Abstract
CONFERENCE PROCEEDING Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies Workshop in Bethesda, MD, USA; December 1–3, 2010
Guest Editors: Arifa Khan (Bethesda, MD), Patricia Hughes (Bethesda, MD) and Michael Wiebe (San Francisco, CA)
Abstract
Detection and identification of pathogens in any given sample without any knowledge about the pathogen requires the combination of sample purification and generic signal amplification. Our universal virus-detection assay combines virus capsid purification with a generic polymerase chain reaction (PCR) optimized for virus-sized genomes. As a general strategy, biochemical and physical purification by targeted digestion of contaminating host nucleic acids precedes nucleic acid extraction. RNA is transcribed into cDNA; cDNA and the DNA are then used as templates for degenerate oligonucleotide primer PCR (DOP-PCR). This PCR uses a pool of related but different primers, combining degenerate and specific annealing conditions. PCR products can be identified by cloning and sequencing, by microarray, or by high-throughput sequencing.
We detected enveloped and non-enveloped DNA and RNA viruses by DOP-PCR in cell culture as well as in clinical samples. The assay has the ability to detect single- and double-stranded genomes, circular genomes, segmented genomes, and linear genomes. Both, large virus genomes such as herpesvirus and very small virus genomes (e.g., circovirus) can be sensitively detected in the same assay. The DOP-PCR assay can be used to identify viruses either directly from biological specimens or from cell culture. A strong feature of the DOP-PCR is its ability to amplify viral sequences without prior knowledge of those sequences, increasing the likelihood of finding mutated or novel viruses. In combination with high-throughput sequencing, the DOP-PCR is a very sensitive tool that can potentially be used for the characterization of a given sample.
- © PDA, Inc. 2011
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