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CrDNA, crRNA, kit and method for detecting plant tomato spotted wilt virus

The technology of tomato spotted wilt virus and kit is applied in the field of molecular biology, which can solve problems such as false positives, and achieve the effects of simple method, simple detection conditions and low cost.

Pending Publication Date: 2022-02-18
TOBACCO RES INST CHIN AGRI SCI ACAD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Isothermal amplification technology does not require expensive equipment, but may produce false positive results under isothermal amplification conditions

Method used

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  • CrDNA, crRNA, kit and method for detecting plant tomato spotted wilt virus
  • CrDNA, crRNA, kit and method for detecting plant tomato spotted wilt virus
  • CrDNA, crRNA, kit and method for detecting plant tomato spotted wilt virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, the design of targeting specific region crRNA

[0040] 1. Design principles of crRNA targeting specific regions

[0041] Since the CRISPR-Cas13a system is a novel targeted DNA gene editing system, in which Cas13a combines with crRNA to form a monitoring complex, the guide region of crRNA recognizes the target DNA with a complementary sequence, and Cas13a degrades the target DNA strand, the crRNA design requires : crRNA includes protein anchor sequence and guide sequence, and the sequence format is: 5`-anchor sequence combined with Cas13a protein-crRNA guide sequence-3`, protein anchor sequence needs to be determined according to Cas13a protein, so that it can be combined with the selected The specific Cas13a protein is matched and bound; the guide sequence is matched with the fragment in the target DNA. The crRNA guide sequence should not be too close to the start codon (ATG); it is 22-24 nucleotides in length.

[0042] With Tomato spotted wilt virus (TS...

Embodiment 2

[0054] Embodiment 2, the kit and method for detecting plant tomato spotted wilt virus

[0055] 1. The composition of the kit

[0056] (A) crRNA (its sequence is shown in SEQ ID NO.1) or crDNA (its sequence is shown in SEQ ID NO.2).

[0057] When the kit is crDNA, the operator needs to first generate RNA from the crDNA fragments under the action of T7 RNA polymerase, recover and purify the crRNA (see Example 1 for specific steps);

[0058] (B) a specific fluorescent probe (its sequence is shown in SEQ ID NO.3), the 5' end of the sequence of the fluorescent probe is marked with a fluorescent group, and the 3' end is marked with a quenching group The fluorescent group includes one of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and TexasRed, and the quenching group includes TAMRA, DABCYL, MGB, BHQ-1, BHQ-2 and one of BHQ-3);

[0059] (C) Cas13a protein, enzyme-free water, DNase inhibitor;

[0060] Preferably, the kit can also include:

[0061] (D) Amplification system: including i...

Embodiment approach

[0102] As another embodiment of the present invention, the kit can also be made into a colloidal gold detection kit;

[0103] The composition of colloidal gold detection kit: detection reagent is the same as embodiment 2, and difference is the specific fluorescent probe in the present embodiment such as any kind of probe shown in SEQ ID NO.15-17, and 3 of described sequence The 'end is labeled with biotin, and the 5' end is labeled with one of FAM, VIC, HEX, TRT, Cy3, Cy5, ROX, JOE and Texas Red; in this embodiment, the 5' end is labeled with FAM. When the detection kit is prepared as a colloidal gold detection kit, the colloidal gold detection kit includes a base plate, a sample pad, a bonding pad, a nitrocellulose membrane and a water-absorbent pad that are bonded to the base plate and overlapped successively; A quality control line is provided on the side of the nitrocellulose membrane close to the binding pad, and a detection line is provided on the side of the nitrocellul...

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Abstract

The invention provides crDNA, crRNA, a kit and a method for detecting plant tomato spotted wilt virus. The nucleotide sequence of the crDNA is as shown in SEQ ID NO.1; the nucleotide sequence of the crRNA is as shown in SEQ ID NO.2; the kit contains the crDNA or the crRNA, a Cas13a protein and a fluorescent probe. The method comprises the following steps of: extracting total RNA of a plant sample to be detected, and acquiring cDNA through reverse transcription; carrying out amplification by taking the cDNA as a template to obtain a DNA amplification product; and using a detection reaction system composed of the amplification product, crRNA, Cas13a protein, the fluorescent probe and enzyme-free water for detection. The crDNA, crRNA, a kit and a method for detecting plant tomato spotted wilt virus provided by the invention have the characteristics of low cost, repeated detection, simple method, high detection speed, sensitivity (the lowest detection limit reaches 10 copies / uL) and specificity.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a crDNA, crRNA, kit and method for detecting plant tomato spotted wilt virus. Background technique [0002] Plant diseases caused by viruses have caused huge economic losses worldwide. Therefore, it is urgent to establish a more efficient and sensitive virus detection method to prevent the occurrence of large-scale epidemics of viral diseases. Currently, strategies for plant virus diagnosis mainly include nucleic acid-based detection methods and antigen-antibody-based detection methods. Nucleic acid-based detection methods are also called molecular biology detection, mainly including polymerase chain amplification reaction (polymerase chain reaction, PCR), real-time fluorescence quantitative PCR (qRT-PCR), nucleic acid sequence-dependent amplification method (nucleic acid sequence -basedamplification, NASBA), high-throughput sequencing, etc. The existing nucleic acid...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12Q1/6804C12N15/113C12N15/11
CPCC12Q1/701C12Q1/6844C12Q1/6804C12N15/1131C12N2310/20C12Q2521/327C12Q2525/151C12Q2525/161C12Q2521/507C12Q2521/107C12Q2522/101C12Q2531/119C12Q2563/107C12Q2565/625C12Q2563/131
Inventor 杨金广张万红蔺忠龙焦玉冰李莹申莉莉王凤龙刘春明夏振远
Owner TOBACCO RES INST CHIN AGRI SCI ACAD
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