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Radix bupleuri capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum and identification method

A technology of DNA fingerprinting and capillary electrophoresis, which is applied in the field of Bupleurum capillary electrophoresis DNA fingerprinting and identification, can solve the problems of reducing the objectivity of analysis results, poor repeatability and stability, and increasing false positive results, etc., to achieve easy automatic analysis and results Objective, Polymorphic Rich Effects

Inactive Publication Date: 2013-05-29
BEIHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] 1. Poor repeatability and stability;
[0011] 2. Although the amplification results are rich in polymorphisms, there is currently a lack of suitable detection methods;
[0012] 3. A large number of amplified bands make it difficult to analyze the results. At present, the commonly used method is to select representative amplified bands in the electrophoretic spectrum and identify them according to the presence or absence of these bands.
However, artificially selecting some amplified bands and discarding other bands reduces the objectivity of the analysis results and increases the probability of false positive results.

Method used

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  • Radix bupleuri capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum and identification method
  • Radix bupleuri capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum and identification method

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Establishment of DNA Fingerprint of Radix Bupleurum by Capillary Electrophoresis

[0035] 1. Bupleurum DNA Extraction

[0036] Take 0.05-0.1 g of Bupleurum (Bupleurum or Bupleurum angustifolia) as reference medicinal material, grind it thoroughly and transfer it to a centrifuge tube, add 2×CTAB (1.4mol / L NaCl, 100mmol / L Tris pH 8.0, 20mmol / L EDTA pH 8.0, 2% PVP, 2% CTAB, 3% β-mercaptoethanol) 200 μL, mix well, keep warm in a water bath at 65°C for 1 hour, and shake continuously during this period. Centrifuge at 12000r / min for 10min at 4°C. Aspirate the supernatant, add an equal volume of chloroform-isoamyl alcohol (24:1) to extract 2-3 times, and centrifuge at 10000r / min for 5min. Add 2 / 3 volume of absolute ethanol to the supernatant, mix well, and place at -20°C for 1 hour to precipitate nucleic acids. Centrifuge at 10,000 r / min for 5 min at 4°C, discard the supernatant to obtain DNA flocculent precipitate. Rinse the precipitate twice with 80% ethanol and absolute ...

Embodiment 2

[0056] Using the Bupleurum (Bupleurum) DNA fingerprint identification method of the present invention for the identification of a certain Bupleurum sample

[0057] Assuming that the existing batch of Bupleurum samples cannot be sure whether it is real Bupleurum, therefore, it is identified according to the following method.

[0058] After the Bupleurum DNA standard fingerprint is established, when a sample is to be identified, the mitochondrial DNA is first extracted by the CTAB method; the extracted DNA is used as a template, and the primers GTTACTCGAG and CCTGAATGCC are used for PCR amplification; the amplified product is Capillary electrophoresis analysis and record the electrophoresis pattern to obtain the DNA fingerprint of the batch of samples to be tested; use the similarity analysis software to compare the similarity between the DNA fingerprint of the sample to be tested and the standard DNA fingerprint of Bupleurum (Bupleurum) Identify the samples to be tested.

[00...

Embodiment 3

[0061] Using the Bupleurum (Bupleurum narrow leaf) DNA fingerprint identification method of the present invention for the identification of a certain Bupleurum narrow leaf sample

[0062] Assuming that the existing batch of Bupleurum angustifolia samples cannot be determined to be real Bupleurum angustifolia, therefore, construct the DNA fingerprints of these samples according to the method of the present invention, and then use the similarity analysis software to compare the DNA fingerprints of the sample to be tested with the The similarity of the DNA standard fingerprint of Bupleurum (Bupleurum angustifolia) is used to identify the samples to be tested according to the similarity.

[0063] Identification results: if the similarity between the two is greater than 95% (including 95%), this batch of samples is the real Bupleurum angustifolia;

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Abstract

The invention relates to the technical field of traditional Chinese medicine identification and particularly relates to a radix bupleuri capillary electrophoresis DNA (Deoxyribonucleic Acid) fingerprint spectrum and an identification method. The radix bupleuri capillary electrophoresis DNA fingerprint spectrum comprises extraction of DNA, PCR (Polymerase Chain Reaction) amplification, analysis of an amplified product, and establishment of the radix bupleuri capillary electrophoresis DNA fingerprint spectrum. The identification method of a radix bupleuri DNA fingerprint comprises the steps of: establishment of the radix bupleuri capillary electrophoresis DNA fingerprint spectrum, establishment of a sample DNA fingerprint spectrum and result identification by utilizing similarity software. The fingerprint spectrum and the identification method can be used for identifying a radix bupleuri sample. The fingerprint spectrum and the identification method can be used for carrying out authenticity identification on radix bupleuri from heredity essence; and the method is rapid, accurate and objective and can be used for identifying the radix bupleuri sample.

Description

technical field [0001] The invention relates to identification technology of Chinese medicinal materials, in particular to a capillary electrophoresis DNA fingerprint and identification method of Bupleurum chinensis. Background technique [0002] Bupleurum is the root or whole plant of Bupleurum chinense DC. and Bupleurum scorzonerifolium Willd., a perennial herb of Umbelliferae. The former is commonly called "Northern Bupleurum" and the latter is commonly called "South Bupleurum". Bupleurum bupleurum is a traditional precious Chinese herbal medicine in my country. It was first recorded in "Shen Nong's Materia Medica" and listed as top grade. "The Classic" says: Bupleurum "maintains confidantes and stomach stagnation, food accumulation, cold and heat pathogens, pushing old to bring about new." In modern times, it is used to disperse and reduce fever, soothe the liver and relieve stagnation, and promote sun and depression. Except for Hainan Province, Hong Kong and Macao Spe...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 苑广信李明成李洪宇王淼张丽华傅桂莲王冰梅
Owner BEIHUA UNIV
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