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Method for authenticating medicinal tetrastigma hemsleyanum Diels et Gilg

The technology of a medicinal plant, Clover, is applied in the direction of biochemical equipment and methods, and the determination/inspection of microorganisms. It can solve the problems of uneven quality, unseen DNA rapid identification technology, and mixed authenticity and high sensitivity. , high specificity, and accurate identification results

Inactive Publication Date: 2013-12-04
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current identification methods of Clover are mainly based on morphology (Huang Chengyong, 2002) and chemical composition (Li Yingqi et al., 2003), and even only rely on empirical judgment, and there is no research report on the establishment of DNA rapid identification technology using modern molecular biology methods.
Therefore, it is necessary to develop a real and reliable DNA molecular marker for rapid identification of Clover, to solve the problems of mixed authenticity and uneven quality in the medicinal material market.

Method used

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  • Method for authenticating medicinal tetrastigma hemsleyanum Diels et Gilg
  • Method for authenticating medicinal tetrastigma hemsleyanum Diels et Gilg
  • Method for authenticating medicinal tetrastigma hemsleyanum Diels et Gilg

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Experimental program
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Effect test

Embodiment 1

[0055] Embodiment 1: Preparation of cloverleaf specific nucleotide sequence

[0056] 1 Genomic DNA extraction

[0057] Genomic DNA of leaf or root samples was extracted by the modified PlantZol method. Proceed as follows:

[0058] (1) Take about 0.05g of dried silica gel leaves, add a small amount of PVP powder, put it into the sample grinding tube, and grind it with BIO101 sample grinding machine, SPEED 4.5, TIME 40s; or add about 0.1g of dry root tuber powder.

[0059] (2) Quickly add 800-1000ul of PlantZol extraction buffer (containing 2% b-mercaptoethanol) preheated at 65°C, and bathe in water at 65°C for 60 minutes.

[0060] (3) Cool to room temperature, add 1000ul of chloroform: isoamyl alcohol (24:1), mix well (slowly invert) for 5-10 minutes, and centrifuge at room temperature at 12000rpm for 5 minutes.

[0061] (4) Aspirate the upper aqueous phase, then add 1000ul of chloroform:isoamyl alcohol (24:1) for extraction, and repeat step (3).

[0062] (5) Aspirate t...

Embodiment 2

[0072] Example 2: Preparation of Clover (length) specific nucleic acid molecular probes Thk1 and Thk2

[0073] On the basis of obtaining the specific nucleotide sequence of Clover, use Primer Premier 5.0 (Vinary Singh, 1998) software to design, and obtain Thk1 and Thk2 (respectively 2 and 3 shown in in the sequence listing) Nucleotide composition and arrangement are good oligonucleotide fragments for the identification of Clover. According to the nucleotide composition arrangement of Thk1 and Thk2 (the nucleotide composition and arrangement shown in the 2 and 3 sequences in the sequence table), it was synthesized on an automatic DNA synthesizer.

Embodiment 3

[0074] Example 3: Identification of Clover (conventional PCR method)

[0075] 1. Extraction of DNA: Genomic DNA of leaves or tubers was extracted by the improved PlantZol method.

[0076] 2. The PCR reaction system is shown in Table 2:

[0077]

[0078] 3. PCR operation: Take two 0.5 ml PCR tubes, add 24 microliters of PCR mixture according to step 2, then add 1 microliter of DNA to one tube, and 1 microliter of PCR mixture (control) to the other tube, and put them in the PCR tube. On the instrument, carry out the PCR reaction according to the following procedures:

[0079]

[0080] Keep warm at 4°C.

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Abstract

The invention provides a method for authenticating medicinal tetrastigma hemsleyanum Diels et Gilg. According to the method for authenticating the medicinal tetrastigma hemsleyanum Diels et Gilg, the medicinal tetrastigma hemsleyanum Diels et Gilg is authenticated through the specificity nucleotide sequence of the tetrastigma hemsleyanum Diels et Gilg, specific primers based on the sequence of the tetrastigma hemsleyanum Diels et Gilg and the PCR technology. The primers designed through the method have high specificity, can increase by about 1800bp strips in tetrastigma hemsleyanum Diels et Gilg with twelve provenances, and cannot react with the DNA of sixteen affinis species and tetrastigma hemsleyanum Diels et Gilg fake product circular soil, and therefore the tetrastigma hemsleyanum Diels et Gilg can be fast and accurately authenticated. The method for authenticating the medicinal tetrastigma hemsleyanum Diels et Gilg can achieve the whole authentication operation with a small number of samples, the authentication result is accurate, and sensitivity is high. The method for authenticating the medicinal tetrastigma hemsleyanum Diels et Gilg is objective, fast and accurate in authentication for the medicinal tetrastigma hemsleyanum Diels et Gilg, and solves the problem that consumers are deceived by counterfeit tetrastigma hemsleyanum Diels et Gilg in the medicine market.

Description

technical field [0001] The invention belongs to the technical field of traditional Chinese medicine identification, and in particular relates to a method for identifying trilobites by using nucleotide sequences and polymerase chain reaction primers. Background technique [0002] Clover ( Tetrastigmahemsleyanum Diels et Gilg), also known as clover clover, is a rare medicinal plant unique to China. It is distributed in the south of the Yangtze River in China and is a commonly used Chinese herbal medicine (Flora of China, 1998). Its tuber root is used as medicine with various functions such as immune regulation, liver protection, anti-tumor, anti-virus, anti-hemorrhagic fever, anti-inflammatory, analgesic and antipyretic. [0003] In recent years, the anti-tumor effect of cloverleaf has been increasingly recognized by scientific research results, the demand for clinical and folk medicine is huge, and the price is rising steadily. However, the supply of Clover basically depen...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 王一涵陈川姜维梅傅承新
Owner ZHEJIANG UNIV
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