The constant threats posed by Influenza and other RNA viruses of pandemic due to antigenic shift or transfer from an animal host, and of season-to-season variation due to antigenic drift can be most effectively countered by pervasive monitoring. Viral monitoring in the clinic and field would be assisted greatly by the availability of microfabricated devices capable of rapid, inexpensive genotyping. To accelerate the emergence of such devices we propose research to engineer and build two prototype microfabricated devices, one to detect influenza A sub-types, and the other to perform single nucleotide polymorphism (SNP) detection to monitor antigenic drift. We will also develop and integrate a purification system to prepare sufficient quantity and purity of RNA material from virus-containing clinical samples for the microfabricated device to perform reverse transcription-PCR (RT-PCR) and genotyping reactions. Specifically, we aim to Aim 1 - Develop a microfabricated device capable of producing viral RNA from biological samples with the levels of purity and concentration required to perform on-chip RT-PCR reactions.
Aim 2 - On a microfabricated device, perform an RT-PCR reaction on HA 1 hemagglutinin domain of influenza A, producing double-stranded complementary DNA.
Aim 3 - On a microfabricated device, perform restriction digestion reactions that can distinguish H1, H3, and H5 types of influenza A. Perform a multiplex reaction that can simultaneously detect RNA from influenza A and another RNA virus, such as influenza B or SARS.
Aim 4 - On a microfabricated device, perform single-nucleotide polymorphisms that can distinguish antigenic drift in the HA1 domain of influenza A.
Aim 5 - Integrate RT-PCR, restriction digestion, and electrophoretic separation onto a single microfabricated device, and integrate this with a system for RNA extraction from clinical, throat-culture, samples.
Showing the most recent 10 out of 20 publications
