金鱼组织中壬基酚的含量检测新方法探讨

Research on a New FQ-PCR Assay for Detection of Nonylphenol in Goldfish

建立一种直接检测环境中痕量壬基酚的外切酶保护-荧光定量PCR方法。首先向含雌激素受体及相关蛋白的细胞溶质中加入壬基酚-丙酮溶液,使受体蛋白活化,从而在体外与含雌激素反应位点的双链结合DNA作用形成壬基酚-雌激素受体-DNA复合物。复合物用核酸外切酶和S1核酸酶消解,去除未受到蛋白质保护的结合DNA。将消解后产物作为模板,进行荧光定量PCR扩增反应,建立壬基酚浓度与标准DNA拷贝数的对数之间的线性关系为：y（壬基酚浓度的对数）=1.637x（标准DNA拷贝数的对数）-9.505。该法检出限为10^-8g/L,用该法对金鱼肝脏组织中壬基酚进行检测,添加回收率水平在98.5%112.2%,变异系数在4.3%以下。

This study aims to establish an Exonuclease Protection-fluorescence quantitative PCR assay for directly detection of the trace nonylphenol in environment.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 research will first add nonylphenol-acetone to solute which contains estrogen receptor and associated protein,and combined to double-strand DNA with estrogen responsive elements in vitro to format the nonylphenol-estrogen receptor-DNA complex.Then these complexes were digested by the exonuclease and S1 nuclease to remove the DNA which is not protected by protein.After the digestion,the complexes can be used as a template for FQ-PCR amplification.The relationship between the logarithm of the concentration nonylphenol and the standard DNA copy number was established,linear equation is： y=1.6455x-9.523.This assay was applied to detect nonylphenol in goldfish,the average recoveries prove to be between 78%-93% and the coefficient variation under 4.3%.