Quantification of Gordonia terrae Bacteriophages using Lysate qPCR
Presentation Type
Thesis
Department
Biology
Location
Walker Conference Center C
Description
Bacteriophages are viruses that infect and replicate only in bacterial cells. Quantitative PCR was used in this experiment to count the number of DNA copies in the phage lysate sample. In this project, the more rapid lysate qPCR method was compared to standard plate count titer assays using the double layer agar method, to match the number of genomic copies with the plaque forming units in the lysate. Initially, multiple primer sets were designed to amplify the best primers and reaction conditions to use for phage detection. Because qPCR is sensitive to contamination, there were multiple controls to ensure no contamination. Subsequently, a standard curve was generated by comparing the phage concentration determined from titer assays with that measured using SYBR Green qPCR. The optimized lysate qPCR method will be a faster method to determine the number of bacteriophage DNA copies in phage lysate, allowing the rapid determination of phage concentration for optical density-based infection growth curve experiments.
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
Quantification of Gordonia terrae Bacteriophages using Lysate qPCR
Walker Conference Center C
Bacteriophages are viruses that infect and replicate only in bacterial cells. Quantitative PCR was used in this experiment to count the number of DNA copies in the phage lysate sample. In this project, the more rapid lysate qPCR method was compared to standard plate count titer assays using the double layer agar method, to match the number of genomic copies with the plaque forming units in the lysate. Initially, multiple primer sets were designed to amplify the best primers and reaction conditions to use for phage detection. Because qPCR is sensitive to contamination, there were multiple controls to ensure no contamination. Subsequently, a standard curve was generated by comparing the phage concentration determined from titer assays with that measured using SYBR Green qPCR. The optimized lysate qPCR method will be a faster method to determine the number of bacteriophage DNA copies in phage lysate, allowing the rapid determination of phage concentration for optical density-based infection growth curve experiments.
Comments
This presentation is currently embargoed. It will be available May 2029.