This R21 application seeks supplemental support to establish mass spectrometric methods for detecting and characterizing viral nucleic acid sequences. Viral sequence targets will be amplified by multiplex PCR in the presence of biotinylated nucleotides using primers labeled with specific photocleavable mass tags. After purification with avidin, amplification products will be cleaved from mass tags by near UV-light. The identity of the viral targets will be revealed by mass spectrometric analysis of the mass tag population. Mass-Tag PCR assays should be more sensitive and rapid than the multiplex PCR assays proposed in our original application (domain specific differential display and consensus PCR) while retaining the advantage of detecting a wide range of viral sequence targets through use of degenerate primers. Single mass tags will be coupled to degenerate primer sets representing regions of relative sequence conservation and used in PCR mass spectrometry experiments to detect related viruses that differ in sequence. Panels of primers specific for different viral taxa will be coupled to individual mass tags and used in PCR mass spectrometry experiments to discriminate between closely related viruses. Amplification products will be validated through direct mass spectrometric sequencing. We will build upon viral sequence databases, primers, and other reagents created under the auspices of AI51292. Assays will be optimized using synthetic viral nucleic acid targets, and applied in programs focused on viral ecology, pathogen discovery, and epidemiology of acute and chronic diseases. Consistent with aims proposed in AI51292, our initial clinical application will be to analyze enteroviral serotypes implicated in human disease in North America. ? ?
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