Method for efficiently and visually discriminating authenticity of Tetrastigma hemsleyanum Diels et Gilg by using DNA mimic enzyme

A cloverleaf, simulating enzyme technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of no verification, time-consuming, etc., avoid gel electrophoresis analysis, and the method is simple , good stability

Active Publication Date: 2016-09-14
ZHEJIANG PHARMA COLLEGE
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  • Application Information

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Problems solved by technology

[0004] Chinese patent application 201310373809.4 discloses a method for identifying the medicinal plant Clover, but this method has false positive signals for some closely related species, and does n

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  • Method for efficiently and visually discriminating authenticity of Tetrastigma hemsleyanum Diels et Gilg by using DNA mimic enzyme
  • Method for efficiently and visually discriminating authenticity of Tetrastigma hemsleyanum Diels et Gilg by using DNA mimic enzyme

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Embodiment 1

[0026] Such as figure 1 As shown, the method for efficiently and visually identifying the authenticity of Clover clover provided by this embodiment includes the following steps:

[0027]Step A, DNA extraction of the genuine and counterfeit Clover: the plant DNA extraction CTAB method is used to extract the DNA of the genuine Clover and the fake Clover; wherein the genuine Clover comes from Zhejiang, Guangxi, Hunan and Jiangxi, etc. The fake Clover is selected from Tuyuaner. Specific steps are as follows:

[0028] (1) 10mL CTAB (cetyltrimethylammonium bromide) separation buffer (CTAB separation buffer is: 2% cetyltrimethylammonium bromide, 20mM ethylenediaminetetraacetic acid disodium salt , 100mM tris-hydrochloric acid (PH=8.0), 1.4M sodium chloride, 0.2% mercaptoethanol) into a 50mL centrifuge tube, and placed in a 60°C water bath to preheat.

[0029] (2) Take 2.0ɡ leaves, place them in a pre-cooled ceramic mortar, pour liquid nitrogen into them, and grind the leaves quick...

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Abstract

The invention discloses a method for efficiently and visually discriminating the authenticity of Tetrastigma hemsleyanum Diels et Gilg by using DNA mimic enzyme. The method comprises the following steps: 1, obtaining the authenticity difference sequence of Tetrastigma hemsleyanum Diels et Gilg, and obtaining an object-to-be-detected PCR product; and 2, carrying out strand displacement cycle signal amplification technology colorimetric visualization on the object-to-be-detected PCR product to discriminate the authenticity of Tetrastigma hemsleyanum Diels et Gilg: carrying out an enzyme digestion strand displacement cycle reaction to generate a large amount of G-rich sequences for forming G-quadruplexes in a system, and forming a G-quadruplex-hemin DNA mimic enzyme in the presence of K<+> and Hemin in order to realize visual discrimination of the authenticity of Tetrastigma hemsleyanum Diels et Gilg, wherein the enzyme has horseradish peroxidase-like activity and can catalyze color development of an ABTS<2->-H2O2 system. The method has the advantages of low cost, simple DNA cultivation technology, good reappearance and good stability, avoids gel electrophoresis analysis to save a lot of time and realize direct use of the method in rapid and visual discrimination of the authenticity of Tetrastigma hemsleyanum Diels et Gilg, and is hopeful to be widely applied in the medicinal material market and the food safety field.

Description

technical field [0001] The invention relates to the field of molecular markers, in particular to a method for efficiently and visually identifying the authenticity of Clover clover by using DNA imitating enzymes. Background technique [0002] Tetrastigma hemsleyanum Diels et Gilg (Tetrastigma hemsleyanum Diels et Gilg) is a perennial herbaceous plant of the genus Tetrastigma hemsleyanum Diels et Gilg. Or the whole herb is used as medicine. It is a commonly used anti-tumor drug. Its main active ingredient has "pro-apoptotic effect" on cell lines such as liver cancer, lung cancer, and blood cancer. , pharyngitis, hepatitis and viral meningitis, topical treatment of snake bites, tonsillitis, cellulitis, bruises. Due to the increasing demand for Clover in the market, excessive exploitation and environmental deterioration, its wild resources are on the verge of extinction and have been included in the "Zhejiang Province First Batch of Germplasm Resources Protection List". In r...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q1/6869C12Q1/6895C12Q2531/119
Inventor 彭昕陈宸杨瑞康李震邱彬
Owner ZHEJIANG PHARMA COLLEGE
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