一种新的检测土壤中壬基酚的荧光定量PCR方法

Developmental fluorescent quantitative PCR assay for non-ylphenol detection in the soil

建立了一种新的外切酶保护-荧光定量PCR方法检测环境激素壬基酚,并将该方法应用于土壤样品中壬基酚的检测。壬基酚可以活化雌激素受体使之与包含特定序列的双链DNA结合,结合的DNA因受到蛋白质的保护可抵抗核酸外切酶Exo Ⅲ的消解而被保留,痕量的保留DNA可通过PCR扩增定量。根据此原理,首先从鱼肝脏中提取含雌激素受体的细胞溶质,与不同浓度的壬基酚结合使雌激素受体活化,形成配体-受体复合物;再设计合成特异性引物序列,采用常规PCR方法扩增制备双链结合DNA;使双链结合DNA与配体-受体复合物反应结合后,用核酸外切酶ExoⅢ和S_1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,从而建立G_1值与壬基酚质量浓度N(g/L)的标准曲线C1=-1.828lgN+11.447。将该方法应用于土壤中壬基酚的检测,最低检测限达到2 pg/g,可用于检测大批量环境样品中壬基酚。

This study is aimed to establish an accurate, sensitive exonuclease protection-fluorescent quantitative PCR assay for nonylphe- nol detection, which can be used to detect nonylphenol in soil sampies. As is known, nonylphenol estrogen is able to help activate the receptor to make it combined with a double-stranded DNA with estro- gen responsive elements. Once combined with the estrogen receptor, such double-stranded DNA tends to get protected by the protein so as to retain it after the digestion of the exonuclease 1H and then get am- plified by FQ-PCR. Based on the above theory, the present study first extracted solution with the estrogen receptor from the fish liver cells, and then incubated it at different concentrations of nonylphenol so as to make it activated. The estrogen receptor and the nonylphenol would then come to form a receptor-ligand complex. And, next, the specific primers could be designed and synthesized, amplified through the conventional PCR with pUCI9 as the template for preparing the so-called double-stranded DNA with estrogen responsive elements. After that, the double-stranded DNA would be bound with the recep- tor-lig complex to result in a receptor-ligand-DNA complex. These receptor-ligand-DNA complexes were digested by the exonuclease 1H and S1 enzyme to remove the DNA without the protein protection. Af- ter the digestion, such receptor-ligand-DNA complexes are likely to get used as a template for FQ-PCR amplification. Thereby the stan- dard curve of the C~ values and the logarithm of the concentration nonylphenol （g/L） can be established, with C~ = -1.8281gN ＋ 11.447. Thus, it can be seen that the method herein described can be applied to detecting nonylphenol in the soil, and the minimum de- tection limit was 2 pg/g. With high sensitivity and low detection bin- it, the method can be used to detect nonylphenol in large sizes and quantities of environmental samples.