Portunidae mitochondrial COI gene universal primer, design and amplification method thereof

A universal primer, swimming crab technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problems of time-consuming, inconvenient, efficient, and inability to identify cephalopod mitochondrial COI genes, etc. Cost saving and simple operation

Inactive Publication Date: 2019-01-18
ZHEJIANG OCEAN UNIV
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  • Abstract
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Problems solved by technology

[0003] On September 29, 2017, the Chinese Patent Office disclosed an invention authorization for a spider mitochondrial genome full-sequence amplification primer and amplification method, the authorization publication number: CN104531688B. Long-PCR (L-PCR) amplification and sequencing

Method used

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  • Portunidae mitochondrial COI gene universal primer, design and amplification method thereof

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Embodiment 1

[0028] 1. Universal primers and design and amplification methods for mitochondrial COI gene of Portunidae

[0029] (1) Collection, identification and preservation of Portunidae samples

[0030] The Portunidae samples used in the examples were all collected from the natural environment in the wild, and all the collected specimens were brought back to the laboratory for information registration, and all were identified with a stereo microscope to determine the species. The identified Portunidae samples were soaked in 100% pure alcohol and stored in a refrigerator at 4°C for later use. The Portunidae species collected include: Pleurotus, Jingying, Spotted, Two-spotted, Swimming, Blue Crab, Scylla serrata, Scylla syringus, Portunus pelagics and Portunus red star.

[0031] (2) Extraction of Mitochondrial COI Gene of Portunidae

[0032] The DNA extraction kit method was used to extract the mitochondrial COI gene of the cephalopod samples to be tested, and the extracted mitochondri...

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Abstract

The invention relates to the research field of cephalopod mitochondrial genome, and especially relates to a portunidae mitochondrial COI gene universal primer, and design and amplification methods thereof. The universal forward primer of mitochondrial COI gene of Portunidae is SEQ ID NO. 1: 5 '- TCNACAAAYCATAAAGAYATYGG-3 ', and that reverse prim is SEQ ID NO. 2: 5'-TANACYTCWGGRTGHCCRAARAAYCA- 3'.The present invention solves the problem that the prior art does not utilize the DNA barcode technology to identify the family Portunidae, and has the following advantages: 1) the universal primer ofmitochondrial COI gene of the family Portunidae provided by the present invention can amplify a plurality of families of Portunidae in batch at one time with high efficiency, and can provide great convenience for the species identification of the family Portunidae; 2) design universal primers in two conservative region of mitochondrial COI gene of family Portunidae to amplify an intermediate mutation sequence, that amplification length is about 600-700 bp, sufficient information sites are used for identification and differentiation of physical evidence, and a sequencing reaction can be complete, the operation is simple, and the cost is saved.

Description

technical field [0001] The invention relates to the field of cephalopod mitochondrial genome research, in particular to a universal primer and a method for designing and amplifying mitochondrial COI gene of Portunidae. Background technique [0002] As an emerging technology for species identification developed in recent years, DNA barcoding (DNA barcoding) technology has received more and more attention. The principle is usually to use a short mitochondrial COI (cytochrome c oxidase subunit Ⅰ) gene sequence for species identification has been widely used in fish, birds, reptiles, mammals and other biological groups, but it is not very common in crabs. Portunidae crabs are rich in resources, with many species, and have extremely high economic value; however, the classification of crabs is mainly based on traditional morphological and behavioral technical means, and phenotypic variation is significantly affected by environmental factors, resulting in phenotypic Uncertainty...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11
CPCC12Q1/6888C12Q2600/156
Inventor 龚理姜辉刘炳舰吕振明刘立芹
Owner ZHEJIANG OCEAN UNIV
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