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Molecular identification method of four major Chinese carps in fresh water

A technology of molecular identification and fresh water, applied in biochemical equipment and methods, microbiological determination/inspection, etc., can solve the problems of poor repeatability and stability of RAPD labeling, and achieve low single-sample cost, low cost, and clear judgment basis Effect

Inactive Publication Date: 2013-09-04
YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

Although the isoenzyme method can also identify species, it requires fresh samples and a sufficient amount of tissue, and it is powerless for tissues preserved in ethanol or when the amount of tissue is insufficient
(3) Molecular marker method: random amplified polymorphic DNA (RAPD) technology is commonly used in species identification. For example, Deng Huai et al. (1998) compared RAPD of 10 common freshwater fishes and found that each fish has its own characteristics. Unique characteristic bands can be used as molecular markers to distinguish species, but RAPD markers have poor repeatability and stability

Method used

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Embodiment Construction

[0018] The present invention will be further described below with reference to specific embodiments.

[0019] 1) Genomic DNA extraction

[0020] Take 32 fin rays of the four major domestic carps and related fish (see Table 1), about 0.2 g each, and wash them twice with sterilized double-distilled water for 15 min each time. Put the washed tissue into a 2 mL EP tube, add 500 μL of HOM buffer (80 mmol / L EDTA, 100 mmol / L Tris-base, 0.5% SDS, pH8.0) and 10 μL of 20 mg / mL Proteinase K, digest at 55°C for about 3 h, and shake gently every 1 h until the digestion is complete. Add 500 μL 4.5 mol / L NaCl solution and 300 μL chloroform, and mix thoroughly for 5 min. Centrifuge at 12000 r / m for 10 min, take the supernatant, add 600 μL of isopropanol (about 0.7 times the volume of the supernatant) to mix, and place on ice for 30 min. Centrifuge at 12000 r / m for 10 min, remove the supernatant, add 1 mL of 75% ethanol pre-cooled at 4°C, and let stand for 5-20 min. Centrifuge at 12000 ...

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Abstract

The invention discloses a molecular identification method of four major Chinese carps in fresh water. The method provided by the invention comprises the following steps: (1) genome DNA extraction; (2) PCR amplification; (3) PCR reaction: the reaction procedure is started with predenaturation at 94 DEG C for 5min, initial denaturation lasts for 5min, then denaturation is carried out at 94 DEG C for 30s, annealing is carried out at 62 DEG C for 30s, extension is carried out at 72 DEG C for 1min, 30 cycles are performed, and after the cycles, extension is carried out again at 72 DEG C for 8min; (4) electrophoresis detection: after PCR, a solution of 2-5 microliters is put on 1% of sepharose gel for electrophoresis, and observation and photographing are carried out; and (5) result judgment. The method provided by the invention has advantages of simple operation, accurate judgment and low cost.

Description

technical field [0001] The invention relates to the field of aquatic science, in particular to a fish identification method. Background technique [0002] Herring ( Mylopharyngodon piceus ), grass carp ( Ctenopharyngodon idellus ) silver carp ( Hypophthalmichthys molitrix ), bighead carp ( Aristichthys nobilis ) belongs to Cyprinidae and Cyprinidae, and is one of the "four major fishes" traditionally farmed in freshwater in China. It is native to the Yangtze River, Pearl River and Heilongjiang River Basin. Some countries have formed natural populations. In recent years, due to drastic changes in the natural environment of the Yangtze River system, the resources of the wild populations of the four major Chinese carps have been seriously disturbed. In order to quickly assess the genetic diversity and resource status of the four major domestic fishes to formulate scientific fishery management measures, it is necessary to establish a rapid and accurate method for identi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 汪登强程晓凤陈大庆刘绍平段辛斌王珂刘明典
Owner YANGTZE RIVER FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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