Transgenic component detection method based on CRISPR Cas enzyme gene editing technology, reaction system and application thereof
A technology of genetically modified components and reaction systems, applied in the field of gene editing, can solve the problems of application, not in plant gene detection, etc.
Pending Publication Date: 2021-12-21
SANYA RES INST OF CHINESE ACAD OF TROPICAL AGRI +1
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Problems solved by technology
At present, the in vitro nucleic acid detection technology based on Cas13a has been initially used in medicine and plant detection, showing high sensitivity and accuracy; the in vitro nucleic acid detection technology based on Cas12a has been initially used in medicine, but has not yet been used in plants. Application in gene detection; neither CRISPR DNA endonuclease nor CRISPR RNA endonuclease has been reported on the application of plant genetically modified components
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[0049] In order to have a further understanding of the technical solution and beneficial effects of the present invention, the technical solution of the present invention and its beneficial effects will be described in detail below in conjunction with the accompanying drawings.
[0050]The object of the present invention is to provide Cas12a enzyme gene editing detection technology efficient RPA isothermal amplification primers and corresponding Cas12a enzyme high-efficiency detection sites of plant materials containing CaMV35S promoter, hygromycin gene, Bar gene, NPT II gene and other plant transgenic components , and use microplate reader, quantitative PCR instrument, and test strip color technology to realize the detection of target genetically modified ingredients, and establish a new detection system for the detection of plant genetically modified ingredients.
[0051] 1. Construction of reaction system
[0052] In order to achieve the above object, the technical scheme o...
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The invention relates to a transgenic component detection method based on a CRISPR Cas enzyme gene editing technology, a reaction system and application thereof. The reaction system is used for detecting plant transgenic ingredients, each part of the reaction system comprises the following components in parts by volume: 2 parts of 10xNE buffer solution 2.1 (50mM sodium chloride, 10mM Tris-hydrochloric acid, 10mM magnesium chloride, 100ug / ml bovine serum albumin, pH 7.9 / 25 DEG C), 1 part of 1uM guide sequence, 1 part of 1uM Lba Cas12a, 1 part of RNA enzyme inhibitor, 1 part of RPA isothermal amplification product of transgenic plant DNA extract or plasmid lysis buffer of transgenic plant, 1 part of reaction marker and 13 parts of water. The invention provides a new technology and method for nucleic acid detection of transgenic plants, certainly promotes the application research of the gene editing detection technology in plant nucleic acid detection, and has important practical significance.
Description
technical field [0001] The invention relates to the technical field of gene editing, in particular to a method for detecting transgenic components, a reaction system and applications thereof based on CRISPR Cas enzyme gene editing technology. Background technique [0002] From 2017 to 2019, the emergence of DNA detection technology DETECTR based on Cas12a (CRISPR DNA endonuclease) and SHERLOCK RNA detection technology based on Cas13a (CRISPR RNA endonuclease) opened up the use of gene editing technology based on nucleic acid As the pioneer of high-throughput, fast, accurate, sensitive, and easy-to-use detection technology, gene editing detection technology is based on efficient nucleic acid detection. Compared with traditional monoclonal antibody protein detection technology, it has unparalleled advantages. Efficient and rapid regulation of diverse plant viruses and transgenic plants is possible. At present, the in vitro nucleic acid detection technology based on Cas13a has...
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IPC IPC(8): C12Q1/6895C12Q1/6844C12N15/11
CPCC12Q1/6895C12Q1/6844C12Q2521/507C12Q2522/101C12Q2521/327C12Q2525/161Y02A50/30
Inventor 赵辉杨小亮段志强李峰郭安平
Owner SANYA RES INST OF CHINESE ACAD OF TROPICAL AGRI