Recombinase Polymerase Amplification for Point-of-Care Diagnosis of Infant HIV-1

描述（由申请人提供）：在撒哈拉以南非洲，超过50%感染人类免疫缺陷病毒1型（HIV-1）的婴儿在2岁前死亡。幸运的是，早期诊断和治疗感染婴儿可显著减缓疾病进展并提高生存率。虽然治疗的可及性正在改善，但目前可用的婴儿HIV-1诊断方法价格昂贵，需要复杂的设备和样品处理。因此，婴儿艾滋病毒-1检测仅限于集中实验室，这延迟了向农村地区及时报告检测结果。可在医疗点或离使用者较近的农村诊所进行的快速和易于使用的婴儿艾滋病毒诊断将缩短检测的周转时间，从而可以更快地开始治疗，并显著改善农村地区艾滋病特异性儿童死亡率。这个拟议项目的目标是创建一种婴儿HIV-1诊断方法，可以检测世界范围内传播的HIV毒株，并且需要最少的基础设施来执行。我们已经确定了可以实现这一目标的技术组合。一种等温扩增方法，重组酶聚合酶扩增（RPA），使用序列特异性引物和探针在~380℃恒温下扩增DNA。扩增子可以通过荧光检测，使用简单的低成本读取器，或通过横向流动条（LFS），减少了对任何复杂设备的需求。设计一种能够诊断所有常见HIV-1亚型的单一RPA HIV-1检测方法是本研究的主要目的。相同的引物和探针序列可以在荧光或LFS格式中使用，为分析使用增加了更大的效用。由于HIV-1是非常多样化的，基因组的保守区域将被识别，RPA引物和探针将被设计并针对不同的病毒模板进行测试。HIV-1 RPA检测将被优化，以高灵敏度和特异性耐受不同HIV亚型的多样性。全血常规用于检测HIV-1 DNA。通常，用滤纸干燥的全血用于稳定婴儿诊断采集的血液。该项目将确定全血或干血斑点（DBS）是否与RPA测定相容，或者捕获淋巴细胞的膜是否会增加测定的敏感性。使用DBS的现有和新型快速DNA提取方法将与RPA分析一起进行评估，以建立具有最佳灵敏度的测试方案。为了消除对动力培养设备的要求，已经确定了在RPA的最佳培养范围内加热的放热化合物，用于LFS RPA格式。拟议项目的总体目标是创建一种基于RPA的HIV-1诊断方法，该方法可以最小限度地或完全不使用仪器；需要最少的样品处理，通过荧光或LFS扩增检测；并且可以在各种资源有限的环境中用于护理点。

DESCRIPTION (provided by applicant): Over 50% of infants in sub-Saharan Africa who are infected with human immunodeficiency virus type 1 (HIV-1) die before 2 years of age. Fortunately, early diagnosis and treatment of infected infants significantly slows disease progression and improves survival. While access to treatment is improving, currently available infant HIV-1 diagnostics are expensive and require complex equipment and sample processing. Therefore, infant HIV-1 testing is restricted to centralized laboratories, which delays the timely reporting of test results to rural areas. A rapid and easy-to-use infant HIV diagnostic that can be performed at the point of care or in rural clinics closer to the user will reduce turn-around time for testing so treatment can start sooner and significantly improve AIDS-specific child mortality rates in rural areas. The goal of this proposed project is to create an infant HIV-1 diagnostic that can detect the strains of HIV that are circulating worldwide and that requires minimal infrastructure to perform. We have identified a combination of technologies that can meet this goal. An isothermal amplification method, recombinase polymerase amplification (RPA), uses sequence-specific primers and probes to amplify DNA at a constant temperature of ~380C. Amplicons can be detected by either fluorescence, using a simple low-cost reader, or by a lateral flow strip (LFS), reducing the need for any complex equipment. It is the primary aim of this proposal to design a single RPA HIV-1 assay that can diagnose all common HIV-1 subtypes. The same primer and probe(s) sequences may be utilized in either the fluorescence or LFS format adding greater utility for assay use. Because HIV-1 is extraordinarily diverse, conserved regions of the genome will be identified and RPA primers and probes will be designed and tested against a panel of diverse viral templates. The HIV-1 RPA assay will be optimized to tolerate diversity across HIV subtypes with high sensitivity and specificity. Whole blood is routinely utilized to detect HIV-1 DNA. Typically, whole blood dried on filter paper is used to stabilize blood collected for infant diagnostics. This project will determine whether whole blood or dried blood spots (DBS) are compatible with the RPA assay or whether membranes that capture lymphocytes increase assay sensitivity. Existing and novel rapid DNA extraction methods using DBS will be assessed in conjunction with the RPA assay to establish a test protocol that gives optimal sensitivity. In order to remove the requirement for powered incubation equipment, an exothermal compound that heats within the optimal incubation range of RPA has been identified for use with the LFS RPA format. The overall goal of the proposed project is to create an HIV-1 diagnostic based on RPA that can be minimally or entirely non- instrumented; requires minimal sample processing, with amplicon detection via fluorescence or LFS; and can be used at the point of care in a variety of resource-limited settings.