Development of a method for quantitative determining PCR products by bioluminescent assay

Objective A bioluminescence-based immunoassay was established for the quantitation of PCR products.Methods It amplified CK19-mRNA by using RT-PCR.The primer in the PCR reaction was labeled with a 5.biotin molecule.A digoxigenin-conjug ated oligonucleotide probe was hybridized to the target biotin-labeled DNA template.The hybridized duplex was captured onto a streptavidin-coated microt iter plate.Digox igenin reacts with digoxigenin-specific antibodies conjugated with the photoprotein aequor in added.The amount of specific DNA captured onto the plate was quantitated by triggering the bioluminescence reaction through the addition of calciumions.Results This technique detected as low as 22 amol/L of amplified CK 19-mRNA products.Conclusion This technique is a sensitive,reliable and non-radioactive method for quantitative detection of trace CK19-mRNA.