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Title page for ETD etd-08312010-164110


Type of Document Dissertation
Author Perez, Jonas William
URN etd-08312010-164110
Title The Design and Characterization of DNA-Based Viral Diagnostics
Degree PhD
Department Chemistry
Advisory Committee
Advisor Name Title
David W. Wright Committee Chair
Charles M. Lukehart Committee Member
Frederick R. Haselton Committee Member
Gary. A. Sulikowski Committee Member
Keywords
  • RSV
  • Virus Detection
  • PCR
  • DNA
Date of Defense 2010-08-13
Availability unrestricted
Abstract
Early detection of pediatric viruses is critical to effective intervention. A successful clinical tool must have a low detection limit, be simple to use and report results quickly. This work describes the integration of new technologies into viral detection to achieve improved diagnostics. One of the methods developed detects viral RNA using DNA hairpin structures covalently attached to a gold filament. In this design, the gold filament serves both to simplify processing and enable fluorescence detection. The approach was evaluated by assaying for the presence of respiratory syncytial virus (RSV) using the DNA hairpin probe covalently attached to a gold-clad filament. The filament functionalized with probes was immersed in a capillary tube containing viral RNA, moved to subsequent capillary tubes for rinsing and then scanned for fluorescence. The lower limit of detection was determined to be 11.9 PFU. This lower limit of detection was ~200 times better than a standard comparison ELISA. The simplicity of the core assay makes this approach attractive for further development as a viral detection platform in a clinical setting.

A second method developed, nanoparticle amplified immuno-PCR (NPA-IPCR), combines antibody recognition of traditional ELISA with a 50-fold nanoparticle valence amplification step followed by amplification by PCR. The assay detects a viral surface protein using an antibody bound to a gold nanoparticle cofunctionalized with thiolated DNA which is a hybridized to tag DNA, giving a tag DNA to antibody ratio of 50 to 1. When virus particles are present, a liquid phase three component “sandwich” complex is formed comprised of the gold nanoparticle construct, virus and an antibody functionalized magnetic particle used for extraction. Post extraction, DNA tags are released upon heating and detected via real-time PCR. The limit of detection of the assay was compared to ELISA and RT-PCR using respiratory syncytial virus infected HEp-2 cells. NPA-IPCR showed a ~4000-fold improvement in the limit of detection compared to ELISA and a 4-fold improvement compared to traditional RT-PCR. With the ability to detect exceptionally low virus concentrations, NPA-IPCR offers a viable platform for the development of an early-stage diagnostic.

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