Developmental Exonuclease Protection Fluorescent Quantitative PCR Assay For Detection Of Nonylphenol

Nonylphenol (NP), one of environmental endocrine disrupting chemicals, has relatively stable chemical properties, easy to enrichment in the sediment and hard to degradate. If NP exposure to the environment, it could lead to the secretion and activity of sex hormone in organism and human being decreased; the count of sperm reduced; abnormalities genital, increased incidence of diseases such as cancer. It not only could reduce the reproductive capacity, makes the offspring's health and survival rate decline, but also may affect the immune system and nervous system of a variety of organisms, lead to bio-genetic mutation and so on. At present, detection methods of nonylphenol are mainly gas chromatography, gas chromatography combined with mass spectrometry, and high-performance liquid chromatography, liquid chromatography combined with mass spectrometry, and later developed into a GC-MS combined with solid-phase extraction and purge-and Trap, immunoassay method and chemiluminescence were occasionally reported. Chromatography method is not only expensive and time-consuming but aslo has cumbersome and lengthy analysis process. In recent years, with the development of biotechnology, a number of nonylphenol biological detection methods were produced. As one of biological detection methods, PCR detection techniques received wide attention because of its high sensitivity and lower detection limit. SYBR Green I fluorescence quantitative PCR is a high-sensitivity quantitative PCR which emerged in recent years. In the field of nonylphenol detection, PCR technology was mostly applicated in detecting estrogenic effects of nonylphenol. The study of directly use PCR to detect Nonylphenol in environmental samples was less reported. In this paper, vitro DNA binding experiments were used, combining with exonuclease digestion, a new fluorescence quantitative PCR method for detection of nonylphenol was established.Estrogen receptor can be activated by nonylphenol so as to combine with a double-stranded DNA which contains estrogen responsive elements. By combined with estrogen receptor, this double-stranded DNA was protected by protein so that it can retain after the digestion of the exonuclease III. This trace retained DNA could be amplified by FQ-PCR. According to this theory, this study first extracted solute containing estrogen receptor from the fish liver cells, and incubated the solute with different concentrations of nonylphenol to make it activation. The combination of estrogen receptor and nonylphenol 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. After that, double-stranded DNA was binding with the receptor-ligand complex to form the receptor-ligand-DNA complex. These receptor-ligand-DNA complexes were digested by exonuclease III and S1 enzyme to remove the DNA without protein protection. After the digestion, these receptor-ligand-DNA complexes can be used as a template for FQ-PCR amplification. Thereby the standard curve of the Ct values and the logarithm of the concentration nonylphenol (g/L) were established. At the same time, the reaction conditions were optimized, detection limit were observed. This method is used to detect the content of nonylphenol in environmental samples. Comparison of the high-performance liquid chromatographythe and FQ-PCR method were taken and their advantages and disadvantages were discussed.The work is summarized as follows:(1) Under nonylphenol exposure conditions, the fish can produce more estrogen receptor. Hydroxyapatite was used to purificat the ligand-receptor complex, and the result shows that it's efficiency. The purification can reduce non-specific amplification in the PCR experiment. SPR was used to identificate the qualitation of combination of ligand-receptor complex. If the concentration of NP in the standard solution within 10^-8-10^-2g/L, There is a linear relationship between the concentration of nonylphenol and the SPR response intensity of receptor-ligand complexes.(2) Using DNA with known copy number to do quantitative standard curve, then according to the standard curve, and the template copy number corresponding to different concentrations of nonylphenol, we get the relationship between nonylphenol concentration and Ct curves which is: Ct =-1.828 * log (nonylphenol concentration) +11.447. The results of analysis shows that if concentration of nonylphenol within 10^-8-10^-2mol/L. the linear relationship is significantly (R²=0.99523). The minimum detection limit of nonylphenol by the fluorescence quantitative PCR method which established in this study is 10^-8mol/L.(3) High-performance liquid chromatography (HPLC) method to detect nonylphenol and comparison with PCR: HPLC was applied to detect nonylphenol, a standard curve of detection were produced. Detection limit were observed. Some environmental samples were analyzed with satisfactory results. Comparison with the fluorescence quantitative PCR method which established in this paper by the first time, the advantages and disadvantages are discussed. This FQ-PCR established here has many advantages such as low detection limit, good reproducibility, and can handle lots of samples at one time.Combined with Exonuclease Protection method, a new kind of nonylphenol detection method-PCR method was established here. This method has high sensitivity, simplely process, can be used as an effective biological screening and quantitative detection method for trace nonylphenol.