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

项目总结 1 Lynntech已经开发出一种方法，可以容易地在基于RNA的噬菌体中识别特定的基因组位点，并 2种病毒。尽管我们开发了一种简单的自动化样本制备设备(SPM)，它将净化 3基因组RNA的下游加工，如逆转录，我们还展示了直接RT-PCR 4利用我们的对流单位放大特定的登革热疫源地。这种对登革热的直接检测消除了 5在诊断登革热感染时需要一个独立的样本制备步骤。此外，我们 6个已经将我们的对流扩增单元转移到执行RNA逆转录(RT)以及DNA 7扩增(PCR)。Lynntech的对流RT-PCR反应已成功识别出 8个MS2噬菌体、两个埃博拉病毒株和四个登革热病毒血清型。对流 9化验的原理是圆柱体两端的两个不同的温度会导致 10两端之间由浮力驱动的稳定循环流动。因此，聚合酶链式反应试剂在圆柱体中流动 11通过允许扩增所需步骤的温度梯度：变性、退火和 12延伸率。对流放大相当吸引人，因为它需要很少的能量。因为试剂 13在温度梯度内循环，不需要温度上升，也不需要电力 冷却，然后加热，反应。因此，对流系统可以由电池供电，并可以提供 15可在护理地点进行RT-PCR的便携装置。此外，这种便携式RT-PCR设备可以相当 16英镑，便宜。 17 Lynntech已经证明了MS2、埃博拉和 18个登革热RNA，采用对流RT-PCR。我们的数据显示，埃博拉病毒和 19登革热病毒。值得注意的是，对于登革热病毒，我们能够很容易地区分我们的 20对流RT-PCR分析，尽管这四种血清型的基因组有60%-70%的同源性。 21此外，我们的对流逆转录聚合酶链式反应也相当敏感。我们能够很容易地检测到不到100 在我们的检测中，登革3型病毒有22个基因组拷贝。这相当于不到1微米的L血浆 23例感染患者。事实上，这些数据强调了我们的对流分析在诊断 24在世界资源有限地区的护理点感染登革热。 25在该计划的第二阶段，Lynntech将进一步将我们的对流放大系统开发成 26个单一、便携、一步式设备，可用于超越传统实验室的限制进行诊断 27登革热感染，以及研究RNA过程。我们将把这个系统转变为一种多重检测 28将在一次反应中识别四种登革热血清型。这项测试将结合我们的便携基因组 29放大模块具有复杂的基于金纳米颗粒的侧向流动系统，可提供护理点 30登革热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.