Deployable Pan-flavivirus and Pan-alphavirus Assays for Screening Pools of Medically Relevant Arthropod

CustomArray produces a field-deployable electrochemical microarray platform that is ideally suited to the multiplexed detection of pathogenic organisms. In this proposal, we will build a complete detection process that will include: Probe design, sample prep, multiplexed PCR amplification, hybridization, detection and data analysis. We have developed a bioinformatic pipeline called GenoTyper that is able to design primers and probes for the simultaneous amplification, detection, interrogation and typing of families of related, rapidly evolving sequence families. We have successfully used this system in the past to detect and type many viral families including, among many others: influenza, alpha-virus, and 16S. In this proposal we will use this bioinformatic system to create an updated pan-flavivirus and pan-alphavirus detection system. This is a proven detection system that relies on primer design that generates broad amplification followed by hybridization probe design and data analysis that is able to interrogate and accurately type the amplified material. Because we can use up to 2240 probes per assay, we are able to offer a resolution and redundancy that is unprecedented in a compact, field-deployable device. We will use our expertise and know-how in sample prep to create a simple method for harvesting viral DNA from arthropod samples. We will incorporate a novel multiplex PCR amplification system that will allow multiple independent PCR amplification reactions to take place in a single-injection device. Hybridization will be performed on our customizable semiconductor-based microarray platform. Hybridization detection will be performed using our electrochemical detection system. The detection device itself is a hand-held product that requires a USB connection, and uses that connection as its sole power source. It can read an entire array in 20 sec. This platform is the only electrochemical detection system of its kind that is as sensitive as fluorescence detection. No other system exists that has such potential to revolutionize the breadth, accuracy and robustness of field-deployable pathogen detection.