Establishment and application of the method for detecting DHBV DNA with fluorescence quantitative PCR / 重庆医科大学学报

ABSTRACT

Objective: To establish the method for detecting duck hepatitis B virus (DHBV) DNA using fluorescence quantitative PCR. Methods: Three primers derived from DHBV DNA S gene were designed . The semi - nested primer was labelled by AmpliSen-sor using coupling reagent.The standard curve of the positive standards of DHBV DNA was established after asymmetric preamplifi-cation ,semi - nested amplification and on - line detetion. 70 samples of duck serum were tested by fluorescent quantitative DHBV DNA PCR method and dot blot hybridization assay using digoxigenin - labelled DHBV DNA probe. The correlation of results between the two methods was analysed. Results: After 30 cycles , amplification products showed two bands about 180bp and 70bp by 2% a-garose gel electrophoresis. The concentration of amplification products was in direct proportion to the initial concentration of the positive standards. The magnitude of detection index was in direct proportion to the amount of amplification products accumulating at the current cycle. The initial concentration of the positive standards was inversely proportional to the number of cycles needed for the amount of amplification products.The correlation coefficient of results was 0. 97 (P