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

摘要 镰状细胞病(SCD)是一种威胁生命的珠蛋白基因遗传性血液疾病，影响超过30万人。 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。儿童早期死亡率 通过建立新生儿筛查计划，在资源丰富的环境中几乎消除了这种情况 能够及时诊断和治疗。相比之下，高达90%的受影响婴儿出生时患有SCD，资源匮乏 设置将在没有收到诊断的情况下在fiVe之前死亡。目前的诊断方法都很昂贵 并需要实验室设备和训练有素的人员，使他们在纳米和技术上无法获得fi 慢性阻塞性肺病负担最重的国家。开发一种快速、低成本、易用的诊断方法 可在护理点(POC)实施的测试可实现早期诊断和及时启动 预防性治疗，到2050年可能挽救5000多万出生时患有SCD的婴儿的生命。许多人在- 昂贵的基于蛋白质的方法已经被开发和试验，但在高胎儿存在的情况下缺乏敏感性。 血红蛋白、最近输血患者的特异性fi差以及缺乏可解释性使这些检测无效。 用于在缺乏fi验证性测试资源的环境中进行新生儿筛查。等温扩增fi阳离子的研究进展 技术和廉价的基于纸张和塑料的诊断平台为克服现有的 限制通过开发一种基于dna的诊断方法来识别！-fi基因编码区的突变。 以准确、低成本的格式检测镰状血红蛋白，可在低资源环境下使用。在协作中 与专门开发低成本诊断平台的工业合作伙伴安信公司合作。有限公司，以及赞助商 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，以及在低资源环境下评估技术，我将开发一种新颖、廉价(&lt；3美元/检测)， DNA扩增fi试验在一个集成平台上检测导致SCD的点突变，并评估 在低资源环境下评估其性能和临床应用。为了实现这一目标，我们的目标是(1)设计 并优化了多重等温fi扩增与横向flow检测相结合的方法，以检测和区分 编码正常的和镰刀状的！-珠蛋白链的基因，并将检测整合到一个纸盒和塑料盒中； (2)开发一种与下游fi扩增相兼容的样品制备方法，用于简单样品- To-Answer Workflow；以及(3)在两项先导性研究中评估绩效和临床效用-一项在德克萨斯州休斯顿和 其中一家在马拉维的利隆圭。这些目标的实现将扩大等温fi扩增方法的能力 为了检测dna中的点突变，提供了一种基于纸和塑料的集成的fi第一次演示。 在低资源环境下进行核酸扩增fi检测，并为基于DNA的 SCD的诊断方法。所开发的技术可以作为整合点突变的平台 检测导致SCD的其他血红蛋白疾病，并使开发和实施 可扩展、廉价的诊断测试是降低SCD儿童早期死亡率的关键一步。

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.