Rapid and Ultrasensitive Direct Detection of RNA for Norovirus Gastroenteritis

 描述（由申请人提供）：本I期申请的目标是开发一种基于纳米颗粒和表面增强拉曼光谱（Sers）的分子检测系统，以直接检测诺如病毒胃肠炎，而无需使用靶扩增技术，如聚合酶链反应或等温扩增。诺如病毒是流行性胃肠炎最常见的病因。根据CDC的说法，诺如病毒是美国食物中毒疾病爆发的最常见原因。诺如病毒爆发可以影响各种环境中的人。例如，军队中爆发的诺如病毒是 定期报告。该病毒影响约2.67亿人，每年造成20多万人死亡;这些死亡通常发生在欠发达国家以及非常年轻、年老和免疫抑制的人群中。诺如病毒感染具有高度传染性，因为感染剂量小于10个病毒颗粒，并且病毒在粪便中脱落至每克106-108个颗粒的高滴度。我们提出使用多功能金包被磁性纳米粒子（AuMNP）和基于SERS的分子检测系统捕获和检测粪便样品中的诺如病毒RNA。样品裂解后，使用AuMNP实现诺如病毒RNA的捕获、纯化和检测，所述AuMNP已经用带正电荷的肽核酸捕获探针（+ve）PNA和固有强拉曼标记物功能化。这些多功能纳米级AuMNP（~50 nm）提供比微米级颗粒或二维传感表面提供的更快速和有效的RNA捕获。在AuMNP上使用拉曼标记（作为单个标签）利用了涂覆在金纳米颗粒上的拉曼报告分子的公认的表面增强特性。与纳米技术专家Chuan-Jian Zhong（SUNY-Binghamton）合作，我们将开发和优化AuMNP以捕获RNA并形成拉曼标签，用于直接识别RNA以进行诺如病毒基因组分型。我们将利用抑制电渗流的微通道电泳进行AuMNPs的分离和磁聚焦用于Sers纳米颗粒检测。我们将与约翰霍普金斯大学的Robert Gilman教授合作，开展原理验证工作，在约120份先前采集并去识别的粪便样本中检测基因组I和II诺如病毒，这些样本已通过实时逆转录PCR（qRT-PCR）进行了表征。这些样品含有低、中、高浓度的病毒以及每组下不同的基因型。我们的平台是独一无二的，因为它使用多功能AuMNP来提供RNA捕获，磁性操作和Sers检测。我们的第一阶段目标是使用低病毒载量样本在30分钟内完成灵敏度和特异性检测。我们的最终目标是将检测方案集成到我们正在开发的样品制备盒中时，在封闭系统中执行所有测定步骤。当完全商业化时，该测定将以自动化方式运行。

 DESCRIPTION (provided by applicant): The goal of this Phase I application is to develop a nanoparticle and surface-enhanced Raman spectroscopy (SERS)-based molecular detection system to directly detect norovirus gastroenteritis without the use of target amplification techniques such as polymerase chain reaction or isothermal amplification. Noroviruses are the most common cause of epidemic gastroenteritis. According to the CDC, noroviruses are the most common cause of foodborne-disease outbreaks in the United States. Norovirus outbreaks can affect people in a variety of settings. For example, norovirus outbreaks in military forces are regularly reported. The virus affects around 267 million people and causes over 200,000 deaths each year; these deaths are usually in less developed countries and in the very young, elderly and immuno-suppressed. Norovirus infections are highly contagious as the infectious dose is less than 10 virus particles and the virus is shed to high titers of 106-108 particles per gram in the feces. We propose to use multifunctional gold coated magnetic nanoparticle (AuMNPs) and SERS-based molecular detection system to capture and detect norovirus RNA in stool samples. After sample lysing, the capture, purification, and detection of norovirus RNA will be achieved using AuMNPs that have been functionalized with positively-charged peptide nucleic acid capture probes (+ve)PNA, and intrinsically-strong Raman labels. These multifunctional nano-sized AuMNPs (~50 nm) provide for a more rapid and efficient capture of RNA than those offered by micron-size particles or 2-dimensional sensing surfaces. The use of Raman labels on the AuMNPs (as a single tag) takes advantage of the well-established surface enhancing characteristics of Raman reporter molecules coated on gold nanoparticles. Working with nanotechnology expert Chuan-Jian Zhong (SUNY-Binghamton), we will develop and optimize the AuMNPs to capture RNA and to form a Raman tag for the direct identification of RNA for norovirus genogroup typing. We will carry out AuMNPs separation and magnetic focusing for SERS nanoparticle detection using microchannel electrophoresis with the electroosmotic flow suppressed. Working with Professor Robert Gilman of Johns Hopkins University, we will carry out proof-of-principle work to detect genogroups I and II noroviruses in about 120 previously collected and de-identified stool samples that have been characterized by real-time reverse transcription PCR (qRT-PCR). These samples contain low, medium, and high concentration of virus as well as different genotypes under each group. Our platform is unique as it uses multifunctional AuMNPs to provide RNA capture, magnetic manipulation and SERS detection. Our Phase I goal is to complete a sensitive and specific assay in 30 min using low viral load samples. Our eventual goal is to perform all the assay steps in a closed system when we integrate the detection scheme into a sample preparation cartridge that we're already developing. The assay, when fully commercialized, will be operated in automated manner.