Lab-in-a-Tube Technology for Genotyping at Point-of-Care

描述(由申请人提供)：该项目的目标是利用IQuum创新的管中实验室(Liat TM)技术开发一种高水平的单核苷酸多态(SNP)多重实时聚合酶链式反应(PCR)分析方法。LIAT技术能够在30分钟内从全血中提取基因组DNA，并使用实时PCR检测序列。LIAT技术是医疗保健的理想选择，因为它将核酸检测的复杂性降至最低。事实上，执行基因组DNA序列检测分析的仪器Liat分子分析仪的操作员只需将血液输入Liar Tubule，并将闭合的Tubule插入仪器。Liat分子分析仪还可以在8分钟内进行多色荧光探针熔融分析。在第一阶段，我们建议使用多色荧光探针熔化作为检测多个SNPs的一种手段来研究分子反转探针分析在实时PCR检测中的适用性。此外，我们建议开发一套具有定义的熔融曲线的杂交标签，以最大限度地提高该检测的多重性。传统的多重实时聚合酶链式反应在一次检测中最多只能检测2个SNP。我们的策略将允许我们在一次检测中获得多达40个SNP。我们估计，多路传输的这种数量级增长将通过1小时的测试完成，该测试可以部署在护理地点。在第二阶段，我们计划开发一种多管Liat分子分析仪，它将有能力在护理点每年获得150万种基因型。此外，我们建议开发40个与动脉血栓形成风险增加相关的SNP的分析方法。静脉血栓每年发生在0.1%的人口中，其最严重的并发症(肺栓塞)在发达国家与中风一样频繁。动脉血栓形成是心肌梗塞、中风和外周动脉疾病的关键组成部分，所有这些疾病每年导致1900万人死亡。在第三阶段，我们将与哈佛医学院和塔夫茨医学院的研究人员合作进行临床试验，以评估与标记物相关的风险。

DESCRIPTION (provided by applicant): The goal of this project is to develop a high-level multiplex real time polymerase chain reaction (PCR) assay for single nucleotide polymorphisms (SNP) using IQuum's innovative Lab-in-a-tube (Liat TM) technology. Liat technology is capable of extracting genomic DNA from whole blood and detecting sequences, using real time PCR, in 30 minutes. Liat technology is ideal for the point of care because it reduces the complexity of nucleic acid tests to a minimum. Indeed, the operator of the Liat Molecular Analyzer, the instrument that performs this genomic DNA sequence detection assay, is only required to input blood into the Liar Tubule and insert the closed Tubule into the instrument. The Liat Molecular Analyzer can also perform multi-color fluorescent probe melting assays in 8 minutes. In Phase I, we propose to investigate the applicability of the molecular inversion probe assay to real time PCR detection using multi-color fluorescent probe melting as a means of detecting multiple SNPs. In addition, we propose to develop a set of hybridization tags with defined melting profiles in order to maximize the multiplexing of this assay. Traditional multiplex real time PCR can only accommodate up to 2 SNPs in one assay. Our strategy will allow us to score up to 40 SNPs in a single assay. We estimate this order of magnitude increase in multiplexing will be accomplished with a 1 hour assay that can be deployed at the point-of-care. In Phase II, we plan to develop a multi-tube Liat Molecular Analyzer that will have the capability of scoring 1.5 million genotypes per year, at the point-of-care. In addition, we propose to develop assays for scoring 40 SNPs that have been associated with increased risk of arterial thrombosis. Venous thrombosis occurs in 0.1 percent of the population each year, and its most severe complication (pulmonary embolism) is as frequent as strokes in developed countries. Arterial thrombosis is a key component of myocardial infarction, stroke and peripheral artery disease, all of which account for >19 million deaths annually. In Phase III, we would collaborate with researchers at Harvard Medical School and Tufts Medical School on clinical trials to evaluate the risk associated with the markers.