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

描述(申请人提供)：低成本的核酸分析，以检测艾滋病毒和乙肝病毒，已经彻底改变了血液供应的筛查。尽管有了新的基因复杂性，传统的[ABO，Rh]血型鉴定仍然在很大程度上是通过80年前的凝集试验进行的。最近，导致标准血型的遗传变异已经被定义，并被证明是相对简单的。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到包括更完整的7个性状：[ABO，Rh，Duffy，Kidd，Kell，Dombrock和Mns]，其潜在的遗传学现已为人所知。这两个平行的趋势表明，基因检测已经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目前的基于核酸的病原体检测大致相同。我们观察到，只要稍加修改，我们为原始血液的基于微阵列的人类白细胞抗原配型开发的一套生化、硬件和软件技术就可以转化为低成本的血型配型技术，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，我们提出了两个具体目标作为里程碑，以证明随后的第二阶段SA1的合理性。13-15个平行进行的聚合酶链式反应的设计和初步验证。7个血液标志物基因之间的所有信息位点将被平行扩增，以产生一个染料标记的扩增子集，准备用于微阵列杂交。SA2.微阵列探针的设计、制造和初步验证。将设计和制造一个输血芯片原型，以询问信息序列变异，这些序列变异定义了7个基因座之间的血型变异。验证将使用来自SA1的扩增DNA。第一阶段的完成将产生一个基于聚合酶链式反应的原型样本标记反应(对原料血起作用)和一个用于DNA血型鉴定的低成本输血芯片芯片原型。第二阶段将侧重于改进聚合酶链式反应和微阵列设计、制造规模扩大、初步聚合酶链式反应试剂盒制造和将这些材料交付给贝塔测试器，包括3-4个顶级血液实验室：将输血芯片的性能与血清学分型以及竞争对手的微阵列和聚合酶链式反应测试进行比较。公共卫生叙事。病原体污染的基因检测已经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的潜在血型检测一直落后。我们在这里提出的一种基于微阵列的血型鉴定方法，将使血型分析现代化，利用二十年的血型遗传学经验：允许以比血清学更完整的方式分析血型抗原变异，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将降低美国的血液供应检测成本，而且我们建议，将在发展中国家实现新一代基于DNA的增强型血型鉴定。 公共卫生相关性：我们在这里提出的一种基于微阵列的血型鉴定方法，将使血型分析现代化，利用二十年的血型遗传学经验：允许以比血清学更完整的方式分析血型抗原变异，成本和技术简化程度与血液病原体筛查一样实用。由此产生的技术不仅将降低美国的血液供应检测成本，而且我们建议，将在发展中国家实现新一代基于DNA的增强型血型鉴定。

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.