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PCR determination method for Chinese medicine or Chinese medicinal crops derived from eukaryote

An identification method and eukaryotic technology, which is applied in the field of PCR identification of traditional Chinese medicine and Chinese medicinal materials derived from eukaryotic organisms, can solve the problems of short fragments, DNA damage, difficult amplification, etc., and achieve low cost, effective identification, Simple operation effect

Active Publication Date: 2011-01-19
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There are only a few thousand copies of rDNA and mtDNA in a single cell. When the DNA content of the sample is abundant and relatively complete, it is very simple and effective to use these sequences to construct detection methods. However, gelatinous substances are a kind of deep processing The product, its DNA damage is extremely serious, its content is extremely small, and its fragments are extremely short. For gelatinous traditional Chinese medicines that have severely damaged DNA due to deep processing and have extremely low content, it is difficult to establish a PCR method based on these two sequences. Clear bands are amplified from the gel-like traditional Chinese medicine DNA extract solution. The actual test results show that if primers are designed according to mtDNA, it is indeed difficult to amplify clear bands from the gel-like material DNA, and they are not suitable for Detection of Deeply Processed Traditional Chinese Medicine
Therefore, only relying on these two commonly used sequences in species analysis cannot meet the needs of colloid detection in practical applications.

Method used

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  • PCR determination method for Chinese medicine or Chinese medicinal crops derived from eukaryote
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  • PCR determination method for Chinese medicine or Chinese medicinal crops derived from eukaryote

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Detection of Donkey-derived Components Contained in Colloidal Chinese Medicines

[0053] 1 Materials and Reagents

[0054] Materials: Donkey-hide gelatin (produced by Shandong Dong'e Co., Ltd.) DNA extraction solution (diluted to 10 ng / μL and 1 ng / μL for use); yellow gelatin (Shandong Dong'e Co., Ltd. Institute uses cowhide boiled) DNA extraction solution (diluted to 10ng / μL and 1ng / μL for use); pig skin glue (made from pigskin in Shandong Dong’a Co., Ltd. Research Institute) DNA extract (diluted to 10ng / μL and 1ng / μL for use); donkey used as a positive control , horse, cow, pig genomic DNA extract (1ng / μL).

[0055] Reagent: PCR premix enzyme Premix Taq (Ex Taq TM Version): (purchased from TAKARA, Cat.No.DRR033A); DNA fluorescent dye 10000×GelRed TM : DNA fluorescent dye (purchased from Biotium, Cat. No. 41000).

[0056] 2 detection method

[0057] Take various gel DNA extracts (10ng / μL) and various meat genome DNA extracts for use, use primers design...

Embodiment 2

[0064] Embodiment 2 detects the bovine-derived components contained in the gelatinous traditional Chinese medicine

[0065] 1 Materials and Reagents

[0066]Materials: Donkey-hide gelatin (produced by Shandong Dong'e Co., Ltd.) DNA extraction solution (diluted to 10 ng / μL and 1 ng / μL for use); yellow gelatin (Shandong Dong'e Co., Ltd. Institute uses cowhide boiled) DNA extraction solution (diluted to 10ng / μL and 1ng / μL for use); pig skin glue (made from pigskin in Shandong Dong’a Co., Ltd. Research Institute) DNA extract (diluted to 10ng / μL and 1ng / μL for use); donkey used as a positive control , horse, cow, pig genomic DNA extract (1ng / μL).

[0067] Reagent: PCR premix enzyme Premix Taq (Ex Taq TM Version): (purchased from TAKARA, Cat.No.DRR033A); DNA fluorescent dye 10000×GelRed TM : DNA fluorescent dye (purchased from Biotium, Cat. No. 41000).

[0068] 2 detection method

[0069] Take various gel DNA extracts (1ng / μL) and various meat genome DNA extracts for use, us...

Embodiment 3

[0075] see results figure 2 , because the primers are designed according to the SINE sequence of ruminants, lane 4 has a positive band, and lane 7 is yellow gelatin, which is boiled from cowhide, so it also has a positive band. It can be seen that although the DNA content obtained from glue-like traditional Chinese medicine is very small , using the primers designed for the SINE sequence of ruminants, the bovine-derived components mixed in glue products can be detected by PCR, but no positive bands are amplified in other samples, indicating that there is no mixed cowhide in donkey-hide gelatin. Since the pair of primers is designed based on the SINE sequence of ruminants, this method can also be used to detect whether other ruminant-derived components such as sheep are mixed in glue products. Embodiment 3 detects the porcine origin component contained in the gelatinous traditional Chinese medicine

[0076] 1 Materials and Reagents

[0077] Materials: Donkey-hide gelatin (pr...

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PUM

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Abstract

The invention provides a PCR identification method derived from eucaryote for Chinese traditional medicine and traditional Chinese medicinal materials. PCR augmentation is processed on DNA samples to obtain augmentation production according to a species-specific primer, wherein, the species-specific primer is designed according to a SINE sequence of eukaryotic genomes. The DNA samples are extracted from the Chinese traditional medicine and traditional Chinese medicinal materials, in particular from the further processing type Chinese traditional medicine. Species categories of eukaryotic species in the further processing type Chinese traditional medicine are estimated by analyzing the augmentation production, and the truth of the identified samples is further estimated. The PCR identification method is suitable for Chinese traditional medicine and traditional Chinese medicinal materials, is particularly suitable for the processing type Chinese traditional medicine, especially for the Chinese traditional medicine which is with extremely little DNA content and extremely short segment due to the further processing. The identification method has the advantages of simple operation, rapidness and sensitivity, low cost and effective truth identification of the Chinese traditional medicine and the traditional Chinese medicinal materials.

Description

technical field [0001] The present invention relates to the technical field of identification of traditional Chinese medicine and Chinese medicinal materials, more specifically, relates to the technical field of identification of traditional Chinese medicine and Chinese medicinal materials derived from eukaryotic organisms, especially refers to the PCR identification method of traditional Chinese medicine and Chinese medicinal materials derived from eukaryotic organisms, very It is suitable for deep processing of traditional Chinese medicine, especially the gelatinous traditional Chinese medicine refined from animal skin and bone, as well as the single medicine in the compound Chinese medicine, unearthed Chinese medicinal materials, etc. Sensitive and rapid identification of Chinese herbal medicines. 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. O...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 周祥山张元兴吕品杨继忠秦玉峰尤金花田守生
Owner EAST CHINA UNIV OF SCI & TECH
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