Kaylin Choe, Laura Carmona, & Dr. James Harber
Keystone bacteria within an oral microbiome can be indicators of gum health and diseases. In 1983, using classical microbiology methods, prominent keystone bacteria, Fusobacterium nucleatum and Streptococcus sanguinis, were identified as major species in dental plaque. Preterm labor, cardiovascular disease, cancers, and neurodegenerative illness are tied to these two keystone species. As a preventative measure for such diseases, there is a need for a device to rapidly assess pathogenic dysbiosis at the point of patient care. The LifeSmile, an oral microbiome diagnostic device, is designed to rapidly generate an “abundance ratio” of the number of Fusobacterium nucleatum, an anaerobe to Streptococcus sanguinis, its facultative partner bacteria. Tooth microbiomes collected from individuals with Next Generation Sequencing (Nextgen) surprisingly had easily measurable levels of the abundance ratio. Nextgen sequencing is not a portable or rapid method. Previously the LifeSmile technology approached the problem of determining the abundance ratio with DNA extraction, LAMP, and CRISPR-Cas12a with two offsite color FRET targets. This portable, palm-sized device, displayed the abundance ratio, indicating positive dysbiosis. We hypothesize that a new compatible technology, CRISPR-Cas12a and Activated Nuclease poly-T Reporter Illuminating Particles (CANTRIP) could be run in parallel in the same reaction, giving two individuals signals (Fusobacterium on CANTRIP red fluorescence channel and Streptococcus on green fluorescence Cas12a offsite cleavage target). The addition of CANTRIP as a second signaling detection assay is hypothesized to serve as a faster, more efficient method to generate the keystone bacteria ratio due to its dual detection system from combined enzymes (CAS12a and terminal deoxynucleotidyl transferase TdT), which generate easily visible green fluorescence and red copper dual signals. The LifeSmile is envisioned to be a robust point-of-care oral healthcare diagnostic device to provide patients with on-demand, preventative assessment of oral symbiosis or dysbiosis.