QUANTITATIVE POLYMERASE-CHAIN-REACTION BASED ON A DUAL-ANALYTE CHEMILUMINESCENCE HYBRIDIZATION ASSAY FOR TARGET DNA AND INTERNAL STANDARD

We have developed a dud-analyte chemiluminescence hybridization assay for quantitative polymerase chain reaction (PCR), The method allows si multaneous determination of both amplified target DNA and internal sta ndard (IS) in the same reaction vessel, The target DNA fi om the sampl e (233 bp) was coamplified with a constant amount of a recombinant DNA IS that had the same size and primer binding regions as the target DN A, differing only by a 24-bp sequence, centrally located, Biotinylated PCR products from target DNA and IS were captured on a single microti ter well coated with streptavidin, The amplified target DNA was hybrid ized with a digoxigenin-labeled specific probe, and the hybrids were d etermined by using antidigoxigenin antibody labeled with aequorin, The amplified DNA IS was hybridized, in the same well, with a fluorescein -labeled probe, and the hybrids were determined by using an antifluore scein antibody conjugated to alkaline phosphatase, Aequorin was measur ed by adding a Ca2+-containing light-triggering solution. Alkaline pho sphatase was measured by using a dioxetane chemiluminogenic substrate. The ratio of the luminescence values obtained from the target DNA and IS amplification products was linearly related to the number of targe t DNA molecules present in the sample prior to amplification. The line ar range extended from 430 to 315 000 target DNA molecules. Average CV s ranged from 7 to 17%, The proposed system is expected to facilitate the automation and routine use of quantitative PCR.