Pertinent Genes for Determination of Prokaryotic Phenotype by PCR
Authors:Joseph Canas, Harold Decastro, Patrick Freymuth, Thedore Peterson, Tiffany Sidwell
Mentor:James Harber, Professor of Biology, Oxnard College
Advances in molecular diagnostics bring the study of genomes to the forefront of life science research. Despite the availability of high-speed DNA sequencing methodologies, there is still a niche need for rapid diagnostic tests using PCR to monitor bacterial identities in real-time. When immediate phylogenetic clinical identification of prokaryotic species is required, molecular strategies have clear speed advantages over biochemical methods, albeit with an important caveat: Most DNA sequencing-based PCR strategies require an extra day and expensive sequencing equipment to identify genera and species based on the 16S region of the bacterial genome. Herein we present experimental data to support an alternative and faster point-of-care diagnostic strategy. This comprehensive real-time polymerase chain reaction (RT-PCR) dichotomous key is based on DNA detection of traditional and time tested discrete biochemical markers to delineate genera of the prokaryotic phylogenetic tree. The taxonomy presented is consistent with the recent release of the human microbiome project. In research data presented herein, the PCR targeting of just a few genes (essential cell wall biosynthesis genes lpxB and kdtA in Gram-negative bacteria, tagB in Gram-positives, and spoA in the spore-forming bacteria) provides more rapid identification of these relevant Genera than either DNA sequencing or biochemical methods. Additional genetic targets presented in this research enhance a second step dichotomous key strategy for specific bacterial species identification. The technology developed in this work has applications toward rapid and disposable RT-PCR diagnostics for field deployable outpatient medical testing devices.