PCR identification method of Chinese medicament turtle-derived component

A technology of source components and identification methods, applied in the field of PCR identification of tortoise-derived components in traditional Chinese medicine, can solve the problems of poor practicability, less DNA content, cumbersome operations, etc., and achieve the effects of low cost, effective identification, and simple operation

Inactive Publication Date: 2010-09-22
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When dealing with samples with different degrees of deep processing, the spectroscopic analysis method needs to make corresponding adjustments to the detection parameters of the instrument. The operation is cumbersome and the practicability is poor.
Most of the reported detection methods based on DNA level are based on mitochondrial DNA, which has a limited copy number and is more suitable for PCR detection of samples with relatively complete DNA preservation or mild damage.
However, for tortoiseshell glue, which has undergone high temperature and high pressure for a long time, resulting in very little DNA content and extremely short deep-processed products, these methods are also slightly insufficient. It has been reported that the PCR method based on 12S rRNA gene cannot be used to identify tortoiseshell glue ( Hsieh HM, Huang LH, Tsai LC, Liu CL, Kuo YC, Hsiao CT, Linacre A, Lee JCI. Species identification of Kachuga tecta using the cytochrome b gene. J Forensic Sci. 2006, 51: 52-56)
In fact, so far, there has not been any effective and generally recognized method that can be used to identify the authenticity of tortoise shell glue at home and abroad.

Method used

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  • PCR identification method of Chinese medicament turtle-derived component
  • PCR identification method of Chinese medicament turtle-derived component
  • PCR identification method of Chinese medicament turtle-derived component

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Detection of turtle-derived components contained in various meat genomes

[0038] 1 Materials and Reagents

[0039] Materials: Genomic DNA extracts (1ng / μL) from various meats including turtles, donkeys, horses, cattle, and pigs.

[0040] 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).

[0041] 2 detection method

[0042] The genomic DNA extracts of various meats were taken for use, and PCR reactions were performed using primers designed according to the SINE sequence Pol III / SINE of Cryptocervica, 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:

[0043] Premix Ex-Taq TM Hot Start Version 12.5 μL, Primer Tortoise up 0.5 μL, Primer Tortoisedown 0.5 μL, Genomic DNA Extract 1 μL, Sterile do...

Embodiment 2

[0048] Embodiment 2 detects the tortoise-derived component contained in the glue class traditional Chinese medicine

[0049] 1 Materials and Reagents

[0050] Materials: tortoise shell glue (produced by Shandong Dong'e Co., Ltd.) DNA extraction solution (10ng / μL); donkey-hide gelatin (produced by Shandong Dong'e Co., Ltd.) DNA extraction solution (10ng / μL); yellow gelatin (Shandong Dong'e Co., Ltd. The research institute uses cowhide boiled) DNA extraction solution (10ng / μL); pig skin glue (Shandong Dong'a Co., Ltd. research institute uses pigskin boiled) DNA extraction solution (10ng / μL).

[0051] 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).

[0052] 2 detection method

[0053] Take various gel DNA extracts (10ng / μL) for use, and use primers designed according to the SINE sequence PolIII / SINE of Cryptocervica for P...

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Abstract

The invention provides a PCR identification method of a Chinese medicament turtle-derived component, which comprises the following steps: a, extracting DNA in a Chinese medicament; b, performing the PCR amplification of the DNA by using a specific primer pair designed according to the SINE sequence of a Cryptodira genome to obtain a amplified product; and c, subjecting the amplified product to electrophoresis, and judging if the DNA has an amplified positive amplified band according to the result of the electrophoresis, wherein if the DNA has the amplified positive amplified band, the Chinese medicament is derived from Cryptodira animals and has a turtle-derived component, and if the DNA has no amplified positive amplified band, the Chinese medicament is not derived from the Cryptodira animals and does not contain any turtle-derived component. The method of the invention is simple, flexible ad quick in operation and low in cost, can effectively identify turtle-derived products, particularly deeply processed tortoise-shell glue which has a very small amount of DNA and extremely short fragments due to deep processing. The method is suitable to be promoted and used in a large scale.

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 tortoise-derived components in traditional Chinese medicine, which is very suitable for deeply processed tortoise shell-derived products, especially for deep-processed tortoise shell-derived products. The tortoise shell glue, which has very little DNA content and very short fragments due to processing, is easy to operate and can be used for sensitive and rapid identification of counterfeit and shoddy Chinese herbal medicines in the market by product inspection departments. Background technique [0002] Tortoise shell, namely the carapace and plastron of the tortoise (Chinemys reevesii), belonging to the traditional Chinese medicine. According to the latest version of the Chinese Pharmacopoeia, taking tortoise shell can nourish y...

Claims

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Application Information

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Patent Type & Authority Applications(China)
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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