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

摘要 镰状细胞病（SCD）是一种危及生命的遗传性血液病！一种影响30多万人的 全球每年的新生儿死亡率超过90%，其中90%以上发生在低收入和中等收入国家。的幼儿死亡 通过建立新生儿筛查项目， 能够及时诊断和治疗。相比之下，在低资源环境中，高达90%的患有SCD的婴儿 五岁之前死亡，从未接受过诊断。目前的诊断方法是昂贵的 并需要实验室设备和训练有素的人员，使他们在财政和技术上无法获得的， SCD负担最重的国家。开发一种快速、低成本和易于使用的诊断方法 可以在护理点（POC）实施的测试可以实现早期诊断和及时启动 预防性治疗，到2050年可能挽救超过5000万出生时患有SCD的婴儿的生命。很多人- 已经开发和试验了昂贵的基于蛋白质的方法，但是在高胎儿期存在下缺乏灵敏度。 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 在缺乏确认性检测资源的环境中进行新生儿筛查。等温扩增技术进展 技术和廉价的纸和塑料为基础的诊断平台提供了一个机会， 通过开发一种基于DNA的诊断方法来识别基因突变的局限性。珠蛋白基因编码 以准确、低成本的形式检测镰状血红蛋白，可用于低资源环境。合作 与专门开发低成本诊断平台的工业合作伙伴Axxin Pty.有限公司，和申办者 莱斯大学和德克萨斯儿童医院的一个团队，专门从事POC诊断开发、儿科 血液学，并在低资源环境中评估技术，我将开发一种新颖，廉价（<3美元/测试）， DNA扩增测试，用于在集成平台上检测导致SCD的点突变，并评估 评估其在低资源环境中的性能和临床实用性。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试验与侧向电泳检测，以检测和区分 基因编码正常和镰状！珠蛋白链，并将测定整合到纸和塑料盒中; (2)开发与简单样品下游扩增兼容的样品制备程序- （3）在两项试点研究中评估性能和临床效用-一项在德克萨斯州休斯顿， 一个在马拉维的利隆圭。这些目标的实现将扩大等温扩增方法的能力 为了检测DNA中的点突变，首次展示了一种基于纸和塑料的 在低资源环境中进行核酸扩增测试，并为基于DNA的 诊断SCD的方法所开发的技术可用作整合点突变的平台 有助于SCD的其他血红蛋白病的检测，并使开发和实施 可扩展的，廉价的诊断测试是减少SCD幼儿死亡率的关键一步。

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.