增RNA，并且可以与最快的PCR分析相匹配(Goldmeyer等人)。2007)，3)它比PCR更耐受碱基变化，4)它对临床标本中发现的扩增抑制剂比PCR更耐受。在这个第一阶段的项目中，我们将探索Zip核酸(ZNA)的潜在应用，以提高我们的艾滋病毒检测的性能(Tang等人。2010)。到研究结束时，我们将完成IsoAmp HIV-1定量检测商业化发布所需的分析研究阶段。在第二阶段，我们将为一项多地点临床研究计划开发一种前IDE，以寻求FDA批准在美国销售。