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Journal of Undergraduate Research

Keywords

real-time polymerase chain reaction, q-PCR, human tick-borne pathogens

College

Life Sciences

Department

Microbiology and Molecular Biology

Abstract

  1. Develop singleplex q-PCR assays to identify the tick-borne pathogens Borrelia burgdorferi, Borrelia hermsii, Bartonella henselae, and Babesia microti. Primer generation software will be used to design primer and probe sequences that will theoretically perform optimally in PCR reactions. All sequences will be subjected to BLAST searches to confirm their specificity for a particular organism. Once primer sequences have been obtained, they will be synthesized by Integrated DNA Technologies, Inc. (IDT). These primers will be evaluated in q-PCR reactions using target DNA from multiple isolates and SyberGreen to detect nucleic acid amplification. Parameters for each reaction will be optimized using these same tools. Once a reaction has been optimized, a specific labeled probe will be synthesized which contains both a fluorescent dye and its specific quencher. This will be used in a Taqman® PCR reaction to detect specific target sequence amplification. The assays will be validated using multiple isolates of each species mentioned above, along with several near-neighbors, in order to determine the specificities and sensitivities of each assay.

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