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Method for identifying and evaluating cordyceps sinensis by using AFLP-DNA fingerprint spectrum

A technology of AFLP-DNA and Cordyceps sinensis, applied in the field of bioengineering, to reduce errors and improve reliability

Active Publication Date: 2016-09-07
NAVY MEDICINE RES INST OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional pharmacognosy identification methods and commonly used chemical analysis methods have major defects, so it is urgent to establish an accurate and efficient evaluation method at the molecular level

Method used

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  • Method for identifying and evaluating cordyceps sinensis by using AFLP-DNA fingerprint spectrum
  • Method for identifying and evaluating cordyceps sinensis by using AFLP-DNA fingerprint spectrum

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] 1. Prepare MseI-C, EcoRI-A, MseI-152, EcoRI-008 primers, the DNA sequences of which are as follows:

[0030] MseI-C: 5'-GATGAGTCCTGAGTAAC-CTT-3';

[0031] EcoRI-A: 5'-GACTGCGTACCAATTCA-AAC-3';

[0032] MseI-152: 5'-GATGAGTCCTGAGTAAC-CTC-3';

[0033] EcoRI-008: 5'-GACTGCGTACCAATT CA-ACG-3'.

[0034] 2. The identification method of Cordyceps sinensis is as follows:

[0035] (1) Obtaining DNA fragments: Take the Cordyceps standard substance (recognized as genuine Cordyceps sinensis) and the Cordyceps sinensis test sample to be tested to establish the enzyme digestion system mixture. The total volume of the enzyme digestion system mixture is 20 μL, including the Cordyceps standard substance Or Cordyceps sinensis sample genome 800ng, 10×NEBbuffer 2μL, EcoRI5.0U, MseI5.0U, 37°C heat preservation enzyme digestion, 80°C heat preservation 20min to stop the reaction, put it on ice immediately, and obtain the Cordyceps sinensis standard product and the Cordyceps sinensis test s...

Embodiment 2

[0041] 1. Prepare MseI-C, EcoRI-A, MseI-152, EcoRI-008 primers, the DNA sequences of which are as follows:

[0042] MseI-C: 5'-GATGAGTCCTGAGTAAC-CTT-3';

[0043] EcoRI-A: 5'-GACTGCGTACCAATTCA-AAC-3';

[0044] MseI-152: 5'-GATGAGTCCTGAGTAAC-CTC-3';

[0045] EcoRI-008: 5'-GACTGCGTACCAATT CA-ACG-3'.

[0046] 2. The identification method of Cordyceps sinensis is as follows:

[0047] (1) Obtaining DNA fragments: Take the Cordyceps standard substance (recognized as genuine Cordyceps sinensis) and the Cordyceps sinensis test sample to be tested to establish the enzyme digestion system mixture. The total volume of the enzyme digestion system mixture is 20 μL, including the Cordyceps standard substance Or Cordyceps to be tested sample genome 700ng, 10×NEBbuffer 1.5μL, EcoRI5.0U, MseI5.0U, 35°C for enzyme digestion, 85°C for 125min to stop the reaction, immediately put on ice to obtain Cordyceps standard and Cordyceps to be tested Genomic DNA fragments;

[0048] (2) Establish the l...

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Abstract

The invention belongs to the technical field of biological engineering, and in particular relates to a method for identifying and evaluating cordyceps sinensis by using an AFLP-DNA fingerprint spectrum. The method comprises the following steps: (1) acquiring DNA fragments, namely, performing dual-enzyme digestion on genomes of a standard cordyceps sinensis product and a cordyceps sinensis sample to be tested by using incision enzymes MseI and EcoRI, so as to obtain genome DNA fragments of the standard cordyceps sinensis product and the cordyceps sinensis sample to be tested; (2) establishing a connection system, namely, adding an EcoRI connector, an MseI connector and a T4DNA ligase into a DNA fragment product, so as to obtain a connection product; (3) performing pre-amplification, namely, diluting the connection product for 5-20 times, adding TaqDNA polymerase, a sample loading buffer, MgCl2, MseI-C and EcoRI-A, and performing pre-amplification on a PCR instrument; (4) performing selective amplification,namely diluting a pre-amplification product by using ddH2O, adding the TaqDNA polymerase, the sample loading buffer, MgC12, MseI-152 and EcoRI-008, and performing selective amplification on the PCR instrument. By adopting the method based on genetic mapping, true and fake cordyceps sinensis can be accurately distinguished.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for identifying and evaluating Cordyceps sinensis by using AFLP-DNA fingerprints. Background technique [0002] Cordyceps sinensis is a medicinal fungus of the genus Cordyceps of the Ergotaceae family that parasitizes on the larvae of the Lepidoptera insects Batmothidae, and has unique medicinal value. Cordyceps sinensis can inhibit the growth of Streptococcus, Bacillus anthracis, Bacillus anthracis and other bacteria, and it is also an anti-cancer active substance, which has a good regulating effect on the endocrine system and nervous system of the human body; Cordyceps acid can change the microcirculation of the human body, and has obvious effects Lowering blood fat and antitussive and expectorant effects; Cordyceps polysaccharide is an immunomodulator, which can enhance the body's resistance to viruses and parasites. However, at present, the qualit...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q2535/138C12Q2525/191C12Q2531/113
Inventor 张阵阵汪家春张淼储智勇栾洁谷爱梅李丹
Owner NAVY MEDICINE RES INST OF PLA