Abstract
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 could sensitively be detected by bioluminescent assay using the firefly luciferase reaction. The detection limit was 10–20 mol/assay and the luminescence 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 Taq I or Apa I enzyme. The reaction products were diluted with assay buffer and transferred to a plate coated with anti FITC IgG. After incubation for 2 h at 37˚C, the plate was washed and reacted with avidin/biotinylated AK, the AK activity was detected by bioluminescence assay using the firefly luciferin/luciferase system. DNA polymorphism types (AA, Aa, aa, TT, Tt, tt) of the vitamin D receptor gene (VDR) could be clearly determined by measuring the bioluminescent intensity or by using photon imaging with a CCD camera.
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Arakawa, H., Kokado, A., Yoshizawa, S. et al. Bioluminescent PCR-RFLP Enzyme-Linked Immunosorbent Assay for Analysis of Vitamin D Receptor Gene Polymorphism. ANAL. SCI. 15, 943–949 (1999). https://doi.org/10.2116/analsci.15.943
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DOI: https://doi.org/10.2116/analsci.15.943