Quantitation of PCR products with chemiluminescence.

Quantitative PCR and reverse transcription PCR (RT-PCR) are widely used in biomedical, industrial and other research applications to determine the number of RNA or DNA molecules of a specific type and/or sequence in a sample of interest. We have developed an assay system to accurately quantitate PCR products that utilizes solid-phase capture and an enzyme-linked chemiluminescent detection method. The entire assay is performed in a single tube or microplate well. Biotinylated PCR products are quantitated by capture onto a streptavidin-coated surface, followed by hybridization of an internal fluorescein-labeled oligonucleotide probe and subsequent detection with an anti-fluorescein-alkaline phosphatase conjugate and CSPD chemiluminescent substrate. Light signal is measured in a luminometer. The assay sensitivity enables accurate quantitation of target DNA because the measurement is performed on product generated during the exponential phase of amplification. The broad dynamic range of the assay, which is greater than three orders of magnitude of PCR product concentration, simplifies the determination of the number of amplification cycles necessary for accurate quantitation of target molecules. The PCR-Light system is an ultrasensitive, non-isotopic and rapid assay for PCR product detection that also has general application to solution hybridization assays and other quantitation methods.