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The goal of this project has been to optimize the Threshold DNA probe assay as a method for quantitating specific sequences after amplification by the polymerase chain reaction. Increasing the probe concentration 5-fold in the standard scheme B assay results in more rapid annealing of the probes to the target DNA, decreasing the overall assay time to less than 1 hour. A second assay format was developed in which the capture reagent and enzyme conjugate are added simultaneously. This scheme A format gives comparable results to those obtained using scheme B, and shortens the assay time to less than 30 minutes. Studies were also initiated to increase the detection sensitivity of the Threshold DNA probe assay by introducing multiple labels into the probe sequence by PCR. Data obtained after the reporting period officially ended indicate that 10 genomic targets can be detected after 25 cycles of PCR by using longer probes containing multiple fluorescein labels.