TIME-RESOLVED IMMUNOFLUOROMETRIC DETERMINATION OF SPECIFIC MESSENGER-RNA SEQUENCES AMPLIFIED BY THE POLYMERASE CHAIN-REACTION

We report a highly sensitive time-resolved immunofluorometric method f or quantification of polymerase chain reaction (PCR)-amplified mRNA se quences. The PCR primers are labeled at their 5' ends, one with biotin and the other with a hapten. The modified primers are incorporated, d uring PCR, in the amplified product. The PCR product is captured, thro ugh its biotin moiety, to a streptavidin-coated solid phase and subseq uently is detected with an alkaline phosphatase-labeled antibody. The phosphate ester of fluorosalicylic acid is used as a substrate. The fl uorosalicylate produced forms a highly fluorescent ternary complex wit h Tb3+-EDTA, which is measured by time-resolved fluorometry. We chose the determination of PCR-amplified chronic myelogenous leukemia-specif ic mRNA as a model system. mRNA molecules corresponding to 0.1 leukemi c cell in the presence of 0.5 million normal cells may be detected (si gnal-to-background ratio of 1.5). The method provides a sensitive and rapid nonisotopic alternative to Southern blot and hybridization with radioactive probes.