Assay formats involving acridinium-ester-labeled DNA probes

We describe the development of several hybridization assay formats involving acridinium-ester-labeled DNA probes. The simplest of these is a homogeneous assay procedure that requires only three steps to complete, including a 5-s detection step. Using this format, we have detected target sequences in the 10(-16) to 10(-17) mol range; when rRNA is the target, this translates to 3000 to 300 bacterial organisms. The entire assay can be carried out in less than 30 min. This is the first homogeneous DNA probe assay to be of practical use in the clinical laboratory, and it represents a major simplification of hybridization formats. We also demonstrate the use of this homogeneous assay format to discriminate single-base differences between two closely related target sequences and to detect DNA as well as RNA target molecules. By combining homogeneous hybrid discrimination with solid-phase separation, we have been able to decrease background readings from unhybridized probe to only a few parts per million. This enhances assay sensitivity about 10-fold, to a range of 10(-17) to 10(-18) mol of target. We are in the process of further improving the performance of these assays.