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Rapid molecular identification method for glycyrrhiza uralensis, glycyrrhiza glabra, glycyrrhiza inflate and hybrid variety thereof

A technology of Ural licorice and licorice flavonoids, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of lack of accurate and efficient molecular identification methods, and achieve the effect of accurate identification

Active Publication Date: 2016-05-04
PEKING UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, for the three important medicinal varieties of licorice, there is still a lack of accurate and efficient molecular identification methods

Method used

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  • Rapid molecular identification method for glycyrrhiza uralensis, glycyrrhiza glabra, glycyrrhiza inflate and hybrid variety thereof
  • Rapid molecular identification method for glycyrrhiza uralensis, glycyrrhiza glabra, glycyrrhiza inflate and hybrid variety thereof
  • Rapid molecular identification method for glycyrrhiza uralensis, glycyrrhiza glabra, glycyrrhiza inflate and hybrid variety thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1, species identification of wild licorice samples.

[0025] 1) Collect 56 samples of wild licorice (all dry roots) from several main production areas of licorice in China. The specific information of the samples is shown in Table 1. The total DNA of the samples was extracted with a plant genomic DNA extraction kit (Tiangen Biochemical Technology Co., Ltd., Beijing), and the specific steps were as follows:

[0026] ① Take 50mg of medicinal material powder in a 1.5mLEP tube, add liquid nitrogen and a small amount of quartz sand, and grind thoroughly with a grinding rod;

[0027] ② Add 700 μL of buffer solution GP1 (containing 0.5% mercaptoethanol) preheated at 65°C to the ground medicinal material powder, mix quickly, and bathe in water at 65°C for 40 minutes;

[0028] ③ Add 700 μL of chloroform, mix thoroughly, and centrifuge at 12000 rpm for 5 min;

[0029] ④ Transfer the aqueous phase to a new centrifuge tube, add 700 μL buffer GP2, and mix well;

[0030...

Embodiment 2

[0046] Embodiment 2, identification of the variety of Radix Glycyrrhiza decoction pieces.

[0047] The experimental method was basically the same as in Example 1, except that the samples were changed from wild licorice samples to commercially available licorice pieces, including 31 batches of raw licorice pieces and 2 batches of processed products (honey roasted). Glycyrrhizae decoction pieces were ground under liquid nitrogen to extract DNA, and PCR amplification and sequence analysis were performed. Table 3 shows the information and identification results of licorice decoction pieces. The results showed that the method could distinguish and identify the source varieties of licorice commercial decoction pieces. The above four SNP sites can be detected in the amplified ITS sequence, and one SNP site can be detected in the ndhC-trnV transcription spacer. Based on this, the identification results of each batch of samples are obtained. According to the determination results, t...

Embodiment 3

[0050] Embodiment 3, the variety identification of Radix Glycyrrhizae seed and seedling.

[0051] The experimental method is basically the same as in Example 1, except that the sample is changed from dried roots of licorice to licorice seeds and germinated licorice seedlings. The results showed that the method can be used for species identification of licorice seeds and seedlings.

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Abstract

The invention discloses a rapid molecular identification method for glycyrrhiza uralensis, glycyrrhiza glabra, glycyrrhiza inflate and a hybrid variety thereof. The rapid molecular identification method comprises the following steps: 1) extracting DNA of a sample; 2) amplifying ITS sequences of the sample by using glycyrrhiza ITS specific primers; 3) determining the basic groups at the 159th locus and the 383th to 385th loci from the 5'end of the ITS sequence: if the 159th locus is C, and the 383th to 385th loci are TGC, then identifying the sample to be the glycyrrhiza uralensis; if the 159th locus has the coexistence of C and T, and the 383th to 385th loci have the coexistence TGC and CAA, then identifying the sample to be the hybrid variety; if the 159th locus is T, and the 383th to 385th loci are CAA, then performing a step 4); 4) performing PCR amplification on the ndhC-trnV internal transcribed spacer sequence of the sample; 5) determining the basic group of the 487th locus from the 5' end of the ndhC-trnV internal transcribed spacer sequence: if the 487th locus is A, then identifying the sample to be the glycyrrhiza glabra; if the 487th locus is T, then identifying the sample to be the glycyrrhiza inflate. According to the rapid molecular identification method, original plants, medicinal materials, seeds, seedlings and the like of glycyrrhiza can be identified accurately; the problems about variety identification, breeding cultivation, germplasm resource development and utilization and the like are effectively solved.

Description

technical field [0001] The invention belongs to the technical field of identification of varieties of Chinese medicinal materials, and specifically relates to the identification of licorice Ural, licorice inflate, licorice glabra, and licorice ural and licorice inflate or glabra with specific single nucleotide polymorphism (singlenucleotidepolymorphism, SNP) sites Samples produced after licorice hybridization were accurately identified and differentiated. Background technique [0002] As one of the most widely used medicinal plants, licorice is recorded in Chinese, American, British, European, and Japanese pharmacopoeias. The 2015 edition of "Chinese Pharmacopoeia" also recorded the dried roots and rhizomes of Glycyrrhizauralensis Fisch., G. glabra L., and G. inflata Bat. as the source of licorice medicinal materials. The main products in my country For ural licorice. However, the distribution of different varieties of licorice varies in different countries and regions. For...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/156
Inventor 叶敏宋玮冯金季帅乔雪王瑛
Owner PEKING UNIV
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