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Characteristic sequence, specific identification primer and identification method for identifying crassostrea sikamea

A technology of Kumamoto oyster and characteristic sequence, which is applied in the fields of molecular biology and protection and application of aquatic germplasm resources, and achieves the effects of simple operation, high specificity and consistent sequencing verification results.

Active Publication Date: 2021-04-09
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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  • Characteristic sequence, specific identification primer and identification method for identifying crassostrea sikamea
  • Characteristic sequence, specific identification primer and identification method for identifying crassostrea sikamea
  • Characteristic sequence, specific identification primer and identification method for identifying crassostrea sikamea

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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, specific identification primer and identification method for identifying crassostrea sikamea. The characteristic sequence is located in a mitochondrial non-coding region between glycine tRNA synthetase and proline tRNA synthetase of the crassostrea sikamea, and the characteristic sequence is a nucleotide sequence as shown in SEQ ID NO.1; a sequence of the specific identification primer comprises an upstream primer MNR-F: 5' -CTGTAAGTATATTTGTCTTCCA-3' ; and a downstream specific primer MNR-S-R: 5' -AGGCTTTCACTCCACTTACT-3' . According to the invention, the crassostrea sikamea can be effectively distinguished from other four drassostrea species, the length of the determined characteristic sequence of the crassostrea sikamea is 660+ / -5 bp, and whether the crassostrea sikamea exists or not can be judged according to the existence and the size of a specific band. In the field of crassostrea sikamea germplasm resource protection and crassostrea sikamea breeding, species identification can be conducted on parent crassostrea sikamea before seed breeding, and it is guaranteed that bred seeds are purebreds and are not affected by other species possibly hybridized with the bred seeds.

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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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2565/125
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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