Eriocheir sinensis plasma germ detecting and identifying technology

A technology of Chinese mitten crab and mitten crab, which is applied to the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve the problems that have not yet reached the level of subspecies differentiation, cannot be effectively distinguished, and the biochemical genetic differences are not significant.

Inactive Publication Date: 2004-07-14
李思发
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The results showed that (1) the biochemical genetic differences among the populations of Liaohe, Yellow River, Yangtze River and Oujiang mitten crabs were not significant, and the differences among populations within genus
(2) The biochemical genetic differences between the Pearl River and Nanliujiang mitten crab populations are not significant, and have not yet reached the level of subspecies differentiation
However, this biochemical genetic analysis method is still unable to effectively distinguish various types of river crabs in the same group (such as distinguishing Liaohe crabs from Yangtze river crabs)

Method used

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  • Eriocheir sinensis plasma germ detecting and identifying technology
  • Eriocheir sinensis plasma germ detecting and identifying technology
  • Eriocheir sinensis plasma germ detecting and identifying technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Genomic DNA extraction

[0058] Cut about 0.2g of crab leg muscle and add 400uL of STE buffer (30mmol / L Tris-HCl, pH8.0, 200mmol / L EDTA, 50mmol / L NaCl). After mixing, add SDS at final concentrations of 1% and proteinase K at 200 μg / mL, and act at 50°C for 5-8 hours. Add an equal volume of saturated phenol, rotate slowly on the rotor for 1 h, centrifuge at 10,000 r for 8 min, absorb the supernatant and add an equal volume of mixed solution (phenol: chloroform: isoamyl alcohol = 25:24:1), slowly rotate for 0.5 h, discard After the lower liquid layer, add an equal volume of chloroform, rotate slowly for 5 minutes, suck and discard the lower liquid layer, add an equal volume of isopropanol or 2 times the volume of absolute ethanol to precipitate DNA, wash with 70% ethanol, and dry. Add 500 μL of TE to dissolve and set aside.

Embodiment 2

[0060] Determination of amplification reaction conditions

[0061] The PCR reaction mixture contains 10mmol / L Tris-HCl, pH9.0, 50mmol / L KCl, 2.0~3.0mmol / L MgCl 2 , 0.001% gelatin, 0.1mmol / L of each dNTP, a primer concentration of 0.2μmol / L, about 125ng of genomic DNA, 1 unit of Taq enzyme, a total reaction volume of 25μL, and 30μL of paraffin oil. The reaction was carried out on a PCR amplification instrument, and the cycle program was as follows: denaturation at 94°C for 5 min; followed by 45 cycles at 94°C for 45 sec, 36°C for 45 sec, and 72°C for 90 sec. After 40 to 45 cycles, extension at 72°C for 5 min. Take 10 microliters of the amplified product, go through 1.5% agarose gel electrophoresis, take pictures after EB staining, and analyze.

Embodiment 3

[0063] Screening of amplification primers

[0064] In this example, a total of 48 random primers (synthesized by Takara Company) were synthesized, which were selected 10-base primers with abundant polymorphisms in the mitten crab genome. First use these 48 primers to amplify a total of 24 individuals of 4 individuals (2 females and 2 males) in 6 populations, and perform statistical analysis on the results. On this basis, find out the primers with population characteristic bands, and then conduct large-scale sample analysis. Each group analyzed 30 individuals, half male and half male.

[0065] (a) Amplification reaction

[0066] The amplification reaction conditions were the same as in Example 2.

[0067] (b) RAPD analysis of genomic DNA

[0068] 48 polymorphic primers were used to amplify 24 individuals of 4 individuals (2 females and 2 males) in each of the 6 populations by PCR. The amplification results were stable and repeatable, with rich primer and template specificity...

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Abstract

The present invention discloses molecular genetic marker method for detecting and identifying plasm germ of Eriocheir sinensis (river crab). The detecting and identifying process includes extracting DNA of river crab; and performing PCR amplification with the DNA as template and primer TGACCCGCCT (SEQ ID No. 1) and amplification product analysis. When the 947 bp band appears in the frequency of 80-100 %, the sample is Yangtse River crab; and when the 947 bp band appears in the frequency of 0-20 %, the sample is Liaohe River crab. The method is used in distinguishing Yangtse River crab from Liaohe River crab fast. The present invention also provides corresponding detection reagent kit.

Description

technical field [0001] The invention relates to the field of quality detection, more specifically, to the germplasm detection and identification technology of Chinese mitten crab (Eriocheirsinensis). Background technique [0002] Chinese mitten crab (Eriocheir sinensis), also known as river crab, belongs to Arthropoda, Crustaceae, Decapoda, Grapsidae, and Eriocheir. A unique aquatic economic animal. In my country, it is distributed as far as 24°N latitude in the south, 42°-43°N latitude in the north, Yalu River Estuary at 124°E in the east, and Shashi, Hubei in the west at 112°E. [0003] Eriocheir sinensis is rich in nutrition. For example, the muscle, liver and gonads of adult crabs in Yangcheng Lake, Jiangsu Province, account for 36.72% of the total body weight, and the four non-edible parts, shell, gills, stomach and heart, account for 53.26% of the total body weight. Another 10.02% is hemolymph and tissue fluid. The edible parts of the female crabs of the first and ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 李思发邹曙明赵金良王成辉蔡完其
Owner 李思发
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