Development Of Fluorescence Quantitative PCR Method For Detection Dibutyl Phthalate

In this study, a novel method to detect Dibutyl phthalate (DBP) using fluorescence quantitative PCR (FQ-PCR) was developed.In this study, when the concentrations of DNA probes were in the range of 109 to 103 copies/μl, the correlation between Ct values and DNA copies was linear. (R2=0.99713).The analysis of melting curve show that Tm value was consistent with the expected, and there was only one remarkable peak, so this standard curve could be used as quantitative standard curve.Extracted Estrogen receptor from the fish liver cells, and incubated the solute with different concentrations of DBP to make it activation. The combination of estrogen receptor and DBP come into receptor-ligand complex. Then, amplified through conventional PCR, specific primers were designed and synthesized in company with pUC19 as template for preparation of double-stranded DNA with estrogen responsive elements. Hydroxyapatite (HAP) was used to purification the ligand-receptor complex, and the Result shows that it's efficiency. These ligand-receptor-DNA complexes were digested by EXOIII and S1 nuclease. S1 stop buffer was added to inactivate S1 nuclease. In this experiment, the relationship between the concentrations of DBP and Ct values were analyzed from the quantitative standard curve and the plot between DNA copies and concentrations of DBP. These Results show that there was linear relationship when DBP concentrations were between 10-5 g/L and 10-10 g/L. When DBP concentration was 10-10 g/L, the Result was positive. So the lowest detection limits of this method 8.439 pg/L for DBP. Furthermore, FQ-PCR was able to detect DBP concentration in water and soil sample. In this study DBP concentration in soil and water can be detected at 0.1357 ug/g and 0.1058μg/L. In this study, SPR was used to detect and identification the quantization of combination of Estrogen receptor and DBP. In this research HPLC method that can be detected lowest DBP concentration at 0.0084μg/L and the linear equation of concentration area standard curves are y =0.00121x-0.05818 (R2=0.99855) and the concentration of DBP in soil samples and water samples are 2.7319 ug/g. and 0.2129μg/L. Compare with the lowest detection limit of HPLC method that can be detected. FQ-PCR could be detected through the concentration of DBP in samples lower than HPLC method.