EVALUATION OF QUANTITATIVE PCR ON T4-LIKE MYOVIRUS COMMUNITIES
Bella (Nhu Trang) Nguyen, David Needham, Jed Fuhrman.
University of Southern California, Los Angeles, CA.
Marine viruses are highly abundant pathogens that rely on host infections for reproduction. Among marine viruses that infect bacteria (bacteriophages) are myoviruses, distinguished by their contractile tails and characterized genetic diversity. T4-like myoviruses contain a gene, g23, used to predict amplification and diversity analysis of sequences from the environment. In order to obtain a closer approximation of the true dynamics of T4-like myovirus groups over time, we developed and evaluated a quantitative polymerase chain reaction (qPCR) assay for T4-like viruses. We tested g23 primers against available T4-like myovirus genomes and observed that 75% of the T4-like isolates would be successfully amplified. Fifteen clones were selected from genomic libraries containing 2 clones per major grouping. Each clone was re-grown, its plasmids quantified and diluted, and subsequent PCR reactions performed. All selected clones amplified as expected with PCR products doubling with each cycle at an average efficiency of 96%. Unexpectedly, a slight positive relationship was observed between PCR fragment size and amplification efficiency, most likely the result of an artifact or unknown bias. Results obtained in this study provided confidence that the qPCR assay would work when applied to environmental samples. Using a standard clone with an average threshold cycle that would most closely approximate real environment samples, our next goal is to couple the qPCR assay with T4-like diversity assessments from the San Pedro ocean time series (SPOT) and ultimately determine how these viruses vary in conjunction with potential host populations.