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Method for detecting phthalic acid esters (PAEs) with molecular beacon fluorescence quantitative PCR

A phthalate ester and molecular beacon technology, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of cumbersome, lengthy, expensive and time-consuming analysis process, and improve the accuracy And the effect of sensitivity, low detection limit and strong specificity

Active Publication Date: 2011-11-02
南通佳环环境技术有限公司
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Problems solved by technology

Chromatographic detection is not only expensive and time-consuming, but also the analysis process is cumbersome and lengthy

Method used

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  • Method for detecting phthalic acid esters (PAEs) with molecular beacon fluorescence quantitative PCR
  • Method for detecting phthalic acid esters (PAEs) with molecular beacon fluorescence quantitative PCR
  • Method for detecting phthalic acid esters (PAEs) with molecular beacon fluorescence quantitative PCR

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Embodiment 1

[0028] 1. Extraction of estrogen receptor and preparation of ligand-receptor complex

[0029] 1. Extraction of estrogen receptor-containing cytosol

[0030] Get the carp domesticated in the water that is 0.01mg / L of phthalate concentration, anesthetize with benzocaine of 5mg / L. Take the liver, wash off the blood outside the liver with 0.15mol / L KCl solution pre-cooled on ice, then blot the surface water with sterilized filter paper, and add HEDG buffer (pre-cooled on ice) was homogenized in a glass homogenizer. The homogenate was centrifuged at 15,000g for one hour, and the supernatant was carefully drawn, which was the cytosol containing receptors, and stored at -80°C after aliquoting.

[0031] 2. Preparation and purification of phthalate-estrogen receptor complex

[0032] Dilute 1g / L of phthalates to 10 -1 g / L-10 -9 g / L, take 10 μL each, add 250 μL of cytosol, and incubate at 20°C for 2 hours with shaking. When the ligand-receptor reaction is over, add 0.2mL of 60% HAP...

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Abstract

The invention relates to a method for detecting PAEs with molecular beacon fluorescence quantitative PCR. The method comprises the following steps that: cytosol which is extracted from carp liver and contains a receptor is combined with PAEs of different concentration to form compounds; the compounds are combined with bound DNA synthesized by specific primers to form ligand-receptor-DNA compounds; the ligand-receptor-DNA compounds are digested by exonuclease III and S1 nuclease; the products of the enzyme digestion are used as templates for molecular beacon fluorescence quantitative PCR amplification, and a regression equation between concentration of added PAEs and copy numbers of an initial template is obtained; according to the regression equation and a quantitative DNA standard curve,the relationship between the concentration of PAEs and Ct values is obtained through linear regression analysis, thereby enabling detection of PAEs of different concentration to be realized. The method provided in the invention has the characteristics of high sensitivity, low detection limit and strong specificity, is applicable to rapid detection of the content of PAEs in mass samples, and has agood application prospect.

Description

technical field [0001] The invention belongs to the detection field of phthalates, and in particular relates to a method for detecting phthalates by molecular beacon fluorescent quantitative PCR. Background technique [0002] Phthalate esters (PAEs) are plasticizers for plastics. They are the most widely produced and widely used synthetic organic compounds in the world. They are widely used in the plastics industry. Such pollutants have entered the environment in large quantities, and generally exist in environmental samples such as soil, sediment, water, organisms, air and atmospheric dust. Due to the widespread existence of PAEs, its impact on the environment has gradually become prominent. Studies have shown that PAEs have various biological toxicity such as acute toxicity, carcinogenicity and teratogenicity. Studies on environmental behavior and ecological effects have shown that the hydrolysis and photolysis rates of phthalates are very slow, and have become a global ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 赵晓祥白舟李颢邵姗珊谢佳
Owner 南通佳环环境技术有限公司
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