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Position: Home > Articles > Development Fluorescent Quantitative PCR Assay for Detecting Phthalates Hubei Agricultural Sciences 2012,51 (4) 184-187

荧光定量PCR法检测邻苯二甲酸酯的研究

作  者:
白舟;李颢;谢佳;赵晓祥
单  位:
东华大学环境科学与工程学院
关键词:
邻苯二甲酸酯;荧光定量PCR;核酸外切酶
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。
译  名:
Development Fluorescent Quantitative PCR Assay for Detecting Phthalates
作  者:
BAI Zhou,LI Hao,XIE Jia,ZHAO Xiao-xiang(College of Environmental Science and Engineering,Donghua University,Shanghai 201620,China)
关键词:
phthalate;fluorescent quantitative PCR;exonuclease
摘  要:
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-receptor-DNA complexes were digested by Exonuclease 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 μg/mL.Due to its good accuracy and specificity,it could be used to detect large amount of environmental samples.

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