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Method for visually detecting phytoplasma based on CRISPR/Cas12a point cutting

A phytoplasma and system technology, applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve problems such as high cost and high dependence, and achieve low cost, reduced dependence, and good application foreground effect

Pending Publication Date: 2021-06-29
HENAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0004] In view of the current situation, the present invention provides a method for visual detection of phytoplasma based on CRISPR / Cas12a point cutting, aiming to solve the problem of high cost, high dependence on laboratory equipment, and requiring deep professional knowledge of detection personnel in traditional phytoplasma detection. problem, enabling visual detection of phytoplasma in the field

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  • Method for visually detecting phytoplasma based on CRISPR/Cas12a point cutting
  • Method for visually detecting phytoplasma based on CRISPR/Cas12a point cutting
  • Method for visually detecting phytoplasma based on CRISPR/Cas12a point cutting

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Embodiment

[0044] A method for visually detecting phytoplasma based on CRISPR / Cas12a point cutting, said method comprising the following steps:

[0045] (1) DNA template primer design for crRNA

[0046] For phytoplasma, RPA primers were designed from conserved regions after multiple sequence alignment of phytoplasma 16S rDNA sequences available in the GenBank database. We generated crRNA to recognize a specific site in the phytoplasma 16S rDNA sequence and shared homology with the fragment amplified by the RPA primer pair ( figure 1 ). The crRNA recognizes a 20bp target sequence adjacent to a TTTN or NAAA site and was designed from highly conserved regions of each virus / virus found in NCBI. Then, using the designed crRNA sequence as a probe, use the BLAST command to screen the available sequences in the NCBI GenBank database to ensure that the crRNA only exists in the phytoplasma 16S rDNA sequence, and there are no highly homologous sequences elsewhere. The designed primers The pair s...

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Abstract

The invention discloses a method for visually detecting phytoplasma based on CRISPR / Cas12a point cutting. According to the method, by designing crRNA aiming at a phytoplasma 16SrDNA sequence conserved region, the incision function of CRISPR / Cas12a protein is activated to cut linker-arm DNA so as to generate a required broken DNA fragment, and multiple phytoplasma are successfully detected by utilizing a nanogold color development technology, which has great significance for realizing rapid visual detection of phytoplasma in the field. The method disclosed by the invention has the characteristics of low cost, simplicity and convenience in operation, high efficiency, high sensitivity, high specificity and the like, naked eye judgment can be realized by color development, and the color development reaction effect is lasting and obvious. The dependence on large instruments and precision signal acquisition instruments in laboratories is reduced, and a new thought and a new choice are provided for detection of phytoplasma.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the detection of phytoplasma by adopting a biological method, and more specifically relates to a method for visually detecting phytoplasma based on CRISPR / Cas12a point cutting. Background technique [0002] Phytoplasma is a kind of mycoplasma that obligately parasitizes the sieve tube cells of plant phloem. Causes diseases of thousands of plants worldwide. After the plant is infected with phytoplasma, it will show abnormal developmental phenotypes such as arbuscules, clusters of leaflets, plant dwarfing, deformed flower organs, and flowers and leaves. In severe cases, the whole plant will die. Because phytoplasma obligately parasitizes the sieve tube cells of plant phloem and cannot be cultured in vitro due to harsh growth conditions, there is still no effective treatment for phytoplasma diseases. Once a plant is infected with phytoplasma, it is difficult to recover the ...

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12N15/11
CPCC12Q1/689C12Q1/6844C12Q2521/507C12Q2522/101C12Q2521/327C12Q2563/155
Inventor 李继东冯建灿陈鹏李奇乘杨琦琪叶霞郑先波谭彬
Owner HENAN AGRICULTURAL UNIVERSITY
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