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

描述（由申请人提供）：本项目的目标是使用IQuum的创新试管实验室（Liat TM）技术开发一种高水平的单核苷酸多态性（SNP）多重真实的实时聚合酶链反应（PCR）检测。Liat技术能够从全血中提取基因组DNA并使用真实的时间PCR在30分钟内检测序列。Liat技术非常适合现场护理，因为它将核酸检测的复杂性降至最低。实际上，Liat分子分析仪（进行该基因组DNA序列检测测定的仪器）的操作者仅需要将血液输入到说谎者小管中并将封闭的小管插入仪器中。Liat分子分析仪还可以在8分钟内进行多色荧光探针熔解分析。在第一阶段，我们建议调查的适用性的分子倒置探针检测真实的时间PCR检测使用多色荧光探针熔解作为检测多个SNP的手段。此外，我们建议开发一组具有定义的熔解曲线的杂交标签，以最大限度地提高该测定的多重性。传统的多重真实的时间PCR在一次测定中只能容纳多达2个SNP。我们的策略将允许我们在一次检测中对多达40个SNP进行评分。我们估计，多路复用的数量级增加将通过可在护理点部署的1小时测定来实现。在第二阶段，我们计划开发一种多管Liat分子分析仪，该分析仪将能够在护理点每年对150万个基因型进行评分。此外，我们建议开发用于对与动脉血栓形成风险增加相关的40个SNP进行评分的测定。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）与发达国家的中风一样频繁。动脉血栓形成是心肌梗死、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.