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PCR identification method for equine derived material in traditional Chinese medicament

An identification method, a technology of traditional Chinese medicine, applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of low DNA content and short fragments, and achieve the effect of simple operation, moderate size and good specificity

Active Publication Date: 2010-08-25
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Colloidal traditional Chinese medicines are mostly processed under high temperature and pressure for a long time, and their DNA content is very small, and the fragments are very short. Traditional molecular markers are difficult to meet the detection needs of such deep-processed products.

Method used

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  • PCR identification method for equine derived material in traditional Chinese medicament
  • PCR identification method for equine derived material in traditional Chinese medicament
  • PCR identification method for equine derived material in traditional Chinese medicament

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Detection of horse-derived components contained in various meat genomes

[0040] 1 Materials and Reagents

[0041] Materials: Genomic DNA extraction solution (1ng / μL) of various meats including donkey, horse, cow, pig, turtle and deer.

[0042] Reagent: PCR premix enzyme Premix Ex-Taq TM Hot Start Version: (purchased from TAKARA, Cat.No.DRR030A); DNA fluorescent dye 10000×GelRed TM : DNA fluorescent dye (purchased from Biotium, Cat. No. 41000).

[0043] 2 detection method

[0044] The genomic DNA extracts of various meats were taken for use, and PCR reactions were carried out using primers designed based on satellite DNA sequences specific to horse germ-derived components, and the primers were dissolved in sterile water to a concentration of 10 μM for use. The specific loading conditions of the 25 μL reaction system are as follows:

[0045] Premix Ex-Taq TM Hot Start Version 12.5 μL, Primer Horse up 0.5 μL, Primer Horse down 0.5 μL, Genomic DNA Extract...

Embodiment 2

[0050] Embodiment 2 detects the horse germ-derived components contained in the gelatinous traditional Chinese medicine

[0051] 1 Materials and Reagents

[0052] Materials: donkey-hide gelatin (produced by Shandong Dong'e Co., Ltd.) DNA extraction solution (10ng / μL); horsehide glue (Shandong Dong'e Co., Ltd. Institute uses horse hide to boil) DNA extraction solution (10ng / μL); yellow gelatin (Shandong Dong'e Co., Ltd. Research Institute uses cowhide to boil) DNA extraction solution (10ng / μL); pig skin glue (Shandong Dong'e Co., Ltd. Research Institute uses pig skin to boil) DNA extraction solution (10ng / μL).

[0053] Reagent: PCR premix enzyme Premix Ex-Taq TM Hot Start Version: (purchased from TAKARA, Cat.No.DRR030A); DNA fluorescent dye 10000×GelRed TM : DNA fluorescent dye (purchased from Biotium, Cat. No. 41000).

[0054] 2 detection method

[0055] Take various gel DNA extracts (10ng / μL) for use, and use primers designed according to the horse breed-specific satellit...

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Abstract

The invention discloses a PCR identification method for an equine derived constituent in a traditional Chinese medicament, which comprises the following steps of : a), extracting DNA from the traditional Chinese medicament; b), performing PCR amplification on the DNA with an equine derived special primer pair designed according to a satellite sequence with equine specificity to form an amplified product; and c), performing electrophresis on the amplified product, judging whether a positive amplified strip is amplified on the DNA or not according to an electrophresis result, wherein if the positive amplified strip is amplified on the DNA, the traditional Chinese medicament is derived from an equine animal and contains equine derived material, and if the positive amplified strip is not amplified on the DNA, the traditional Chinese medicament is not derived from the equine animal and does not contain any equine derived constituents. The method of the invention is simple, sensitive and rapid in operation and low in cost, can effectively identify an equine derived product, is particularly suitable for the identification of a deeply processed horsehide gelatin which has low DNA content and very short fragment caused by deep processing, and is suitable for massive popularization and application.

Description

technical field [0001] The present invention relates to the technical field of traditional Chinese medicine, more specifically, to the technical field of identification of traditional Chinese medicine, in particular to a PCR identification method for horse-derived ingredients in traditional Chinese medicine, which is very suitable for deeply processed horse-derived products, especially for Due to deep processing, horsehide glue with very little DNA content and very short fragments is easy to operate and can be used for sensitive and rapid identification of fake and shoddy Chinese herbal medicines in the market by product inspection departments. Background technique [0002] my country's traditional Chinese medicine is extensive and profound, and there are many kinds of substances that can be used as Chinese medicine. One of them is glue-like Chinese medicine made from animal skin and bone, including donkey-hide gelatin, tortoise shell glue, and deer horn glue. These gelatino...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 周祥山张元兴吕品秦玉峰尤金花田守生
Owner EAST CHINA UNIV OF SCI & TECH
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