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Method for detecting nonyl phenol by exonclease protection fluorescent quantitative PCR

A fluorescence quantification and nonylphenol technology, which is applied in the direction of fluorescence/phosphorescence, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of low sensitivity and high false positive rate, and achieve high sensitivity and detection limit Low, the effect of improving accuracy and sensitivity

Inactive Publication Date: 2010-04-07
DONGHUA UNIV
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Problems solved by technology

The traditional enzyme protection analysis method only uses exonuclease ExoIII to analyze the bound DNA directly through agarose gel electrophoresis, which has low sensitivity. Using it in conjunction with PCR will easily lead to a high false positive rate

Method used

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  • Method for detecting nonyl phenol by exonclease protection fluorescent quantitative PCR
  • Method for detecting nonyl phenol by exonclease protection fluorescent quantitative PCR
  • Method for detecting nonyl phenol by exonclease protection fluorescent quantitative PCR

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Embodiment 1

[0027] 1. Extraction of nonylphenol receptor and preparation of receptor-ligand complex

[0028] 1. Extraction of cytosol containing nonylphenol receptors

[0029] Goldfish with similar body length and weight were first sterilized with 5% saline solution, and then domesticated in the laboratory for a week with tap water that had been naturally dechlorinated for 3 days. During the domestication process, feed once a day and aerate with a sand head. One week later, nonylphenol was added to make the concentration of nonylphenol in the water 0.01mg / L. During the feeding process, the static fluid replacement method was adopted and replaced every 24 hours. After one month of domestication, the goldfish were taken out and anesthetized with 5 mg / L benzocaine. 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 ho...

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Abstract

The invention relates to a method for detecting nonyl phenol by exonclease protection fluorescent quantitative PCR. The method comprises the following steps: combining the characteristic primer with DNA, nonyl phenol receptor extracted from fish liver and nonyl phenol with different concentrations to form a composite; ensuring the dissociative DNA not to be augmented by resolving the compound by nucleic acid exonclease III and S1 nucleic acid exonclease; taking the product after enzyme cutting as a template, and carrying out fluorescent quantitative PCR augmentation to obtain a regression equation adding nonyl phenol concentration and copy number of an initial template; utilizing quantitative DNA standard curve and linear regression analysis to obtain the relationship between nonyl phenol concentration and a Ct value; and carrying out the detection on the nonyl phenol with different concentrations. The invention has the characteristics of high sensitivity and low detection limit, and can be used for rapidly detecting the content of the nonyl phenol in a large quantity of samples.

Description

technical field [0001] The invention relates to the field of environmental estrogen detection, in particular to a method for detecting nonylphenol by fluorescent quantitative PCR with exonuclease protection. Background technique [0002] Nonylphenol (nonylphenol, NP) is one of the endocrine disrupting chemicals (Endocrine Disrupting Chemicals, EDCs). Research by Staples and Banat et al. in 1999 and 2000 (Chemosphere) respectively proved that nonylphenol is stable in the environment, not easy to degrade, and easy to enrich through the biological chain. Due to its stable chemical properties and ultra-trace level in the environment, making it extremely difficult to detect. At present, the detection method of nonylphenol is mainly chromatographic method. However, chromatography is not only expensive and time-consuming, but also the analysis process is cumbersome and lengthy. In recent years, with the development of biotechnology, many nonylphenol biological detection methods ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/44G01N21/64
Inventor 赵晓祥邓琴
Owner DONGHUA UNIV
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