Rapid and Ultrasensitive Direct Detection of RNA for Norovirus Gastroenteritis

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 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.