Bioluminescent PCR-RFLP enzyme-linked immunosorbent assay for analysis of vitamin D receptor gene polymorphism

We developed a sensitive and rapid PCR-RFLP ELISA using acetate kinase (AK) and firefly luciferase as a detection system. AK used as a label enzyme co uld sensitively be detected by bioluminescent assay using the firefly lucif erase reaction. The detection limit was 10(-20) mol/assay and the luminesce nce was stable for 48 h. FITC-labeled sense primer and biotin labeled anti sense primer were used for PCR amplification of the vitamin D receptor gene . After PCR, the products were digested with Tag I or Apa I enzyme. The rea ction products were diluted with assay buffer and transferred to a plate co ated with anti FITC IgG. After incubation for 2 hat 37 degrees C, the plate was washed and reacted with avidin/biotinylated AK, the AK activity was de tected by bioluminescence assay using the firefly luciferin/luciferase syst em. DNA polymorphism types (AA, Aa, aa, TT, Tt, tt) of the vitamin D recept or gene (VDR) could be clearly determined by measuring the bioluminescent i ntensity or by using photon imaging with a CCD camera.