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Method for identifying Catha edulis based on micro molecular marker and application

A molecular marker, chaat grass technology, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. Accurate identification of species, etc., to achieve the effect of high sensitivity, good specificity, and improved success rate

Active Publication Date: 2020-05-12
NANJING FOREST POLICE COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Usually illegally smuggled into the country are the dried leaves of khat grass (such as figure 1 As shown), such leaves are specially baked and dried. If the shape is complete, the species can be identified through traditional morphological identification. However, in order to increase the difficulty of identification, criminal suspects usually smash the leaves (such as image 3 As shown), the morphology cannot be identified at all, so DNA molecular identification is the best way to identify its species
However, at present, there is no universal DNA barcode for plant molecular identification like animals, and each plant group has its own particularity. Even the relatively common molecular markers such as mat K and rbc L, because their fragment lengths are all greater than 700bp, are suitable for Such baked branch and leaf fragments are not suitable for samples with a large amount of DNA degradation, and the target fragments often cannot be successfully amplified.
This also seriously hinders the accurate identification of its species, which brings troubles to the crackdown on drug original plant smuggling crimes

Method used

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  • Method for identifying Catha edulis based on micro molecular marker and application
  • Method for identifying Catha edulis based on micro molecular marker and application
  • Method for identifying Catha edulis based on micro molecular marker and application

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Experimental program
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Embodiment 1

[0063] Development of miniature molecular markers for chhatella species identification based on the chloroplast genome

[0064] 1.1 Acquisition of chloroplast genome sequence

[0065] The chloroplast genome data of khat grass were retrieved from Genbank, and the nucleotide sequences of its mat K, rbc L and trn H-psb A genes were extracted.

[0066] 1.2 Design of micromolecular marker primers

[0067] The mat K, rbc L and psb A genes were the target fragments, and Primer 3.0 software was used for primer design. The principles of primer design are: product size <500bp, primer length 18-22bp, GC content 40%-60%, annealing temperature 50-60°C, difference between upstream and downstream primer Tm values ​​less than 5°C, primer pairs on the same target fragment , There is at least 50 bp base sequence coverage between the amplified products.

[0068] 1.3 DNA extraction

[0069] Select the standard sample of khat grass stored in the herbarium of the Forest Public Security Judicial...

Embodiment 2

[0076] Using 11 pairs of micro-molecular marker primers designed and developed, a total of 11 suspected drug original plant khat grass samples in four suspected illegal drug smuggling cases were used for PCR amplification, and the amplified products were subjected to Sanger bidirectional sequencing. Such as image 3 As shown, the leaves of 11 samples were broken, and the morphology could not be recognized at all.

[0077] The PCR reaction system is: a total volume of 25 μL, containing 12.5 μL of Premix Taq polymerase, 4 μL of template DNA (10 ng / μL), 1 μL of upstream and downstream primers, ddH 2 O 6.5 μL.

[0078] The PCR amplification program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 30 s, 38 cycles; extension at 72°C for 10 min.

[0079] Figure 4 Genomic DNA agarose gel electrophoresis images of 11 samples of khat grass samples. It can be seen from the pictures that a large amount of DNA has...

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Abstract

The invention discloses a method for identifying Catha edulis based on a micro molecular marker and application, and particularly relates to development and application of a group of Catha edulis molecular identification DNA bar code marker primers. The micro molecular marker for identifying the species of the Catha edulis as a drug original plant is developed based on the chloroplast genome of the Catha edulis, 11 pairs of designed and developed micro molecular marker primers can be used for identifying the dry leaves of the Catha edulis with seriously degraded DNA, the specificity is good, the sensitivity is high, the reliability is high, and the method can be applied to evidence identification of cases such as illegal transaction and smuggling of the Catha edulis as the drug original plant, and striking of criminal activities on the drug original plant is enhanced favorably.

Description

technical field [0001] The invention belongs to the technical field of species identification, and in particular relates to the development and application of miniature molecular marker primers for molecular identification of khat grass. Background technique [0002] Catha edulis, also known as Qiaocha, Yemen tea, Arabic tea, Ethiopian tea, etc., is native to East Africa and the Arabian Peninsula, and is now widely distributed in African countries such as the Arabian Peninsula and Ethiopia. Chhat grass contains the excitatory chemical substance cathinone, which has a stimulating effect on the central nervous system of the human body. Long-term chewing will lead to addiction. It is also known as "East African poppy" and has serious social hazards. [0003] In recent years, the illegal smuggling of khat grass, the original drug plant, has occurred frequently. Its appearance resembles tea and is highly camouflaged. As for whether the illegally smuggled khat grass is the origin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12N15/11
CPCC12Q1/6895
Inventor 陈云霞南程慧薛晓明谢春平吴显坤黄娅琳郭海涛蒋敬吉小林
Owner NANJING FOREST POLICE COLLEGE