The main objective of Phase I of the """"""""Rapid, Sensitive Chemiluminescent Detection of Infectious Diseases with Enzyme Labeled DNA Probe"""""""" SBIR contract is to optimize the sensitivity of membrane based DNA probe hybridization assays for Herpes Simplex Virus I. Several concerns which are known to limit assay sensitivity will be addressed. These concerns are: non-specific background, type of membrane and enhancement of the chemiluminescent signal. The most important factor in the assay sensitivity limitation is the background, which is caused by nonspecific binding of alkaline phosphatase labeled probe to membrane surfaces. In addition, other reagents such as BSA, etc., also contain minute quantities of alkaline phosphatase contamination. In order to control this background we will test various materials such as gelatin, casein and synthetic polymers which are suited for background blocking. The final choice of the optimal blocking reagent will be very critical in background reduction. In addition, various nylon membranes obtained from commercial vendors will be evaluated for background reduction and chemiluminescent signal enhancement parameters. The best membrane and blocking reagent(s) will be subsequently evaluated in the total hybridization assay and its sensitivity will be compared to the commercial colorimetric systems. The optimum chemiluminescent assay will be further evaluated for the shortest turnaround time. It is our objective to develop a system which will allow us to obtain very sensitive detection results in less than one hour.
