An Integrated Isothermal Nucleic Acid Test for Improved Sickle-Cell Diagnosis at the Point-of-Care

用于改进镰状细胞病床边诊断的集成等温核酸测试

• 批准号: 10490967
• 负责人: Megan Ming Chang
• 金额: $ 3.62万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-04-21
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10490967
• 关键词: Address; Affect; Africa South of the Sahara; Age; Agreement; Alleles; Antibody Specificity; Biological Assay; Blood specimen; Caring; Chemicals; Clinical; Collaborations; Country; Cytolysis; DNA; DNA amplification; Data; Detection; Development; Diagnosis; Diagnostic; Diagnostic Procedure; Diagnostic tests; Diarrhea; Differentiated Gene; Disease; Early Diagnosis; Early treatment; Fetal Hemoglobin; Fingers; Future; Genes; Genetic; Genomic DNA; Genotype; Globin; Goals; Gold; HIV; Health; Hematological Disease; Hemoglobinopathies; High Pressure Liquid Chromatography; Hospitals; Human Resources; Improve Access; Industrialization; Infant; Inherited; Institutional Review Boards; Isoelectric Focusing; Knowledge; Laboratories; Lateral; Life; Malaria; Malawi; Measures; Methods; Mutate; Mutation; Mutation Detection; Neonatal Screening; Newborn Infant; Nucleic Acid Amplification Tests; Paper; Patients; Pediatric Hematology; Pediatric Hospitals; Performance; Pilot Projects; Point Mutation; Polymerase; Preparation; Preventive treatment; Procedures; Proteins; Protocols documentation; Reaction; Research; Resource-limited setting; Resources; Rice; Sampling; Savings; Sickle Cell; Sickle Cell Anemia; Sickle Hemoglobin; Special Hospitals; Specificity; Techniques; Technology; Testing; Texas; Time; Universities; Whole Blood; Work; amplification detection; base; clinically relevant; cost; cost effective; design; detection assay; diagnostic platform; disease diagnosis; early childhood; equipment training; experience; experimental study; genotyped patients; improved; isothermal amplification; laboratory equipment; low and middle-income countries; mortality; novel; pathogen; performance tests; point of care; point-of-care diagnostics; prevent; reagent testing; recombinase; screening; screening program; sickling; treatment program; virtual

Abstract Sickle-cell disease (SCD) is a life-threatening inherited blood disorder of the !-globin gene that affects over 300,000 newborns globally each year, over 90% of which occur in low- and middle- income countries. Early childhood mortality has been virtually eliminated in high-resource settings through the establishment of newborn screening programs that enable timely diagnosis and treatment. In contrast, up to 90% of affected infants born with SCD in low-resource settings will die before age five without ever receiving a diagnosis. Current diagnostic methods are expensive and require laboratory equipment and trained personnel, making them financially and technically inaccessible to the countries with the greatest burden of SCD. The development of a rapid, low-cost, and easy-to-use diagnostic test that can be implemented at the point-of-care (POC) could enable early diagnosis and prompt initiation of preventative treatment, potentially saving the lives of over 50 million infants born with SCD by 2050. Many in- expensive protein-based methods have been developed and piloted, but lack of sensitivity in the presence of high fetal hemoglobin, poor specificity in patients recently transfused, and lack of interpretability make these tests ineffective for newborn screening in settings that lack resources for confirmatory testing. Advances in isothermal amplification techniques and inexpensive paper- and plastic- based diagnostic platforms offer an opportunity to overcome existing limitations through the development of a DNA-based diagnostic that identifies the mutation in the !-globin gene encod- ing for sickled hemoglobin in an accurate, low-cost format that can be used in low-resource settings. In collaboration with an industrial partner that specializes in developing low-cost diagnostic platforms, Axxin Pty. Ltd., and a sponsor team based at Rice University and Texas Children's Hospital that specializes in POC diagnostic development, pediatric hematology, and evaluating technologies in low-resource settings, I will develop a novel, inexpensive (<$3/test), DNA amplification test to detect the point mutation responsible for SCD on an integrated platform, and eval- uate its performance and clinical utility in a low-resource setting. To accomplish this goal, we aim to (1) design and optimize a multiplexed isothermal amplification assay with lateral flow detection to detect and differentiate the genes encoding for normal and sickled !-globin chains, and integrate the assay into a paper- and plastic- cartridge; (2) develop a sample preparation procedure that is compatible with downstream amplification for a simple sample- to-answer workflow; and (3) evaluate performance and clinical utility in two pilot studies — one in Houston, TX and one in Lilongwe, Malawi. Completion of these aims will expand the ability of isothermal amplification methods to detect point mutations in DNA, provide the first demonstration of an integrated paper- and plastic- based nucleic acid amplification test in a low-resource setting, and provide proof-of-concept data for a DNA-based approach to diagnosing SCD. The technology developed can be used as a platform to incorporate point-mutation detection of other hemoglobinopathies that contribute to SCD, and enable the development and implementation of a scalable, inexpensive diagnostic test that is a critical step towards reducing early childhood mortality from SCD.

抽象的 镰状细胞疾病（SCD）是一种威胁生命的遗传性血液疾病！ 每年的新生儿全球，其中90％在低收入的幼儿死亡率中 在高资源环境中，实际上已经消除了新生儿筛查计划的建立 相比之下，启用及时的诊断和信任。 设置将在五岁之前就死亡，而无需接受诊断的诊断方法。 并需要实验室设备和训练有素的人员，使他们 SCD负担最大的国家。 可以在卡点（POC）上实施的测试可以实现地球诊断并提高创新 预防性治疗，有可能在2050年挽救超过5000万患有SCD的婴儿的生命。 昂贵的基于蛋白质的方法已经开发和试验，但是在存在高胎儿的情况下缺乏灵敏度 血红蛋白，最近输血的患者的特异性较差，缺乏可解释性使得测试无效 对于缺乏确定测试的资源的新生儿筛查。 技术和廉价的纸张和基于塑料的诊断平台提供了克服现有的 通过开发基于DNA的诊断的局限 在合作的情况下，以精确的低成本格式中插入了igkklethemglobin，该格式可用于低资源设置。 与专门开发低成本诊断平台的工业合作伙伴，Axxin Pty。 莱斯大学和德克萨斯儿童医院的团队专门从事POC诊断开发 血液学和评估技术环境中的技术，我将开发出一种新颖的，便宜的（<$ 3/test）， DNA扩增测试以检测负责集成平台上SCD的点突变，并进行评估 在低资源环境中，其性能和临床实用程序以实现这一目标。 并优化具有侧流检测的多路复用等级放大测定法，以检测和区分您 用于正常和谨慎的基因！ （2）制定样品制备程序，该程序与简单样品的下游放大器兼容 待交付的工作； 马拉维的一个在利隆圭的目标。 为了检测DNA中的点突变，提供了综合纸和塑料的首次演示 在低资源设置中的核酸扩增测试，并为基于DNA的概念证明数据 诊断SCD的方法可以用作融合点声称的平台 检测有助于SCD的其他血红蛋白疾病，并能够开发和实施 可扩展，廉价的诊断测试，以降低SCD的幼儿迫切性。