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.
