The Transfusion Chip: A Simple, Low Cost Microarray for DNA Based Blood Typing

描述（由申请人提供）：用于检测 HIV 和 HBV 的低成本核酸分析彻底改变了血液供应筛查。尽管出现了新的基因复杂性，传统的血型分型 [ABO、Rh] 在很大程度上仍然是通过 80 年前的凝集测试的变化进行的。最近，产生标准血型的遗传变异已经被定义，并且被证明相对简单。与此同时，研究表明，为了提高输血质量，历史标记集可能会扩大，以包括更完整的 7 个性状：[ABO、Rh、Duffy、Kidd、Kell、Dombrock 和 MNS]，这些性状的潜在遗传学现已已知。这两个平行的趋势表明，基因检测已成为血型筛查的未来：但前提是基因检测的成本和技术简单程度与凝集或当前基于核酸的病原体检测大致相同。我们观察到，经过微小的修改，我们为基于微阵列的原血 HLA 分型开发的生化、硬件和软件技术套件可以转化为低成本的血型分型技术，我们称之为“输血芯片”。在这个为期 6 个月的第一阶段计划中，我们提出了 2 个具体目标作为里程碑，以证明后续第二阶段的合理性。 SA1。并行进行 13-15 个 PCR 反应的设计和初步验证。 7 个血液标记基因组中的所有信息位点将被并行扩增，以生成可用于微阵列杂交的染料标记的扩增子组。 SA2。微阵列探针设计、制造和初步验证。将设计和制造输血芯片原型，以询问信息序列变异，这些序列变异定义了 7 个基因座组之间的血型变异。验证将使用 SA1 中的扩增 DNA。 与后续第二阶段的关系。第一阶段的完成将产生基于 PCR 的样品标记反应原型（适用于原血）和用于基于 DNA 血型分型的低成本输血芯片微阵列原型。第二阶段将重点关注 PCR 和微阵列设计的完善、生产规模扩大、初步 PCR 试剂盒制造以及将这些材料交付给由 3-4 个顶级血液实验室组成的 beta 测试人员：将输血芯片的性能与血清学分型以及竞争性微阵列和 PCR 测试进行比较。公共卫生叙述。针对病原体污染的基因检测彻底改变了血液供应的安全性，但由于成本和复杂性，基于类似 DNA 的基础血型检测却相对滞后。我们在这里提出的基于微阵列的血型分型方法，将通过利用二十年的血型遗传学，使血型分析现代化：允许以比血清学更完整的方式分析血型抗原变异，其成本和技术简单程度与血液病原体筛查一样实用。由此产生的技术不仅会降低美国血液供应检测的成本，而且我们建议，还将在发展中国家实现新一代增强型、基于 DNA 的血型分型。 公共健康相关性：我们在这里提出的基于微阵列的血型分型方法，将通过利用二十年的血型遗传学经验，使血型分析现代化：允许以比血清学更完整的方式分析血型抗原变异，其成本和技术简单程度与血液病原体筛查一样实用。由此产生的技术不仅会降低美国血液供应检测的成本，而且我们建议，还将在发展中国家实现新一代增强型、基于 DNA 的血型分型。

DESCRIPTION (provided by applicant): Low-cost nucleic acid analysis, to detect HIV & HBV, has revolutionized screening of the blood supply. In spite of that new genetic sophistication, traditional blood group typing [ABO, Rh] is still performed, for the most part, via variations upon 80-year-old agglutination testing. Recently, the genetic variation which gives rise to the standard blood groups has been defined, and shown to be relatively simple. In parallel, it has been shown that, to enhance transfusion quality, the historical marker set might be expanded to include a more complete panel of 7 traits: [ABO, Rh, Duffy, Kidd, Kell, Dombrock & MNS] for which the underlying genetics are now known. These two parallel trends suggest that genetic testing has emerged as the future of blood group screening: but only if the genetic test can be delivered at roughly the same cost and level of technical simplicity as agglutination, or the current panel of nucleic acid based pathogen tests. We have observed that, with minor modification, the suite of biochemical, hardware & software technologies that we have developed for microarray based HLA-typing on raw blood could, instead, be transformed into low-cost technologies for blood-group typing, which we refer to as "The Transfusion-Chip". In this 6-month Phase I plan, we propose 2 Specific Aims as Milestones to justify a subsequent Phase II. SA1. Design & preliminary validation of 13-15 PCR reactions to be performed in parallel. All informative sites among the set of 7 blood marker genes will be amplified in parallel, to generate a dye-labeled amplicon set that is ready to be used for microarray hybridization. SA2. Microarray probe design, fabrication &preliminary validation. A Transfusion-Chip prototype will be designed and fabricated, to interrogate the informative sequence variations which define blood group variation among the set of 7 loci. The validation will employ amplified DNAs from SA1. Relation to a Follow-on Phase II. Completion of Phase I will yield a prototype PCR-based sample labeling reaction (that works on raw blood) and a prototype low-cost Transfusion-Chip microarray for DNA based blood-typing. Phase II will focus on refinement of the PCR and microarray designs, manufacturing scale-up, preliminary PCR kit fabrication and delivery of those materials to beta testers, comprising 3-4 top blood labs: to compare Transfusion-Chip performance to serological typing and to the competing microarray and PCR tests. Public Health Narrative. Genetic testing for pathogen contamination has revolutionized the safety of the blood supply, yet analogous DNA based testing of the underlying blood groups has lagged-behind, due to cost and complexity. A microarray-based approach to blood group typing that we propose here, will allow blood type analysis to be modernized, by exploiting two decades worth of blood group genetics: allowing analysis of blood group antigen variation, in a way that is more complete than can be obtained by serology, at a cost and level of technical simplicity that is as practical as that of blood pathogen screening. The resulting technology will not only reduce the cost of blood supply testing in the US, but we propose, will enable a new generation of enhanced, DNA-based blood group typing in the developing world. PUBLIC HEALTH RELEVANCE: A microarray-based approach to blood group typing that we propose here, will allow blood type analysis to be modernized, by exploiting two decades worth of blood group genetics: allowing analysis of blood group antigen variation, in a way that is more complete than can be obtained by serology, at a cost and level of technical simplicity that is as practical as that of blood pathogen screening. The resulting technology will not only reduce the cost of blood supply testing in the US, but we propose, will enable a new generation of enhanced, DNA-based blood group typing in the developing world.