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

实验室外 RNA 分析的一步式 POC 样本到应答流程

基本信息

批准号：
9201510
负责人：
JOHN E MUELLER
金额：
$ 83.35万
依托单位：
LYNNTECH, INC.
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-09-15 至 2018-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9201510
关键词：
Africa Bacteriophages Biological Assay Central America Cessation of life Clinical Complementary DNA DNA Data Dengue Dengue Infection Dengue Virus Detection Development Devices Diagnosis Diagnostic Disease Documentation Ebola virus Enterobacteria phage MS2 Environment Far East Genome Genomics Goals Gold Hand Hawaii Health Professional Heating Human Laboratories Lateral Morbidity - disease rate One-Step dentin bonding system Patients Phase Plasma Preparation Process RNA RNA Processing RNA Viruses RNA analysis Ramp Reaction Reagent Reporting Resources Reverse Transcription Risk Sampling Sensitivity and Specificity Serotyping Signal Transduction South America Specificity System Temperature Testing Time United States Virus Visual amplification detection associated symptom base clinically relevant design diagnostic assay flu genomic RNA instrumentation nanoparticle next generation nucleic acid purification point of care point-of-care diagnostics programs prototype statistics tool

项目摘要

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.

项目概要 1 Lynntech 开发了一种流程，可以轻松识别基于 RNA 的噬菌体中的特定基因组位点尽管我们开发了一种简单的自动化样品制备装置 (SPM)，可以纯化 2 种病毒。 3 基因组RNA用于下游处理，例如反转录，我们还演示了直接RT-PCR 4 使用消除我们的对流单位放大特定的登革热基因座，这直接检测登革热。 5 诊断登革热感染时需要独立的样品制备步骤。 6 已将我们的对流放大单元转变为执行 RNA 逆转录 (RT) 以及 DNA Lynntech 的对流 RT-PCR 反应已成功鉴定出 7 扩增 (PCR) 内的基因组位点。 8 个 MS2 噬菌体、两种埃博拉病毒株和四种血清型登革热病毒。 9 分析基于这样的原理：圆柱体两端的两个不同温度将导致 10 浮力驱动的两端之间的稳定循环流因此，圆筒中的PCR试剂将流动。 11 通过温度梯度允许扩增所需的步骤：变性、退火和 12 伸长率相当有吸引力，因为它需要很少的能量。 13 温度梯度内检修，无需升温，无需电源 14.冷却，然后加热，反应因此对流系统可以由电池供电并且可以提供。 15 种在现场进行 RT-PCR 的便携式装置此外，这种便携式 RT-PCR 设备非常实用。 16 便宜。 17 Lynntech 已证明 MS2、埃博拉病毒和使用对流 RT-PCR 检测 18 登革热 RNA，我们的数据表明对埃博拉病毒和 19 登革热病毒值得注意的是，对于登革热病毒，我们能够轻松区分我们的四种血清型。 20 对流 RT-PCR 检测，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 检测非常灵敏，我们能够轻松检测到少于 100 个。我们的样本中含有 22 个登革热 3 病毒的基因组拷贝，这相当于少于 1 µL 血浆的检测量。事实上，这些数据强调了我们的对流检测对 23 名感染患者的诊断的适用性。 24 世界资源有限地区的护理点登革热感染情况。 25 在该计划的第二阶段，Lynntech 将进一步开发我们的对流放大系统 26 个单一、便携式、一步式装置，可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变为多重检测。 28 将在一次反应中识别四种登革热血清型该测定将结合我们的便携式基因组。 29 个放大模块，配有先进的基于金纳米颗粒的侧流系统，可提供即时护理 30 登革热病毒的检测和分析鉴定。