Time-resolved immunofluorometric determination of specific mRNA sequences amplified by the polymerase chain reaction

We report a highly sensitive time-resolved immunofluorometric method for quantification of polymerase chain reaction (PCR)-amplified mRNA sequences. The PCR primers are labeled at their 5' ends, one with biotin and the other with a hapten. The modified primers are incorporated, during PCR, in the amplified product. The PCR product is captured, through its biotin moiety, to a streptavidin-coated solid phase and subsequently is detected with an alkaline phosphatase-labeled antibody. The phosphate ester of fluorosalicylic acid is used as a substrate. The fluorosalicylate produced forms a highly fluorescent ternary complex with Tb(3+)-EDTA, which is measured by time-resolved fluorometry. We chose the determination of PCR-amplified chronic myelogenous leukemia-specific mRNA as a model system. mRNA molecules corresponding to 0.1 leukemic cell in the presence of 0.5 million normal cells may be detected (signal-to-background ratio of 1.5). The method provides a sensitive and rapid nonisotopic alternative to Southern blot and hybridization with radioactive probes.