摘要
建立了一种利用外切酶保护-荧光定量PCR检测环境激素邻苯二甲酸酯的方法。将从鱼肝脏中提取含雌激素受体的细胞溶质,与不同浓度的邻苯二甲酸酯结合,采用常规PCR方法扩增制备双链结合DNA,将其与配体-受体复合物反应的结合物,用核酸外切酶ExoⅢ和S1核酸酶处理,消解游离DNA,并以消解后产物作为模板,进行荧光定量PCR扩增反应,建立Ct值与邻苯二甲酸酯质量浓度C(g/L)的对数标准曲线:Ct=-0.273 lg(C)+6.320。将该方法应用于水样中邻苯二甲酸酯的检测,最低检测限达到10～100μg/mL。该法准确度高、抗干扰性强、适用于检测大批量环境样品中邻苯二甲酸酯。
An exonuclease protection-fluorescent quantitative PCR assay for detecting dimethyl phthalate in water was established. Solution containing estrogen receptor from liver cells in carp was prepared, and then combined with different concentration of PAEs. The double-stranded DNA was amplified by conventional PCR and bind with the ligand-receptor complex to form the ligand-receptor-DNA complex. These ligand-reeeptor-DNA complexes were digested by Exonuelease III and S1 to remove the free state of DNA. These ligand-receptor-DNA complexes were used as templates in fluorescent quantitative PCR. The standard curve of the Ct values and the logarithm of the concentration （C,g/L） was established for phthalate, Ct=-0.273 lg （C） ＋ 6.320. This method was applied for the detection of phthalate in water; And the minimum detection limit was 10-100 ug/mL. Due to its good accuracy and specificity, it could be used to detect large amount of environmental samples.
出处
《湖北农业科学》
北大核心
2012年第4期823-826,共4页
Hubei Agricultural Sciences
基金
上海市基础研究重点项目(09JC1400600)
上海市重点学科建设项目(B604)
