We have developed technology for the rapid, ultrasensitive detection of infectious agents. We call it the peroxidase chain reaction - PxCR. Unlike PCR, PxCR amplifies signal rather than targets, thereby avoiding all the problems associated with contamination of samples, reagents, and work spaces by amplified DNA. Because signal is amplified, PxCR detects not only DNA and RNA, but also proteins and infectious particles - viruses, bacteria, and other microorganisms. In PxCR, targets are captured onto a biosensor, tagged with a biotinylated probe, and then coated with streptavidin-conjugated horseradish peroxidase (SA-Px). By cycling sensors between two solutions, one containing a biotinylated substrate for the enzyme and the other more SA- Px, PxCR amplifies the amount of peroxidase on a sensor. Detection occurs either in real-time as sensors report the amount of deposited enzyme and substrate or, with even greater sensitivity, by incubation of sensors in a traditional staining substrate like diaminobenzidine. All operations are performed robotically, completely hands-off. Because capture of targets by the biosensor relies on the biochemistry of binding-hybridization for nucleic acids;antibody affinities for proteins and infectious particles sample preparation is minimal. For RNA, it could merely be solubilization to release genomic RNA from a virus. For proteins and infectious particles with exposed epitopes, preparation could be as simple as dilution into the loading solution. Specificity is high like PCR because detection requires that targets bind to two separate probes - a capture probe which is tethered onto the biosensor and a biotinylated tagging probe. We have shown show that PxCR detects 100 copies of protein, DNA, and RNA. In this proposal we focus on using PxCR to develop diagnostics for dengue virus.
The Specific Aims are to: (1) Develop pan and serotype specific PxCR assays for dengue viral RNA using oligonucleotide probes. (2) Develop serotype specific PxCR assays for the detection of dengue viral particles using antibodies to the envelope protein. (3) Develop a PxCR assay for the detection of pan-dengue NS1 protein. (4) Determine the sensitivity and specificity of PxCR assays for the detection of viral RNA, NS1 protein, and viral particles in human blood. (5) Assess performance of PxCR assays in de-identified, blinded clinical samples obtained from patients in the viremic phase of dengue infection. (6) Assess dengue virus PxCR assays using serum samples obtained as part of a phase I trial of a live attenuated tetravalent dengue vaccine. PxCR has the potential to revolutionize molecular detection. It complements PCR in that PxCR provides the sensitivity and speed for protein detection that PCR offers for nucleic acids. PxCR goes further in that the same platform detects proteins, DNAs, RNAs and infectious particles. In principal, oligosaccharides could also be detected using lectin probes. By developing and optimizing PxCR technology, this proposal will significantly expand our ability to rapidly detect a broad range of biodefense and emerging pathogens as well as their toxins.
This project aims to develop new and broadly applicable tools that would be used not only for the early diagnosis of infectious disease but also for the very sensitive detection of biological warfare agents which could be used for bioterrorism. Like the technological spin-offs from space exploration, the technology that will be developed by this project will make a broad impact on public health, including the ability for physicians to make much more accurate and speedy diagnoses in their offices without needing to wait days for lab results.
