Innovative DNA for Highly Multiplexed Molecular Diagnostics

Firebird has developed chemistry to support ultra-highly multiplexed PCR amplification (>>50 targets already demonstrated) of nucleic acids with insignificant risk of dropouts and other arti­facts that would generate erroneous results. In parallel, Siomyx’s founder, Arjang Hassibi, has developed an instrument based on a CMOS chip[i] that can operate in the field to (a) perform multi­plexed PCR using Firebird’s chemistry, and (b) deliver to the user a report, a correspond­ingly high multiplexed readout indicating the presence/absence of each target, at least 256 tar­gets with fourfold redun­dancy. The targets may be, for example, bacterial or fungal pathogen DNA.Combining the chemistry and the instrument is the goal of this Phase I SBIR project. This com­bination will support fast and highly multiplexed “omics” assays in far forward use in ultra-low resource environments. Our Phase I metric is a 100x multiplex with less than 50% variation in signals for analytes presented in equal amounts, a statement that corresponds to negligible dropouts or false calls, In Phase II, the platform will be developed to meet criteria needed to get certifi­cation of waiver (COW) status from clinical laboratory improvement amendments (CLIA)The chemistry and the instrument concept have both already been applied in other contexts. These include assays that received COWs and used in CLIA labs. Thus, the AEGIS reagents to be used in this project supported a 2003 SARS assays that received FDA approval in the US, and comparable regulatory approval in Canada and China.[ii] AEGIS supported diagnostic products with over $1 billion in life­time sales to monitor viral load in the serum of patients infected with HIV, hepatitis B, and hepatitis C viruses.[iii] These personalized the care of those patients in a colla­boration with Bayer Diagnostics and Siemens Diagnostics. Other products with approval were used in genomic medicine to advise cystic fibrosis parents,[iv] and in respiratory disease panels.[v]As Specific Aims, we will (1) Develop a prototype that combines a CMOS chip with Firebird's reagents to detect at least 100 targets related to sepsis. (2) The prototype will be a breadboard device with metricked specifications. (3) Phase I will demonstrate the feasibility of combining Firebird’s reagents with a CMOS chip, where the proof of concept metric is the simultaneous assay of 100 “omics” tar­gets with less than 50% variance in output signal of equal amounts of nucleic acid target. (4) The Phase I deliverable will thus show a feasible route to develop wide multiplexing capabil­ity with high precision as compared to traditional next generation sequencing platforms. This metric will be used/ tested from the beginning of protocol design.