A PCR identification method between the germplasm of flounder and its close relative alien species

A technology of brown flounder and close relatives, applied in the field of PCR identification, can solve the problems of unseen species identification technology of brown flounder and Atlantic flounder, and few patents for detection and identification of biological germplasm resources, and achieve simple technology Ease of operation, speed up the detection process, and high specificity

Inactive Publication Date: 2017-02-22
INST OF OCEANOLOGY - CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there are not many patents on the detection and identification of biological germplasm resources, and there are no reports on the species identification technology of brown flounder and Atlantic flounder

Method used

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  • A PCR identification method between the germplasm of flounder and its close relative alien species
  • A PCR identification method between the germplasm of flounder and its close relative alien species
  • A PCR identification method between the germplasm of flounder and its close relative alien species

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Mitochondrial genomic DNA extraction:

[0024] Using the back muscle material of brown flounder and its close relative species Atlantic flounder as raw materials, take 100 mg each and treat them with sterile water, grind them into powder in liquid nitrogen, put them into a 1.5ml centrifuge tube, Refer to the refined version of the Molecular Cloning Experiment Guide (J. Sambrook, D.W. Russell) and use the standard phenol / chloroform extraction method to extract the mitochondrial genomic DNA from the raw materials, and set them aside.

[0025] 2. Specific primer design:

[0026] Based on the complete mitochondrial sequences and partial control region sequences of flounder and Atlantic flounder published in the GENBANK database, DNASTAR8.0 software was used for homologous sequence comparison, and PRIMER5.0 software was used to assist in the design of specific primers.

[0027] 2.1 Amplification primers:

[0028] (1) The name and sequence of the amplification primers fo...

Embodiment 2

[0048] Using the DNA of the back muscle of the juvenile flounder and its close relative alien species Atlantic flounder as a template, based on the PCR reaction conditions in Example 1, and using the common cultured flounder species turbot as a negative control, the PCR reaction The results showed that the Atlantic flounder sample had specific amplification positive bands at 422bp and 201bp, the brown flounder sample had a positive amplification band at 422bp, and the turbot sample had no corresponding specific band (see figure 1 ).

[0049] From the above, it can be known that the F / R of the primer of the mitochondrial DNA control region 1 The combination can amplify both Atlantic flounder and brown flounder, primers F / R 2 The combination only had a species-specific amplification effect on Atlantic flounder, so there were differences in PCR amplification bands between brown flounder and Atlantic flounder, while F / R 1 and F / R 2 The combination is non-specific for other flo...

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Abstract

The invention relates to a qualitative detection method for identifying species by using a molecular biological technique, and in particular relates to a PCR identification method for paralichthys olivaceus and germ plasm of affinis alien species of paralichthys olivaceus. Three specifically marked primers F, R1 and R3 are adopted to amplify mitochondrial DNA genes of paralichthys olivaceus and affinis alien species paralichthys dentatus of paralichthys olivaceus, and the paralichthys olivaceus is distinguished from paralichthys olivaceus according to amplified specific fragments. By adopting the method, a rapid, economic and reliable molecular biological technique is provided for seedling qualitative identification of paralichthys olivaceus and paralichthys dentatus which cannot be morphologically identified in the early development period, intelligent support is provided for enhancement and releasing identification and alien species invasion prevention of paralichthys olivaceus seedlings, and meanwhile, the quality detection development of the bastard halibut seedling industry is accelerated.

Description

technical field [0001] The invention relates to a qualitative detection method for species identification by molecular biology technology, in particular to a PCR identification method for germplasm of flounder and its close relative alien species by using molecular biology technology. Background technique [0002] Atlantic flounder (Paralichthys dentatus) belongs to the order Pleuriformes, Flounderidae, and genus Flounder. It is mainly distributed along the Atlantic coast of North America. It is a cold-water economic fish. Brown flounder (P. olivaceus) and Atlantic flounder are closely related species, and they are very similar in biomorphology. They are mainly distributed in the Northwest Pacific coast and are important species of flounder in Asia. In order to change the single cultured species of flounder in my country, Atlantic flounder was introduced into my country from the United States for the first time in 2002 for artificial cultivation and breeding. In the breedin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCY02A90/40
Inventor 肖永双李军肖志忠马道远徐世宏刘清华
Owner INST OF OCEANOLOGY - CHINESE ACAD OF SCI
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