High-performance real-time quantitative RT-PCR using lanthanide probes and a dual-temperature hybridization assay

We report a time-resolved fluorescence-based, homogeneous approach for multiplex, real-time or end-point detection of PCR products. Signal generation consists of PCR associated digestion of a 5'-labeled oligonucleotide probe, rapid cooling of the reaction mixture, and hybridization of undigested probe oligonucleotides with a complementary, shorter probe that incorporates a quencher at its 3' end. The signal coming from intact fluorescent probe molecules is, thus, quenched. The fluorophores we have used are environmentally sensitive lanthanide chelates. Their signals can be measured in a time-resolved manner that eliminates most of the unspecific fluorescent background. Signal-to-noise ratios are further enhanced by the environmental sensitivity of these chelates; they exhibit a higher fluorescence intensity when free in solution than when coupled to intact probe molecules. Because of the minimal background fluorescence, the signal-to-noise ratios are higher and threshold cycles are lower than those obtained using conventional TaqMan probes. The multiplexing capacity of the assay chemistry is demonstrated through simultaneous amplification and detection of prostate specific antigen (PSA) cDNA and an internal standard mRNA (mmPSA) using probes labeled with terbium and europium. The applicability of the assay chemistry to routine clinical diagnostics is demonstrated through absolute quantification of PSA mRNA in peripheral blood.