SBIR-STTR Award

Fabrico Technology and Professor Andrew Ellington of the University of Texas at Austin, proposes to develop an in-field detection platform based on a novel variation of loop-mediated isothermal amplification (LAMP). The proposed reaction works by utilizing a patent pending oligonucleotide strand displacing (OSD) probe which functions for LAMP in much the same way as a TaqMan probe functions for qPCR. This OSD innovation overcomes a major flaw in LAMP-only based diagnostics by greatly reducing the likelihood of producing false positives. The proposed LAMP-OSD device will distinguish between viable and non-viable coliform bacteria andE coli by comparing ratiometrically, the stable and short-lived RNA markers that degrade before and after bacteria death. LAMP-OSD allows for highly specific Yes/No answers with minimal processing time and complexities. A number of LAMP devices and assay form factors have been demonstrated but little to no work has been done using RNA markers to differentiate between viable from non-viable organisms into a LAMP-OSD device which is one of the key technical challenges and goals of this proposal effort.

Fabrico Technology, Dr. Sanchita Bhadra and Professor Andrew Ellington of the University of Texas at Austin, have successfully demonstrated the proof-of-concept of an unparalleled, first-in-class in-field nucleic acid diagnostic platform for distinction of viable from non-viable E. coli and coliforms in <5 h. This platform is based on a novel variation of the Ellington patent-pending loop-mediated isothermal amplification (LAMP) - oligonucleotide strand displacing (OSD) assay, which is capable of not only exquisitely sequence-specific analyte detection but also analyte copy number estimation with only single endpoint visual 'yes/no' readout of presence or absence of signal. By comparing ratiometrically the increase in nucleic acid analyte copies in live bacteria and lack thereof in dead counterparts our thresholded RT-LAMP-OSD System allows for highly specific 'Yes/No' answers with minimal processing time, and technical and infrastructure complexities.The Phase I demonstrated that the thresholded RT-LAMP-OSD assay can meet the detection limit of 1 CFU/100 mL and is highly specific to only coliforms and E. coli.In Phase II, we propose to optimize and validate specificity of our thresholded RT-LAMP-OSD System by testing against background bacteria, test for chlorine-stressed E. coli and coliform, and prepare an EPA ATP application for independent laboratory verification of the system.