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: 8488405
• 负责人: David Scott Boyle
• 金额: $ 37.86万
• 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 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8488405
• 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.

描述(申请人提供)：撒哈拉以南非洲地区感染人类免疫缺陷病毒1型(HIV-1)的婴儿中，超过50%的婴儿在2岁之前死亡。幸运的是，感染婴儿的早期诊断和治疗显著减缓了疾病的发展，提高了存活率。虽然获得治疗的机会正在改善，但目前可用的婴儿艾滋病毒-1诊断方法价格昂贵，需要复杂的设备和样本处理。因此，婴儿艾滋病毒-1检测仅限于中央实验室，这推迟了向农村地区及时报告检测结果。一种快速且易于使用的婴儿艾滋病毒诊断可以在护理地点或在离使用者更近的农村诊所进行，这将减少检测的周转时间，因此治疗可以更早开始，并显着提高农村地区特定于艾滋病的儿童死亡率。这一拟议项目的目标是创建一种婴儿艾滋病毒-1诊断方法，该诊断方法可以检测在全球范围内传播的艾滋病毒毒株，并且只需要最少的基础设施就可以执行。我们已经确定了能够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)，使用序列特异的引物和探针在~380℃的恒温下扩增DNA。扩增产物可以通过使用简单的低成本阅读器的荧光检测，也可以通过横向流动试纸(LFS)检测，从而减少了对任何复杂设备的需要。这项建议的主要目的是设计一种单一的RPA HIV-1检测方法，可以诊断所有常见的HIV-1亚型。相同的引物和探针(S)序列可以在荧光或LFS格式中使用，增加了更大的分析用途。由于HIV-1病毒非常多样化，将识别基因组的保守区域，并将设计RPA引物和探针，并针对一组不同的病毒模板进行测试。HIV-1RPA检测将进行优化，以高灵敏度和特异性耐受不同HIV亚型的多样性。全血通常被用来检测HIV-1DNA。通常，在滤纸上干燥的全血被用来稳定为婴儿诊断收集的血液。该项目将确定全血或干血斑(DB)是否与RPA分析兼容，或者捕获淋巴细胞的膜是否提高了分析灵敏度。使用DBS的现有和新的快速DNA提取方法将与RPA分析一起进行评估，以建立一种提供最佳灵敏度的测试方案。为了消除对动力孵化设备的要求，已经确定了一种在RPA的最佳孵化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，这种方法可以最小限度地或完全不使用仪器；需要最少的样本处理，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的护理点使用。