P. Cosme, E. Valliant, M. Medina-Rivera, C. Batt, A.D. Strickland
Keywords: lab-on-a-bubble, total coliform, E. coli, detection, LAMP, PCR
Summary:iFyber LLC is currently developing a platform technology for capture, concentration and detection of analytes using buoyant microsphere reagents and Lab-on-a-Bubble (LoB™) assays. Reminiscent of magnetic capture assays, LoB assays offer the convenience of a no-way assay, with the added benefit of being independent of volume – where magnetic capture is typically limited to small volumes due to limitations in magnetic field. iFyber’s assay for total coliforms is highlights the benefits of LoBs, as typical EPA standards for detection bacteria typically requires 100 mL sample volumes. Our results show that the LoB assay can differentially detect E. coli and total coliform, which circumvents the current challenges with detecting these organisms at a level of 1 CFU per 100 mL in less than 8 hours. In this study, we show positive results in less than 8 hours by making use of novel microsphere reagents for capture and concentration of the target cells, and coupling these reagents with loop-mediated amplification (LAMP) or real-time polymerase chain reaction (qPCR) of coliform and E.coli genetic markers with fluorescence detection. The microspheres concentrate the target cells by at least 1000 fold, where they are further enriched with growth medium, and their respective genetic targets are amplified through LAMP. Currently a benchtop prototype assay and system exists and a Phase II DoD SBIR is helping with assay optimization, kit development, and development of a alpha prototype system that will be validated next to currently approved EPA test methods for eventual approval through the EPA program for Alternative Test Procedures (ATP).