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Method for identifying Rhizoma Corydalis Repentis

A Corydal Corydalis, a specific technology, applied in the field of identification of whole-leaf Corydalis Corydalis, can solve the problems that have not yet been seen such as rapid identification of the authentic DNA of Corydalis Corydalis, and achieve the effect of solving the problem of mixing and using less samples

Inactive Publication Date: 2012-01-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] So far, there have been many reports on the research on the phytochemistry and medicinal value of Corydalis Corydalis in the world, but there has been no research report on the rapid identification of the authentic DNA of Corydalis Corydalis

Method used

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  • Method for identifying Rhizoma Corydalis Repentis
  • Method for identifying Rhizoma Corydalis Repentis
  • Method for identifying Rhizoma Corydalis Repentis

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Example 1: Preparation of whole-leaf Corydalis specific nucleotide sequence

[0041] 1. Genomic DNA Extraction

[0042] Leaf genomic DNA was extracted using the modified CTAB method (Doyle, 1991). Proceed as follows:

[0043] (1) Take about 0.05 grams of silica gel dried leaves, add a small amount of PVP powder, put it into the sample grinding tube, and grind it with BIO101 sample grinding machine, SPEED 4.0, TIME 20s.

[0044] (2) Quickly add 800-1000ul of 2×CTAB extraction buffer (containing 2% b-mercaptoethanol) preheated at 65°C, shake well and transfer to a 5ml centrifuge tube, then wash the grinding sample with 800-1000ul 2×CTAB tube, transferred to the same 5ml tube, 65. C water bath for 30 minutes.

[0045] (3) Cool to room temperature, add an equal volume of chloroform: isoamyl alcohol (24:1), mix well (slowly invert) for 15-30 minutes, and centrifuge at room temperature at 10,000 rpm for 10-15 minutes.

[0046] (4) Aspirate the upper aqueous phase, add 1 / 10...

Embodiment 2

[0060] Example 2: Preparation of whole-leaf Corydalis specific nucleic acid molecular probes Repens31 and Repens32

[0061] On the basis of obtaining the specific nucleotide sequence of the whole leaf Corydalis, utilize Primer Primer 5.0 (Vinay Singh, 1998) software to design, draw Repens31 and Repens32 (respectively 18-40bp shown in 1 in the sequence listing and The nucleotide composition and arrangement of 476-495bp) is a good oligonucleotide fragment for identifying the whole leaf Corydalis. According to the arrangement of the nucleotide composition of Repens31 and Repens32 (the nucleotide composition and arrangement shown in the 2 and 3 sequences in the sequence listing), it was synthesized on an automatic DNA synthesizer.

Embodiment 3

[0062] Embodiment 3: the identification of whole leaf Corydalis Corydalis (conventional PCR method)

[0063] 1. Extraction of DNA: The modified CTAB method was used to extract the total plant DNA.

[0064] 2. The PCR reaction system is:

[0065]

[0066] 3. PCR operation: take two 0.5 ml PCR tubes, add 22.5 microliters of PCR mixture according to step 2, then add 2.5 microliters of DNA to one tube, and 2.5 microliters of PCR mixture (control) to the other tube, and put On the PCR instrument, carry out the PCR reaction according to the following procedures:

[0067]

[0068] The results of PCR amplification were detected by electrophoresis on 1.5% agarose gel containing 0.1% EB. For electrophoresis results see figure 2 and image 3 . figure 2 It shows that there are bands around the molecular weight of 600bp in the whole leaf of Corydalis Corydalis, image 3 It shows that Corydalis has no bands around the molecular weight of 600bp. It shows that if Repens31 and R...

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Abstract

The invention discloses a nucleic acid probe and a method for identifying the nucleotide sequence of Corydalis repens, and belongs to the technical field of identification of Corydalis repens by using a molecular biological mehtod. The invention provides the Corydalis repens specific nucleotide sequence and the nucleic acid probe originated from the sequence, as well as the method for identifyingCorydalis repens with the probe. The method provided by the invention has the advantages of small sample needed for the whole operation, accurate identification and high sensitivity, provides an objective, rapid and accurate method for identifying the genuineness of the medical herb of Corydalis repens, and solves the puzzle of mixing of the Corydalis and Corydalis repens in the market of medicalherbs.

Description

technical field [0001] The invention belongs to the technical field of identifying whole-leaf Corydalis by means of molecular biology methods, and in particular relates to a nucleotide sequence, nucleic acid molecular probe and method for identifying whole-leaf Corydalis. technical background [0002] Corydalis yanhusuo is one of the famous traditional Chinese medicine "Eight Flavors of Zhejiang". At present, the authentic product of Corydalis yanhusuo refers to Corydalis yanhusuo. In addition, some other plants of the same genus also replace Corydalis yanhusuo in some areas, but the price and efficacy are the same as Corydalis yanhusuo. There is a big difference. Among them, whole-leaf Yanhusuo is the most common substitute and confusion in the raw material market. At present, there are varieties of confusion in the cultivation of Chinese herbal medicines, and there are quality problems in the raw material market. The scientific identification of varieties and the genetic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 陈川赵云鹏宗敏陈炳龙傅承新
Owner ZHEJIANG UNIV
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