Method for identifying composition species of mixed traditional Chinese medicine powder by utilizing denaturing gradient gel electrophoresis (DGGE)

A technology of traditional Chinese medicine powder and species, which is applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of high price, high technical threshold, and difficulty in popularization and use, so as to reduce experimental costs, lower technical thresholds, repeat sex high effect

Inactive Publication Date: 2016-12-07
ZHONGSHAN ZHONGZHI PHARMA GRP
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AI Technical Summary

Problems solved by technology

However, its technical defect is that DNA barcoding is only suitable for the genetic identification of single Chinese medicines, and cannot analyze materials mixed with multiple species, such as Chinese medicine powder mixtures
Technical defects: second-generation sequencing requires the use of high-end sequencers, and the data obtained from sequencing requires professional kno...

Method used

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  • Method for identifying composition species of mixed traditional Chinese medicine powder by utilizing denaturing gradient gel electrophoresis (DGGE)
  • Method for identifying composition species of mixed traditional Chinese medicine powder by utilizing denaturing gradient gel electrophoresis (DGGE)
  • Method for identifying composition species of mixed traditional Chinese medicine powder by utilizing denaturing gradient gel electrophoresis (DGGE)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Identification of Yupingfeng powder prescription mixed traditional Chinese medicine powder

[0031] 1 Select and prepare samples

[0032] Taking Yupingfeng powder (drug composition: Astragalus, Fangfeng, Atractylodes macrocephala) as an example, each medicinal material is separately crushed into fine powder, mixed with equal weight and uniform, and used as an experimental sample.

[0033] 2 Genomic DNA extraction (modified CTAB method)

[0034] Weigh 100 mg of the sample, put it into a 2.0 mL centrifuge tube, add 2 Φ5 mm sterilized steel balls, shake it with a biological sample homogenizer at 6.0 m / s for 30 s, and repeat the shaking twice with an interval of 30 s. Add 1000 μL of CTAB free buffer (100 mM Tris-HCl, 20 mM EDTA, 1.4 M NaCl) to the centrifuge tube, shake vigorously, rotate and mix for 5 min, centrifuge at 1,3000 rpm for 3 min, discard the supernatant; repeat this step 1-2 times. Add 800 μL of CTAB buffer solution (3% CTAB, 100 mM Tris-HCl, 20 mM...

Embodiment 2

[0051] Example 2: Identification of Ginseng, Panax notoginseng, Radix Ginseng and Platycodon grandiflora mixed Chinese medicine powder (using ITS2 as a common primer)

[0052] 1. Select and prepare samples

[0053] Taking ginseng, Panax notoginseng, Radix Ginseng and Platycodon grandiflora as examples, each herb was separately crushed into fine powder, mixed with equal weights and evenly used as experimental samples.

[0054] 2. Genomic DNA extraction (modified CTAB method)

[0055] Weigh 100 mg of the sample, put it into a 2.0 mL centrifuge tube, add 2 Φ5 mm sterilized steel balls, shake it with a biological sample homogenizer at 6.0 m / s for 30 s, and repeat the shaking twice with an interval of 30 s. Add 1000 μL of CTAB free buffer (100 mM Tris-HCl, 20 mM EDTA, 1.4 M NaCl) to the centrifuge tube, shake vigorously, rotate and mix for 5 min, centrifuge at 1,3000 rpm for 3 min, discard the supernatant; repeat this step 1-2 times. Add 800 μL of CTAB buffer solution (3% CTAB, 1...

Embodiment 3

[0072] Example 3: Identification of Ginseng, Panax notoginseng, Radix Ginseng and Platycodon grandiflora mixed Chinese medicine powder (using trnL as a common primer)

[0073]The primers in Example 2 were replaced by trnL, and the PCR amplification system was: 12.5 μL of Taq DNase master mix, primer GCtrnL_F (5`-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGCGAAATCGGTAGACGCTACG-3`) and reverse primer: trnL_R (5`-CCATTGAGTCCTGCACCTATC-3` ) each 1.0 μL, genomic DNA obtained in step 2 2.0 μL, ddH 2 O to make up the volume to 25.0 μL. Reaction program: 95°C, 2min; 95°C, 30s, 50°C, 30s, 72°C, 50s, 40 cycles; 72°C, 5min. The resulting electrophoresis of the first round of PCR amplification products is as follows: Figure 7 Shown; DGGE analysis results are shown in Figure 8 Shown; The electrophoresis of the second round of PCR amplification products is shown in Figure 9 shown. The results were all 100% similarity by gene bank comparison.

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Abstract

The invention relates to identification of composition species of mixed traditional Chinese medicine powder. A technology comprises the steps of extracting genome DNAs of the mixed traditional Chinese medicine powder, and using a specific primer modified by a specific GC clamp structure to perform polymerase chain reaction (PCR) amplification; causing PCR amplification products to undergo electrophoretic separation and dyeing in denaturant gel, performing denaturing gradient gel electrophoresis (DGGE) analysis to obtain DGGE denaturing gel electrophoresis bands; cutting a single DNA band, recycling DNAs, and performing second round PCR amplification; causing an obtained DNA sequence to undergo sequence splicing and cutting, and submitting the DNA sequence to a public database to perform sequence alignment, and taking highest sequence similarity species as identification results, so as to distinguish species composition of the mixed traditional Chinese medicine powder. According to the method, DNA bar codes and a DGGE technology are combined, the shortcoming that the DNA bar codes cannot be used for identifying the mixed traditional Chinese medicine powder is overcome, and identification of composition species of the mixed traditional Chinese medicine powder or a compound preparation and traditional Chinese medicine adulteration identification are performed effectively. The method has the advantages of being quick, accurate, high in repeatability and low in cost and the like.

Description

technical field [0001] The invention relates to the gene identification technology of mixed traditional Chinese medicine powder. Especially the species analysis of herbal mixed traditional Chinese medicine powder. Background technique [0002] At present, in addition to the traditional appearance and physical and chemical identification methods, the identification methods of Chinese herbal medicines also include DNA barcode identification methods. This technology can identify species through a recognized and relatively short DNA sequence in the genome, which is fast, accurate and repeatable. It has high advantages and is widely used in the species identification of medicinal plants. After more than ten years of accumulation, the Institute of Traditional Chinese Medicine of the China Academy of Traditional Chinese Medicine has completed the DNA barcode research of more than 8,000 kinds of Chinese herbal medicines and their counterfeit products, and created the "Chinese herba...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 郑夏生陈士林成金乐赖智填
Owner ZHONGSHAN ZHONGZHI PHARMA GRP
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