SBIR-STTR Award

Fluorogenic minor groove binder probes containing the MGB(tm) ligand at the 5'-end, an Eclipse(r) Dark Quencher and a fluorophore have been shown not to be degraded by 5'-nuclease activity. These probes, with excellent specificity and low fluorescent background in solution, fluoresce on hybridization to their complementary targets. It is proposed to immobilize this type of probe on a solid support in a model microwell system. The immobilized probe fluoresces upon hybridization to its unlabeled target. The Phase I goal is therefore to a) develop a model solid phase DMA probe system where the probe fluoresces upon hybridization to its complementary unlabeled target and b) to evaluate the ability of the probe solid support to efficiently detect and discriminate nucleic acid targets from closely related infectious organisms. Probes with high signal to noise (>100) in solution, will be immobilized through an optimized linker and attachment position on the probe to the solid support with proven chemistries developed at Epoch Biosciences. The surface of the solid support will be modified to improve hybridization kinetics and reduce non-specific binding. The goal is to achieve signal to noise ratios on the solid support similar to that obtained in solution. The solid support will be used to demonstrate the differentiation of unlabeled ssDNA amplicons generated from Salmonella serovars with no, one and two mismatches and to compare the results to that obtain with a similar probe in a homogeneous assay. During Phase II the results of Phase I will be further optimized if necessary and transferred to proposed commercial product lines, a glass solid support and a microfluidic cartridge that contains probes complementary to about 20 targets. Different infectious disease panels will be developed after assessment of market acceptance potential. The panels will be used in the two product formats. These will include panels to address enteric and hepatic respiratory and viral diseases.

Thesaurus Terms:
biohazard detection, biosensor, communicable disease diagnosis, diagnosis design /evaluation, fluorescent dye /probe, microorganism classification, nucleic acid probe, nucleic acid quantitation /detection DNA, Salmonella, bacterial disease, chemical conjugate, chemical stability, gastrointestinal infection, liver infection, microfluidics, oligonucleotide, rapid diagnosis, respiratory infection, virus disease bioengineering /biomedical engineering, biotechnology, bioterrorism /chemical warfare

Bacterial and viral meningitis and encephalitis is a major cause of morbidity and mortality in all societies of the world. The rapid progression of symptoms and potentially devastating effect of these diseases necessitate early recognition and immediate treatment. The large number of potential organisms involved, the fact that some infections are self-limiting while others may have potentially fatal outcome, complicates diagnosis and treatment. The goal of the proposed work is to develop an assay that will simultaneously detect all the common meningitis and encephalitis pathogens typically seen in a medical facility in the US. The potential pathogens will be amplified in a multiplexed PCR from a single cerebral spinal fluid (CSF) sample and detected on an array containing pathogen-specific probes that fluoresce on hybridization. In Phase I we have shown that a Pleiades probe immobilized to a hydrogel electronic NanoChip array cartridge fluoresces when hybridized to its complementary amplified target with signal to background ratios significantly better than any comparable immobilized probe. It was shown that the immobilized probe could distinguish between match, single-mismatch and double-mismatched amplified targets. In Phase II the common meningitis and encephalitis pathogens responsible for disease in the US will be identified. Primer pairs will be developed for amplification of each meningitis and encephalitis target, multiplexed to allow the detection of each target in a single CSF amplified sample. The proposed Phase II work will allow the simultaneous detection of about 90% of the pathogens typically seen in a US medical facility in contrast to about 45% of the organisms currently confirmed clinically. The proposed meningitis and encephalitis panel will be the first available in a clinical laboratory to assist diagnosis and treatment. The goal of the proposed Phase II project is to develop an array for the rapid detection of the common pathogens involved in meningitis and encephalitis in the US. This will enable the rapid differentiate of organisms that cause self-limiting disease from those with potentially fatal outcome