Location
Nessmith-Lane Atrium
Session Format
Poster Presentation
Research Area Topic:
Natural & Physical Sciences - Biology
Co-Presenters and Faculty Mentors or Advisors
Marina Eremeeva (Georgia Southern University)
Abstract
Background: Ehrlichia chaffeensis, E. ewingii, E. muris and E. canis are known to cause febrile illnesses in humans and dogs. These pathogens are transmitted by ticks, and infection is established following invasion of the human or dog white blood cells. Ehrlichia chaffeensis infections of humans have a fatality rate up to 5%. Ehrlichia ewingii displays antigenic cross-reactivity with E. chaffeensis; however, laboratory case confirmation is difficult because E. ewingii is not cultivable so antigens and genetic data are hard to acquire. Our project will develop approaches and tools to characterize E. ewingii surface protein gene homologous to the major 120 kDa (gp120) diagnostic protein antigen gene of E. chaffeensis using a targeted RNA bait protocol.
Methods: A multiple sequence alignment was performed for the gp120 gene region of E. chaffeensis, E. canis, and E. muris using MEGA6. Primers were designed for long-range tile amplification of 900-3,000 base pair fragments of E. chaffeensis and E. canis. Temperature gradient PCR was used to select optimal amplification conditions for each of the targeted fragments. Identity of the amplified fragments was confirmed by sequencing. Selected primers were used to generate biotinylated RNA baits representing different sites of the target region.
Results: Once optimized PCR conditions were established for amplification of homologous DNA, primers were tested to determine their performance in amplifying homologous and heterologous DNA from E. chaffeensis, E. canis and E. muris. A recombinant plasmid for each overlapping fragment was constructed and used as a template to produce biotinylated baits.
Conclusions: These experiments established a proof of principle that RNA baits can be produced using recombinant plasmid containing PCR fragment of Ehrlichia surface antigen gene. The bait will be used in pull down experiments to recover and clone the analogous genetic fragments of E. ewingii from tick and dog samples known to be infected with this pathogen.
Keywords
Georgia Southern University, Research Symposium, Molecular tools, Human ehrlichiosis
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Presentation Type and Release Option
Presentation (Open Access)
Start Date
4-16-2016 10:45 AM
End Date
4-16-2016 12:00 PM
Recommended Citation
Mears, Megan, "Developing Molecular Tools for Diagnosis of Human Ehrlichiosis" (2016). GS4 Georgia Southern Student Scholars Symposium. 4.
https://digitalcommons.georgiasouthern.edu/research_symposium/2016/2016/4
Included in
Developing Molecular Tools for Diagnosis of Human Ehrlichiosis
Nessmith-Lane Atrium
Background: Ehrlichia chaffeensis, E. ewingii, E. muris and E. canis are known to cause febrile illnesses in humans and dogs. These pathogens are transmitted by ticks, and infection is established following invasion of the human or dog white blood cells. Ehrlichia chaffeensis infections of humans have a fatality rate up to 5%. Ehrlichia ewingii displays antigenic cross-reactivity with E. chaffeensis; however, laboratory case confirmation is difficult because E. ewingii is not cultivable so antigens and genetic data are hard to acquire. Our project will develop approaches and tools to characterize E. ewingii surface protein gene homologous to the major 120 kDa (gp120) diagnostic protein antigen gene of E. chaffeensis using a targeted RNA bait protocol.
Methods: A multiple sequence alignment was performed for the gp120 gene region of E. chaffeensis, E. canis, and E. muris using MEGA6. Primers were designed for long-range tile amplification of 900-3,000 base pair fragments of E. chaffeensis and E. canis. Temperature gradient PCR was used to select optimal amplification conditions for each of the targeted fragments. Identity of the amplified fragments was confirmed by sequencing. Selected primers were used to generate biotinylated RNA baits representing different sites of the target region.
Results: Once optimized PCR conditions were established for amplification of homologous DNA, primers were tested to determine their performance in amplifying homologous and heterologous DNA from E. chaffeensis, E. canis and E. muris. A recombinant plasmid for each overlapping fragment was constructed and used as a template to produce biotinylated baits.
Conclusions: These experiments established a proof of principle that RNA baits can be produced using recombinant plasmid containing PCR fragment of Ehrlichia surface antigen gene. The bait will be used in pull down experiments to recover and clone the analogous genetic fragments of E. ewingii from tick and dog samples known to be infected with this pathogen.
