This Small Business Innovation Research Phase I project aims to develop a novel probe labeling method to increase the sensitivity of in situ hybridization assays. Although this probe labeling method can be used for a variety of RNA and DNA assay formats, this proposal will focus ondeveloping the technology for use with viral assays, where there is an urgent need for signal amplification technologies. Current methods for detecting viral infected cells include in situ PCR and fluorescence in situ hybridization. In situ PCR, however, is prone to several artifacts, such as non-specific target amplification and amplicon leakage out of the cell. Signal amplification methods, such as tyramide signal amplification (TSA), have increased the sensitivity of in situ hybridization assays, however, use of TSA increases assay complexity by adding several procedural steps. Oligonucleotide probes modified by enzymatic """"""""tailing"""""""" increase the sensitivity of in situ hybridization assays, however, this modification complicates labeling procedures and increases production costs. Furthermore, use of these probes is limited due to licensing issues. This proposal will combine a novel probe labeling method with image scanning cytometry to develop sensitive in situ hybridization assays for detecting rare viral infected cells. ? ? ? ?
