One Step, POC Sample to Answer Process for RNA Analysis Outside the Laboratory

项目摘要 1 Lynntech开发了一种方法，可以很容易地识别基于RNA的噬菌体中的特定基因组位点， 2种病毒。虽然我们开发了一种简单的自动化样品制备装置（SPM）， 3基因组RNA的下游加工，如逆转录，我们也证明了直接RT-PCR 4使用我们的对流单元扩增特定的登革热位点。这种对登革热的直接检测消除了 5在诊断登革热感染中需要独立的样品制备步骤。另外我们 6已经将我们的对流扩增单元转变为进行RNA逆转录（RT），以及DNA 7扩增（PCR）。Lynntech的对流RT-PCR反应已经成功地识别了 8 MS 2噬菌体、两种埃博拉病毒株和四种登革热病毒血清型。对流 9测定法是基于这样的原理，即在圆柱体的端部处的两个不同温度将导致 10在这些端部之间的浮力驱动的稳定循环流。因此，圆筒中的PCR试剂将流动 11通过温度梯度，允许扩增的必要步骤：变性，退火，和 12伸长。对流放大是相当吸引人的，因为它需要很少的功率。因为试剂 13在温度梯度内循环，因此不需要温度斜升并且不需要功率来 14冷却，然后加热反应。因此，对流系统可以由电池供电， 15种便携式工具，用于在护理点进行RT-PCR。此外，这种便携式RT-PCR设备可以相当 16便宜 17 Lynntech已经证明了MS 2、埃博拉病毒和其他病毒的逆转录和随后的cDNA扩增 18登革RNA，使用对流RT-PCR。我们的数据表明，埃博拉病毒和 19登革热病毒值得注意的是，对于登革病毒，我们能够很容易地区分四种血清型， 20对流RT-PCR测定，尽管这四种血清型的基因组具有60-70%的同源性。 21此外，我们的对流RT-PCR测定是相当敏感的。我们能够轻易地检测到少于100个 在我们的测定中，22个登革3型病毒的基因组拷贝。这相当于来自一个人的血浆少于1微升。 23感染者事实上，这些数据强调了我们的对流分析对诊断的适用性 24世界上资源有限地区的护理点登革热感染。 25在该计划的第二阶段，Lynntech将进一步发展我们的对流放大系统， 26个单一的便携式一步单元，可以超越传统实验室的范围进行诊断 27登革热感染，以及研究RNA过程。我们将把这个系统转换成一个多重检测系统， 28将在单一反应中鉴定四种登革热血清型。这个检测将联合收割机结合我们的便携式基因组 29具有复杂的基于金纳米颗粒的侧向流系统的扩增模块，以提供即时护理 30登革病毒的检测和鉴定试验。

PROJECT SUMMARY 1 Lynntech has developed a process to readily identify specific genomic loci in RNA-based bacteriophage and 2 viruses. Although we developed a simple, automated sample preparation device (SPM) that will purify 3 genomic RNA for downstream processing, such as reverse transcription, we also demonstrated direct RT-PCR 4 amplification of a specific dengue locus using our convective units. This direct detection of dengue eliminates 5 the need for an independent sample preparation step in the diagnosis of dengue infection. In addition, we 6 have transitioned our convective amplification units to perform RNA reverse transcription (RT), as well as DNA 7 amplification (PCR). Lynntech's convective RT-PCR reaction has successfully identified genomic loci within the 8 MS2 bacteriophage, two strains of the Ebola virus, and the four serotypes of the dengue virus. The convective 9 assay is based on the principle that two different temperatures at the ends of a cylinder will result in a 10 buoyancy-driven steady circulatory flow between those ends. Thus, PCR reagents in the cylinder will flow 11 through a temperature gradient allowing the necessary steps for amplification: denaturation, annealing, and 12 elongation. Convective amplification is rather appealing in that it requires very little power. Because reagents 13 circulate within a temperature gradient, there is no need for temperature ramping and power is not needed to 14 cool, and then heat, the reaction. So the convective system can be powered by batteries and can provide a 15 portable means to perform RT-PCR at the point-of-care. In addition, this portable RT-PCR device can be quite 16 inexpensive. 17 Lynntech has demonstrated the reverse transcription and subsequent cDNA amplification of MS2, Ebola, and 18 dengue RNA, using convective RT-PCR. Our data indicated excellent specificity for both the Ebola virus and 19 the dengue virus. Notably, for the dengue virus, we were able readily distinguish the four serotypes in our 20 convective RT-PCR assay, despite the fact that the genomes of these four serotypes share 60-70% homology. 21 In addition, our convective RT-PCR assay was quite sensitive. We were able to easily detect less than 100 22 genomic copies of the dengue 3 virus in our assay. This would equate to less than 1 µL of plasma from an 23 infected patient. Indeed these data underscore the applicability of our convective assay to the diagnosis of 24 dengue infection at the point-of-care in resource-limited regions of the world. 25 During Phase II of this program, Lynntech will further develop our convective amplification system into a 26 single, portable, one-step unit that can be used beyond the confines of the traditional laboratory to diagnose 27 dengue infection, as well as to study RNA processes. We will transition this system to a multiplex assay that 28 will identify the four dengue serotypes in a single reaction. This assay will combine our portable genome 29 amplification module with a sophisticated gold-nanoparticle-based lateral flow system to provide a point-of-care 30 detection and identification assay for the dengue virus.