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PCR method for rapid identification of four types of blue crabs of scylla

A mud crab, rapid technology, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of poor labeling repeatability, difficult to master, low identification accuracy, etc., to improve efficiency and accuracy, Simple to use effects

Inactive Publication Date: 2010-08-04
EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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Problems solved by technology

However, this method is only suitable for professionals who are very familiar with the phenotypic characteristics of the four blue crabs, and it is not easy for those who are not specialized in the taxonomic research of blue crabs
In addition, Klinbunga et al. (2000) reported RAPD molecular markers that can identify three species of blue crabs, but due to the instability of the RAPD marker itself, the repeatability of the marker is poor, so the accuracy of identification is not high

Method used

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  • PCR method for rapid identification of four types of blue crabs of scylla
  • PCR method for rapid identification of four types of blue crabs of scylla
  • PCR method for rapid identification of four types of blue crabs of scylla

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Embodiment 1

[0041] 1. Extraction of Genomic DNA of 4 Scyllas

[0042]Cut a small amount of muscle tissue (about 100-150 mg) from blue crabs and place it in 500 μl tissue extract solution (10 mmol / L Tris-CI, pH 8.0; 100 mmol / L LEDTA, pH 8.0; 100 mmol / L NaCl; 0.5% SDS) Cut into pieces, add proteinase K (final concentration 20mg / ml) and RNase A (final concentration 100μg / ml), mix thoroughly, digest at 55°C for 2-3 hours until clear; mix with phenol: chloroform: isoamyl alcohol solution (volume ratio 25:24:1) twice; then precipitate DNA with 2 volumes of absolute ethanol, wash with 70% ethanol and dry naturally, then dissolve in 50 μL TE (10 mmol / L Tris-HCl, pH8.0; 10mmol / L EDTA, pH8.0); Finally, the DNA concentration was diluted to 100ng / μl and stored at -20°C for later use;

[0043] 2. Mitochondrial COI gene sequence analysis and species-specific primer design

[0044] Download the COI sequences of four kinds of blue crabs from the public database GenBank, use DNAMAN 4.0 biological softwa...

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Abstract

The invention relates to a PCR method for the rapid identification of four types of blue crabs of Scylla, comprising the following steps: extracting the genome DNA of the four types of the blue crabs; (2) designing a species specific amplification primer according to the nucleotide sequence of the chondriosome (COI) gene of the four types of the blue crabs; (3) carrying out PCR amplification on the genome DNA of the four types of the blue crabs; and (4) utilizing 1.5% of agarose gel electrophoresis to detect PCR products, so as to rapidly identify the four types of the blue crabs according to the different sizes of the products. The method has simple and safe operation, clear amplification stripe and true and reliable results, and can rapidly identify the four types of the blue crabs, thus being widely used in the fileds of protection and management of blue crab germ plasm resources, inheritance breeding and the like.

Description

technical field [0001] The invention belongs to the field of species identification of the genus Scylla, a marine economic crab, and particularly relates to a PCR method for rapidly identifying four species of Scylla genus. Background technique [0002] The genus Scylla belongs to the phylum Arthropoda, the class Crustacea, the order Decapoda, and the family Portunidae, including 4 species: Scylla paramamosain and Scylla serrata. (Scyllaserrata), Scylla olivacea and Scylla tranquilbarica are mainly distributed in the tropical, subtropical and temperate regions of the Pacific Ocean and the Indian Ocean. In my country, it is mainly distributed in the southeast coastal provinces, with the most in Fujian, Guangdong and Hainan. It is one of the important cultured crabs in my country. In order to identify and protect the resources of blue crabs widely distributed in my country, Ma Lingbo et al. (2006) used the mitochondrial COI gene sequence to study the phylogenetic relationship...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 马洪雨马凌波马春艳
Owner EAST CHINA SEA FISHERIES RES INST CHINESE ACAD OF FISHERY SCI
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