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 镰状细胞病 (SCD) 是一种危及生命的 !-珠蛋白基因遗传性血液疾病，影响超过 300,000 人 全球每年都有新生儿出生，其中 90% 以上发生在低收入和中等收入国家。幼儿死亡率 通过建立新生儿筛查计划，在资源丰富的环境中，这种情况实际上已被消除 以便及时诊断和治疗。相比之下，在资源匮乏的地区，高达 90% 的受影响婴儿出生时患有 SCD 人们将在没有得到诊断的情况下在五岁之前死亡。目前的诊断方法价格昂贵 并需要实验室设备和训练有素的人员，这使得他们在财政和技术上无法获得 SCD 负担最重的国家。开发快速、低成本且易于使用的诊断方法 可以在护理点 (POC) 进行的测试可以实现早期诊断并迅速启动 到 2050 年，预防性治疗可能会挽救超过 5000 万出生时患有 SCD 的婴儿的生命。 昂贵的基于蛋白质的方法已经开发出来并进行了试点，但在高胎儿风险的情况下缺乏敏感性 血红蛋白、最近输血患者的特异性差以及缺乏可解释性使得这些测试无效 在缺乏确认检测资源的环境中进行新生儿筛查。等温扩增的进展 技术和廉价的纸质和塑料诊断平台提供了克服现有技术的机会 通过开发基于 DNA 的诊断方法来识别 !-珠蛋白基因编码中的突变，从而克服局限性。 以准确、低成本的形式获取镰状血红蛋白，可用于资源匮乏的环境。合作中 与专门开发低成本诊断平台的工业合作伙伴 Axxin Pty. Ltd. 和赞助商 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、儿科 血液学，并评估资源匮乏环境中的技术，我将开发一种新颖的、廉价的（<3 美元/测试）， DNA 扩增测试，在集成平台上检测导致 SCD 的点突变，并进行评估 在资源匮乏的环境中评估其性能和临床实用性。为了实现这一目标，我们的目标是 (1) 设计 并优化多重等温扩增检测和侧流检测，以检测和区分 编码正常和镰状 !-珠蛋白链的基因，并将检测整合到纸质和塑料盒中； (2) 开发一个与简单样品下游扩增兼容的样品制备程序 回答工作流程； (3) 评估两项试点研究的性能和临床效用——一项在德克萨斯州休斯顿， 其中之一位于马拉维利隆圭。这些目标的完成将扩展等温扩增方法的能力 检测 DNA 中的点突变，首次演示了基于纸和塑料的集成 在资源匮乏的环境中进行核酸扩增测试，并为基于 DNA 的核酸扩增测试提供概念验证数据 诊断 SCD 的方法所开发的技术可用作整合点突变的平台 检测导致 SCD 的其他血红蛋白病，并能够制定和实施 可扩展、廉价的诊断测试是降低儿童早期猝死死亡率的关键一步。