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The Characteristic Sequence, Specific Identification Primer and Identification Method of Oyster Kumamoto

A Kumamoto oyster and identification method technology, applied in biochemical equipment and methods, microbe determination/inspection, DNA/RNA fragments, etc., to achieve consistent sequencing verification results, easy operation, and high specificity

Active Publication Date: 2022-02-22
浙江万里学院宁海海洋生物种业研究院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved by the present invention is, aiming at the mitochondrial non-coding region between the glycine tRNA synthetase and the proline tRNA synthetase of oyster, propose a kind of characteristic sequence, specific identification primer and identification method for identifying Kumamoto oyster, which can Play an important role in the protection of Kumamoto oyster germplasm resources, artificial propagation and related biological research

Method used

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  • The Characteristic Sequence, Specific Identification Primer and Identification Method of Oyster Kumamoto
  • The Characteristic Sequence, Specific Identification Primer and Identification Method of Oyster Kumamoto
  • The Characteristic Sequence, Specific Identification Primer and Identification Method of Oyster Kumamoto

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Embodiment 1

[0026] a) Sample collection

[0027] The long oysters cultured in the Qingdao sea area, the Fujian oysters cultured in the Fujian sea area, the near river oysters and Hong Kong oysters cultured in the Guangdong sea area, and the Kumamoto oysters cultured in the Ningbo sea area of ​​Zhejiang were collected. 30 oysters were randomly sampled from each location, and the adductor muscles were released after dissection. Put it into a cryopreservation tube, freeze it in liquid nitrogen and store it in a -80°C refrigerator;

[0028] b) Extraction of DNA

[0029]1) Take about 20 mg of tissue from each oyster individual in a 1.5 mL sterilized centrifuge tube, and use a DNA kit to extract DNA to obtain a DNA stock solution;

[0030] 2) Perform 1.2% agarose gel electrophoresis on the extracted DNA stock solution to detect the integrity of the DNA, and use Nanodrop to detect the concentration and purity of the DNA;

[0031] 3) Dilute the extracted DNA stock solution to 1-10ng / μL with ste...

Embodiment 2

[0039] a) Sample collection

[0040] Collect intertidal oyster samples from three bays along the coast of Zhejiang—Xiangshan Bay, Sanmen Bay, Yueqing Bay, and Zhangzhou, Fujian. 30 oysters were randomly selected from each location. After the oysters were dissected, the adductor muscles were quickly frozen in liquid nitrogen at -80°C Store in refrigerator for subsequent verification;

[0041] b) Extraction of DNA and molecular identification of oyster species

[0042] Use the DNA kit to extract DNA according to the instructions. After checking the purity and concentration of the DNA, perform MNR and COI sequence amplification. After the PCR product is sequenced, the NCBI Blast is used to determine the oyster species, mainly Kumamoto oyster and Fujian oyster (Yueqing Bay only takes Kumamoto oyster Oyster);

[0043] c) PCR amplification verification of specific primers

[0044] For oysters from the above four locations, 3 oysters were randomly selected from each species for th...

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Abstract

The invention discloses a characteristic sequence for identifying Kumamoto oyster, specific identification primers and an identification method, wherein the characteristic sequence is located in the mitochondrial non-coding region between glycine tRNA synthetase and proline tRNA synthetase of oyster, and the characteristic sequence is The nucleotide sequence shown in SEQ ID NO.1; the sequence of the specific identification primer is: upstream primer MNR-F: 5'-CTGTAAGTATATTTGTCTTCCA-3'; downstream specific primer MNR-S-R: 5'-AGGCTTTCACTCCACTTACT ‑3'. The present invention can effectively distinguish Kumamoto oyster from other four species of Pyramid oyster, the determined characteristic sequence length of Kumamoto oyster is 660±5bp, and whether it is Kumamoto oyster can be judged by the existence and size of the specific band. In the field of Kumamoto oyster germplasm resource protection and Kumamoto oyster breeding, species identification can be carried out on broodstock before seed breeding to ensure that the breeding seed is pure and not affected by other species that may hybridize with it.

Description

technical field [0001] The invention relates to the protection and application fields of molecular biology and aquatic germplasm resources, in particular to a characteristic sequence for identification of Kumamoto oyster (Crassostrea sikamea), a specific identification primer and an identification method. Background technique [0002] Oysters, commonly known as oysters, oysters, oyster yellows, etc., are distributed all over the world, with an annual global output of more than 6 million tons. They are important marine cultured shellfish. my country is rich in oyster resources and has a variety of cultured species. The cultured species in Liaoning and Shandong provinces in the north are mainly long oysters (Crassostrea gigas), Fujian provinces are Fujian oysters (C.angulata), Guangdong and Guangxi are Hong Kong oysters (C.hongkongensis) and Jinjiang Oyster (C. ariakensis). Zhejiang Province has a vast sea area, and there are important breeding bays such as Xiangshan Port, Yue...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686
Inventor 刘圣薛清刚徐洪强柳敏海林志华
Owner 浙江万里学院宁海海洋生物种业研究院
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