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

重组酶聚合酶扩增用于婴儿 HIV-1 的护理点诊断

• 批准号: 8210785
• 负责人: David Scott Boyle
• 金额: $ 39.5万
• 依托单位: PATH
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8210785
• 关键词: 2 year old; Acquired Immunodeficiency Syndrome; Africa; Africa South of the Sahara; African; Age; Aliquot; Antibodies; Area; Biological Assay; Blood; Caring; Child; Child Mortality; Childhood; Client; Clinic; Collection; Communicable Diseases; Complex; DNA; DNA amplification; Data; Detection; Diagnosis; Diagnostic; Disease Management; Disease Progression; Distant; Early Diagnosis; Early treatment; Effectiveness; Equipment; Fluorescence; Fred Hutchinson Cancer Research Center; Genome; Genomics; Goals; HIV; HIV-1; Health; Health Services Accessibility; Heating; High temperature of physical object; Humidity; Infant; Infant Mortality; Infection; Laboratories; Lateral; Lymphocyte; Malaria; Maternal antibody; Membrane; Methods; Modeling; Paper; Patient Care; Performance; Polymerase; Positioning Attribute; Power Sources; Prevention; Process; Protocols documentation; Reader; Reagent; Reporting; Research; Research Infrastructure; Resources; Rural; Rural Health Centers; Sampling; Sensitivity and Specificity; Site; Source; Specificity; Specimen; Spottings; Sterility; Technology; Temperature; Test Result; Testing; Time; Tuberculosis; Vaccination; Vertical Disease Transmission; Viral; Water; Whole Blood; Work; antiretroviral therapy; base; cost; design; experience; follow-up; improved; instrument; meetings; mortality; novel; pandemic disease; point of care; programs; rapid diagnosis; recombinase; rural area; sample collection; tool

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. PUBLIC HEALTH RELEVANCE: Currently many infants infected with HIV-1 die due to lack of diagnosis and treatment, especially in rural areas that have limited access to testing facilities. Therefore, the rapid diagnosis of HIV-1 is a critical factor for successful disease management of infected infants. This proposal describes several nascent technologies that can be used to develop a rapid and easy-to-use HIV-1 diagnostic tool for use at the point of care in low-resource settings.

描述（由申请人提供）：在撒哈拉以南非洲地区，超过 50% 的感染 1 型人类免疫缺陷病毒 (HIV-1) 的婴儿在 2 岁之前死亡。幸运的是，受感染婴儿的早期诊断和治疗可以显着减缓疾病进展并提高生存率。虽然获得治疗的机会正在改善，但目前可用的婴儿 HIV-1 诊断价格昂贵，并且需要复杂的设备和样本处理。因此，婴儿HIV-1检测仅限于集中实验室，这延迟了检测结果及时向农村地区报告的情况。快速且易于使用的婴儿艾滋病毒诊断可以在护理点或靠近用户的农村诊所进行，这将减少检测的周转时间，从而可以更快地开始治疗，并显着降低艾滋病相关的儿童死亡率在农村地区。该拟议项目的目标是创建一种婴儿 HIV-1 诊断方法，可以检测在世界范围内传播的 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。重组酶聚合酶扩增 (RPA) 是一种等温扩增方法，使用序列特异性引物和探针在约 380°C 的恒温下扩增 DNA。扩增子可以使用简单的低成本阅读器通过荧光检测，也可以通过侧流试纸条 (LFS) 检测，从而减少对任何复杂设备的需求。该提案的主要目的是设计一种可以诊断所有常见 HIV-1 亚型的 RPA HIV-1 检测方法。相同的引物和探针序列可用于荧光或 LFS 格式，为测定使用增加更大的实用性。由于 HIV-1 极其多样化，因此将鉴定基因组的保守区域，并针对一组不同的病毒模板设计和测试 RPA 引物和探针。 HIV-1 RPA 检测将经过优化，能够以高灵敏度和特异性耐受不同 HIV 亚型的多样性。全血通常用于检测 HIV-1 DNA。通常，在滤纸上干燥的全血用于稳定为婴儿诊断而收集的血液。该项目将确定全血或干血斑 (DBS) 是否与 RPA 检测兼容，或者捕获淋巴细胞的膜是否可以提高检测灵敏度。使用 DBS 的现有和新型快速 DNA 提取方法将与 RPA 测定结合进行评估，以建立提供最佳灵敏度的测试方案。为了消除对动力孵化设备的需求，已确定在 RPA 最佳孵化范围内加热的放热化合物可与 LFS RPA 格式一起使用。拟议项目的总体目标是创建一种基于 RPA 的 HIV-1 诊断方法，该方法可以最少或完全非仪器化；需要最少的样品处理，通过荧光或 LFS 进行扩增子检测；并且可以在各种资源有限的环境中在护理点使用。 公共卫生相关性：目前，许多感染 HIV-1 的婴儿因缺乏诊断和治疗而死亡，特别是在检测设施有限的农村地区。因此，HIV-1的快速诊断是感染婴儿成功疾病管理的关键因素。该提案描述了几种新兴技术，可用于开发快速且易于使用的 HIV-1 诊断工具，供资源匮乏地区的护理点使用。