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）：超过50％的撒哈拉以南非洲婴儿感染了人类免疫缺陷病毒1型（HIV-1）在2岁之前死亡。幸运的是，感染婴儿的早期诊断和治疗显着减慢了疾病的进展并改善了存活率。虽然可以使用治疗，但目前可用的婴儿HIV-1诊断很昂贵，需要复杂的设备和样品处理。因此，婴儿HIV-1测试仅限于集中实验室，这延迟了对农村地区的测试结果的及时报告。可以在医疗点或更接近用户的农村诊所进行的快速易用的婴儿艾滋病毒诊断，将减少测试的转折时间，因此治疗可以更快地开始，并显着提高农村地区的艾滋病特定儿童死亡率。该提议的项目的目的是创建一个婴儿HIV-1诊断，该诊断可以检测到全球流通的艾滋病毒菌株，并且需要最少的基础设施才能执行。我们已经确定了可以满足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〜380C的恒定温度下扩增DNA。可以使用简单的低成本读取器或横向流动条（LFS）来检测扩增子，从而减少了对任何复杂设备的需求。该提案的主要目的是设计一个可以诊断所有常见HIV-1亚型的RPA HIV-1分析。可以在荧光或LFS格式中使用相同的底漆和探针序列，从而增加了更大的实用程序以供测定使用。由于HIV-1非常多样化，因此将鉴定出基因组的保守区域，RPA引物和探针将针对各种不同的病毒模板进行设计和测试。 HIV-1 RPA测定法将被优化，以耐受艾滋病毒亚型的多样性，具有高灵敏度和特异性。全血被通常用于检测HIV-1 DNA。通常，在滤纸上干燥的全血被用来稳定用于婴儿诊断的血液。该项目将确定全血或干血点（DBS）是否与RPA分析兼容，或者捕获淋巴细胞的膜是否会增加测定敏感性。现有和新型的快速DNA提取方法使用DBS将与RPA分析一起评估，以建立具有最佳灵敏度的测试方案。为了消除对电动孵化设备的需求，已经确定了在RPA最佳孵育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基于RPA的HIV-1诊断，该诊断可能是最小或完全非仪器的；需要最小的样品处理，并通过荧光或LFS检测扩增子。并且可以在各种资源有限的设置中在护理点使用。 公共卫生相关性：目前，由于缺乏诊断和治疗，许多感染了HIV-1死亡的婴儿，尤其是在获得测试设施有限的农村地区。因此，HIV-1的快速诊断是成功疾病治疗感染婴儿的关键因素。该建议描述了几种新生技术，可用于开发一种快速且易于使用的HIV-1诊断工具，以便在低资源设置的护理点使用。