A kind of identification method of bletilla striata

A technology of Bletilla striata and Bletilla striata, which is applied to biochemical equipment and methods, measurement/inspection of microorganisms, etc., can solve problems such as low drying rate, wild resources of Bletilla striata that cannot meet market demand, and difficulty in distinguishing Bletilla striata purple or Bletilla striata yellow, etc. To achieve the effect of effective identification method

Active Publication Date: 2021-01-26
GUIZHOU MEDICAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, the wild resources of Bletilla striata are far from meeting the market demand, and the price is gradually rising.
[0004] Although several species of Bletilla striata can be used as Chinese medicinal materials, the 2015 edition of "Chinese Pharmacopoeia" stipulates that the Chinese medicinal material Bletilla striata is the dried tuber of the orchid plant Bletilla striata, that is, Bletilla striata, and it is difficult to distinguish it from the dried tuber. It is still Bletilla striata, plus the tuber cell tissue of Bletilla striata is relatively loose, and the drying rate is relatively low
Therefore, the development of identification methods that can identify Bletilla striata and Bletilla striata is very important to ensure the quality of medicinal materials, but there is no relevant literature report

Method used

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  • A kind of identification method of bletilla striata

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] A method for identifying Bletilla striata, comprising the following steps:

[0023] a. Select 3 Bletilla striata strains and 3 Bletilla striata strains with the same year, same growth conditions and same plant size, and use the CTAB method to extract individual genomic DNA respectively; then use amplification primers DUAN5: 5'-TAGTAACGGCGAGCGAAC-3', DUNA65 '-GGCATAGTTCACCATCTTTC-3' was amplified by PCR, and the size of the amplified product was 760bp; the amplification conditions were pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 58°C for 45 s, and extension at 72°C for 1 min , according to this cycle 30 times, and finally extended at a temperature of 72°C for 10 min, and stored at a temperature of 4°C; the initial reaction system for PCR amplification is: 30 μL system containing 6 μL of 5×Taq buffer, 2.4 μL of dNTPs, 1 μL of each primer, and 1 μL of primers. Final concentration 0.4μM, template 1μL, template final concentration about 6...

Embodiment 2

[0028] A method for identifying Bletilla striata, comprising the following steps:

[0029] a. Select 60 Bletilla striata individuals with the same planting time, including 2 Bletilla striata chrysalis;

[0030] b. Using the CTAB method to extract 60 individual genomic DNAs respectively;

[0031] c. Use amplification primers DUAN5: 5'-TAGTAACGGCGAGCGAAC-3' and JDHH: 5'-TTATCAGCTTCCTTGCGCCCG-3' for PCR amplification; PCR amplification conditions are: pre-denaturation temperature 94°C, time 5min; denaturation temperature 94°C, Time 1min; annealing temperature 54°C, time 45s; extension temperature 72°C, time 1min; press this cycle 30 times, finally extend at 72°C for 10 minutes, store in refrigerator at 4°C; PCR amplification system is 30μL system: which contains 5×Taqbuffer 6μL, dNTPs 2.4μL, upstream and downstream primers 1μL each, primer final concentration 0.4μM, template 1μL, template final concentration about 60ng, Taq enzyme 0.15μL, add ddH 2 0 to 30 μL;

[0032] d. Perf...

Embodiment 3

[0034] A method for identifying Bletilla striata, comprising the following steps:

[0035] a. Select 100 Bletilla striata individuals with the same planting time, including 5 Bletilla striata chrysalis;

[0036] b. Using the CTAB method to extract 100 individual genomic DNAs respectively;

[0037]c. Use amplification primers DUAN5: 5'-TAGTAACGGCGAGCGAAC-3' and JDHH: 5'-TTATCAGCTTCCTTGCGCCCG-3' for PCR amplification; PCR amplification conditions are: pre-denaturation temperature 94°C, time 5min; denaturation temperature 94°C, Time 1min; annealing temperature 54°C, time 45s; extension temperature 72°C, time 1min; press this cycle 30 times, finally extend at 72°C for 10 minutes, store in refrigerator at 4°C; PCR amplification system is 30μL system: which contains 5×Taqbuffer 6μL, dNTPs 2.4μL, upstream and downstream primers 1μL each, primer final concentration 0.4μM, template 1μL, template final concentration about 60ng, Taq enzyme 0.15μL, add ddH 2 0 to 30 μL;

[0038] d. Car...

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Abstract

The invention discloses a method for identifying bletilla striata, which belongs to the technical field of identification of traditional Chinese medicinal materials. The method comprises the following steps: selecting a batch of different types of bletilla striata and bletilla striata, using the CTAB method to extract individual genome DNA of bletilla striata, and then designing The primers are used to amplify, clone, sequence and compare the DNA sequence of the D1-D3 region of the Bletilla striata ribosomal large subunit gene to determine the SNP site to be tested; then design specific PCR amplification primers for the SNP site to be tested Carry out PCR amplification, verify the SNP site, and obtain standard data; finally, use PCR amplification primers to amplify the DNA of the sample to be tested, and determine whether the target band of the amplified product is in the PCR amplification electrophoresis. The genotype of the SNP site of the test sample is compared with the standard data, so as to determine whether the sample to be tested is Bletilla striata yellow or Bletilla striata purple.

Description

technical field [0001] The invention relates to the technical field of authenticity identification of Chinese medicinal materials, in particular to an identification method of Bletilla striata chrysalis. Background technique [0002] Bletilla striata is a species of Orchidaceae, Bletilla striata. According to the records of "Flora of China", there are about 6 species of bletilla striata in the world, which are distributed in the northern part of Myanmar in Asia, through my country to Japan. There are 4 kinds of Bletilla striata produced in China, namely Bletilla striata, Bletilla striata North China, Bletilla striata small and Bletilla striata yellow. The Bletilla striata varieties currently cultivated in my country include Bletilla striata purple, Bletilla striata yellow and a small amount of Bletilla striata. [0003] Bletilla striata is used medicinally from dried tubers, which has the effects of astringent and hemostasis, clearing away heat and promoting dampness, reduc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895
CPCC12Q1/686C12Q1/6895C12Q2600/156C12Q2565/125
Inventor 刘红美张洁刘显义张婷婷曾丽娜
Owner GUIZHOU MEDICAL UNIV
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