Quadruple-analyte chemiluminometric hybridization assay. Application to double quantitative competitive polymerase chain reaction

We developed a highly sensitive quadruple-analyte chemiluminometric hybridization assay for simultaneous quantification of four nucleic acid sequences. The targets are amplified by the polymerase chain reaction (PCR) and captured to microtiter wells coated with streptavidin. The immobilized fragments are hybridized with specific probes containing a sequence complementary to the target and a sequence or a hapten that allows linkage with a chemiluminescent reporter. We prepared a mixture of four reporters conjugated to complementary oligonucleotides or antihapten antibodies. The reporters were aequorin(dT)(30), galactosidase-oligonucleotide, horseradish peroxidase-antifluorescein, and alkaline phosphatase-antidigoxigenin conjugates. The four chemiluminescent reactions were triggered sequentially. The signals were linearly related to the concentration of target sequences. The entire quadruple-analyte bioluminometric hybridization assay is complete in 75 min. We have demonstrated the applicability of the proposed assay to high-throughput quantitative competitive PCR of two target sequences in the presence of the corresponding competitors. The assay is universal since the same reporter conjugates can be used for multianalyte quantification of any sequences with properly designed probes.