The last decade has witnessed substantial improvement in the development of parallel screening technologies designed to detect many types of biological threats. One type of screening technology has concentrated on mitigating threats caused by viral Biothreat agents by using multiplex PCR followed by hybridization onto optically encoded beads from Luminex. However, the rather small number of usable bead optical codes (~30-40), and the delicate flow cytometer based detector, prevent the detection of all viral threats in one panel or use of the screen outside of a controlled laboratory environment. To address these issues, a system based on Parallume (www.parallume.com) optical encoding, which can support thousands of optical codes, will be used to encode beads that will be read by a completely portable, battery-powered reader. The final goal will be to develop a screening panel capable of detecting, differentiating and identifying all known viral families in any location by using a portable detection system. In collaboration with scientists at Lawrence Livermore National Laboratory (LLNL), Parallel Synthesis Technologies, Inc. (PSTI) will formulate a Parallume-encoded bead set containing 15 optical codes which will be used in a direct comparison against a 15-bead Luminex-based Viral Screening Panel (VSP) already in use at LLNL to detect human influenza viruses. Using biotinylated primers designed by the LLNL Bioinformatics Group, PCR for 15 regions of viral genomes are performed in a single reaction. A target region of each PCR product contains a sequence which is hybridized to a capture probe on the optically encoded bead. By treating the PCR product after hybridization with a streptavidin-phycoerythrin (SAPE) conjugate to label the biotinylated primers, the presence of the viral genome is detected by a serially diluted, threshold SAPE level on an encoded bead as compared to the controls. In the Phase I effort, Parallel and LLNL will compare the state-of-the-art Luminex system with the Parallume encoding technology by characterizing the same reactions on a divided PCR product mixture. The Parallel samples will be characterized using Parallume beads and Parallel's robust Multiplex Assay Reader System (MARS), which has replaced the fragile flow cytometer and lasers with micromachined silicon bead localization slides and super-bright signage LEDs, and the measurement rate, accuracy, precision and cost of the two systems directly compared. The combination of the Parallel and LLNL technologies could allow all known viruses to be screened anywhere in one PCR reaction.
This innovation seeks to create a viral screening panel (VSP) whereby a human, animal, or plant sample containing DNA is subjected to a simultaneous screen for multiple pathogens (viruses in the current application) within a single vial. Limited virus detection technologies exist, but are hampered by technical limitations not possessed by the proposed VSP. This novel VSP proposes a method for expanding the number of viruses currently detectable in a single screen and adds to the potential functionality of the panel by being able to incorporate other screens (e.g., bacterial and other non-viral pathogens) into this single, multiplexed panel. This technology also offers a dramatically less-expensive reader (VSP analysis equipment) to accompany this new product.
