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Molecular specific labeled primer and method for identifying eriocheir sinensis

A technology of Chinese mitten crab and marker primer, which is applied to DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of destruction of spawning ground, destruction of living environment of Chinese mitten crab, and decline of Chinese mitten crab population, etc. Achieving the effect of high sensitivity and simple method

Inactive Publication Date: 2018-03-27
ZHENJIANG HUADA DETECTION CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the destruction of spawning grounds due to the construction of dams in the upper reaches of the Yangtze River; the invasion of alien species and water pollution have severely damaged the living environment of Chinese mitten crabs, so the population of Chinese mitten crabs has been declining

Method used

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  • Molecular specific labeled primer and method for identifying eriocheir sinensis
  • Molecular specific labeled primer and method for identifying eriocheir sinensis
  • Molecular specific labeled primer and method for identifying eriocheir sinensis

Examples

Experimental program
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Effect test

Embodiment 1

[0034] (1) Extraction of Genomic DNA of Eriocheir sinensis to be tested

[0035] The tissue samples of Eriocheir sinensis were taken, shredded and extracted with a genomic DNA extraction kit, and the obtained DNA was detected by electrophoresis using 1.5% agarose gel, and the DNA concentration was detected by an ultraviolet spectrophotometer, and stored in a -20°C refrigerator for later use.

[0036] (2) Design and synthesize specific PCR amplification primers

[0037] The specific primers based on Eriocheir sinensis were designed and synthesized. The upstream primer PXF: 5'-ACTTGGTGGTTTAAACTTAACCC-3' and the downstream primer PXR: 5'-ATTGAATTAGTATAGAAAGGGAG-3' were designed by technicians and synthesized by the primer design company.

[0038] (3) PCR amplification of molecular specific marker primers (PXF / PXR)

[0039] 25 μL of PCR reaction system: 2.5 μL of 10×PCR Buffer, 0.5 μL of dNTPS, 0.2 μL of Taq (5U / μl), MgCl 2 1.5 μL, PXF primer (10 μM) 1 μL, PXR primer (10 μM) 1 μ...

Embodiment 2

[0045] The method of Example 1 is used to detect three kinds of crabs: Chinese mitten crab, Japanese mitten crab, and swimming crab. The sampler records and numbers the species according to the specific information, and carries out the experiment when the experimenter does not know the name of the species. The results show that Only one kind of crab can amplify a specific DNA band with a molecular weight of about 200bp, and the other two crab samples cannot amplify, and the test results are completely consistent with the information recorded by the sampler. It can be shown that the nucleic acid molecule primer of the present invention has extremely high specificity, and therefore can be used for rapid identification of Chinese mitten crabs.

Embodiment 3

[0047]Two groups of muscle tissue samples were selected from crab tissue samples and preserved in 95% alcohol. The selected muscle tissues belong to two groups of crabs of different species, one group is Chinese mitten crab (5 samples), and the other group is other species (5 samples). No. The experiment operator conducted the experiment without knowing the species of the two groups of samples. Adopt the same experimental method as Example 1 to carry out the experiment, the PCR product sequencing result shows that wherein 5 samples of a group can amplify the specific DNA band of about 200bp to be the tissue sample of Chinese mitten crab, and the 5 samples of another group The first sample was a tissue sample of a non-Eriocheir sinensis, and the experimental results were completely consistent with the information recorded by the sampler.

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Abstract

The invention discloses a molecular specific primer and a method for identifying eriocheir sinensis. The molecular specific primer for identifying eriocheir sinensis has a forward primer PXF shown asSEQ ID NO: 1, and a reverse primer PXR shown as SEQ ID NO: 2. By utilizing the molecular specific labeled primer PXF / PXR, eriocheir sinensis can be subjected to rapid molecular identification by a conventional PCR method. The method is simple, is capable of finishing detection with PCR technology and is an effective assistance for identifying eriocheir sinensis through morphological characteristics.

Description

technical field [0001] The invention relates to a molecular-specific marker primer for identifying Chinese mitten crabs, and a method for quickly identifying Chinese mitten crab specimens by using the specific primers. Background technique [0002] Chinese mitten crab (scientific name: Eriocheir sinensis) inhabits freshwater lakes and rivers. Every year from June to July, newborn young crabs go up the river and enter fresh water, and live on the banks of rivers and lakes. They like to live in burrows, or hide in gravel, water and grass. When digging holes, it mainly relies on a pair of chelicerae, and the legs only play an auxiliary role. Feeds on aquatic plants, benthic animals, organic debris and animal carcasses. When feeding, it catches it with its chelicerae, and then sends the food to the mouth. When the nutritional conditions are good, the weight of young crabs can reach 50 to 70 grams, the maximum can reach 150 grams, and the gonads are mature, and they can parti...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
Inventor 崔海月耿小燕张继敏蒋寿佳游欣欣王敏
Owner ZHENJIANG HUADA DETECTION CO LTD
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