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: 8290288
• 负责人: David Scott Boyle
• 金额: $ 39.67万
• 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/8290288
• 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（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，感染婴儿的早期诊断和治疗可显著减缓疾病进展并提高存活率。虽然获得治疗的机会正在改善，但现有的婴儿艾滋病毒-1诊断费用昂贵，需要复杂的设备和样本处理。因此，婴儿艾滋病毒-1检测仅限于集中实验室，这延误了及时向农村地区报告检测结果。在护理点或在离使用者较近的农村诊所进行快速和易于使用的婴儿艾滋病毒诊断，将减少检测的周转时间，从而可以更快地开始治疗，并大大降低农村地区艾滋病儿童死亡率。该项目的目标是开发一种婴儿HIV-1诊断方法，可以检测出在世界范围内传播的HIV病毒株，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。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约380 ℃的恒定温度下扩增DNA。扩增子可以通过荧光检测，使用简单的低成本读取器，或通过侧流条（LFS），减少对任何复杂设备的需要。本提案的主要目的是设计一种可诊断所有常见HIV-1亚型的单一RPA HIV-1检测试剂盒。相同的引物和探针序列可以以荧光或LFS形式使用，增加了测定用途的更大效用。由于HIV-1是非常多样化的，将确定基因组的保守区域，并设计RPA引物和探针，并针对一组不同的病毒模板进行测试。HIV-1 RPA检测试剂盒将进行优化，以耐受HIV亚型的多样性，并具有高灵敏度和特异性。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收集的血液。本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增加检测灵敏度。将结合RPA测定评估使用DBS的现有和新型快速DNA提取方法，以建立提供最佳灵敏度的测试方案。为了消除对动力孵育设备的要求，已确定在RPA的最佳孵育范围内加热的暴露化合物用于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，该方法可以最低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在护理点使用。