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

描述（由申请人提供）：在该I期项目中，我们提出研究一种称为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，所述阳离子精胺部分增强引物和探针在核酸靶标附近的有效浓度。 据报道，该特性可提高RT-PCR的速度和灵敏度（Moreau et al. 2009）。 ZNA与Taqman检测格式兼容（巴黎等人，2010）。 我们预计这种检测技术将进一步加快我们的RNA检测试验，并提高我们的准确性。 我们将比较ZNA与LNA DNA双标记（Tong et al. 2008，Li et al. 2010）和CPT探针的性能。 在研究结束时，我们将完成Isopropyl HIV-1定量检测试剂盒商业放行所需的分析研究阶段。 我们的第一阶段研究计划包括四个目标： 1.设计并测试针对HIV-1所有亚型的ZNA引物， 2.设计和测试ZNA taqman和ZNA循环探针用于HIV检测， 3.开发从干血斑（DBS）和干血浆中提取RNA的简单工作流程 点（DPS），以及 4.使用ZNA技术结合以下技术测试检测的灵敏度和特异性： 一组HIV-1分离株中的异丙肾上腺素。 在第一阶段结束时，我们将准备确定最佳的探针技术，以开发一种用于美国和国外商业销售的商业Isoprotein HIV-1定量检测。 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的类型。 在第二阶段，我们将为多中心临床研究计划开发一个预IDE，以寻求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行。 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标准护理。 HIV定量检测依赖于使用聚合酶链反应（PCR）的高通量系统，并且都受到一个主要限制：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，由于经济驱动因素，使用低成本的CD 4+监测检测（Rodriguez et al. 2005），尽管在治疗失败的情况下，CD 4+监测滞后于病毒RNA载量的上升（Vaidya et al. 2010）。 BioHessels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，可以解决这个问题。 该技术与PCR病毒载量检测方法相比有4个优点：1）它依赖于低成本的工具（1/10的成本PCR机器），2）它可以扩增RNA比DNA更快，可以匹配最快的PCR检测（Goldmeyer等，2007），3）它比PCR更能容忍引物和探针中的碱基变异，和4）它比PCR更耐受在临床样品中发现的扩增抑制剂。 在该I期项目中，我们将探索Zip核酸（ZNA）的潜在应用，以提高我们的HIV检测试剂盒的性能（Tang et al. 2010）。 在研究结束时，我们将完成Isopropyl HIV-1定量检测试剂盒商业放行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预IDE，以寻求FDA批准在美国销售。

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.