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GRNA for targeting pathogen gene RNA and C2c2-based pathogen gene detection method and detection kit

A pathogen, c2c2 technology, applied in the direction of DNA / RNA fragments, genetic engineering, DNA preparation, etc.

Pending Publication Date: 2019-02-01
GUANGZHOU PLUSLIFE TECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] The applicant found that there is no relevant report on the pathogen gene detection method based on the CRISPR-C2c2 system

Method used

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  • GRNA for targeting pathogen gene RNA and C2c2-based pathogen gene detection method and detection kit
  • GRNA for targeting pathogen gene RNA and C2c2-based pathogen gene detection method and detection kit
  • GRNA for targeting pathogen gene RNA and C2c2-based pathogen gene detection method and detection kit

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Experimental program
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preparation example Construction

[0125] 2. Preparation of target RNA gRNA preparation of target nucleotides:

[0126] extract

[0127] Target nucleotides are amplified by PCR, recombinase polymerase amplification (RPA), NASBA isothermal amplification or, loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA) and nickase amplification reaction (NEAR) to amplify target DNA. Gel separation and purification (using the MinElute gel extraction kit (Qiagen) kit), the purified dsDNA was incubated with T7 polymerase at 30°C overnight (using the HiScribe T7 Quick High Yield RNA Synthesis kit (New England Biolabs) kit), and then using MEGAclear Transcription Clean-up kit (Thermo Fisher) was used to purify RNA to obtain target nuclear RNA.

[0128] NASBA isothermal amplification

[0129] At 4°C, configure the amplification system as follows:

[0130]

[0131]

[0132] The above mixed system was placed at 65°C for 2 minutes; then at 41°C for...

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Abstract

The invention provides a gRNA for targeting a pathogen gene RNA, and the invention also provides a human pathogen gene detection method and detection kit based on a clustered regularly interspaced short palindromic repeat (CRISPR)-C2c2 system. The detection method combines the advantages of the gRNA in targeting to recognize a pathogen gene transcript RNA (target RNA sequence) and the characteristic that when the CRISPR-C2c2 complex detects the target RNA sequence, the complex cleaves a reporting RNA with a detection marker to release detectable signals, the CRISPR-C2c2 system is applied to pathogen gene detection, and has high sensitivity and high accuracy. The detection method and the detection kit have great commercial application value.

Description

technical field [0001] The invention relates to the field of gene detection and gene modification, and relates to a gRNA for specifically targeting pathogen gene RNA, and a pathogen gene detection method and detection reagent based on a clustered regularly interspaced short palindromic repeat sequence (CRISPR) system Cassette; in particular to a gRNA for specifically targeting pathogen resistance genes and / or specific gene RNA, and a pathogen resistance gene and / or based on a clustered regularly interspaced short palindromic repeat (CRISPR) system Specific gene detection methods, detection kits. Background technique [0002] Clustered regularly interspaced short palindromic repeat system (clustered regularly interspaced shortpalindromic repeat; CRISPR-associated, CRISPR-Cas) is an important immune defense system for archaea and bacteria to resist virus and plasmid infection, and is used to resist foreign genetic material. Invasions, such as phage viruses and foreign plasmid...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/74C12Q1/6876
CPCC12N15/102C12N15/113C12N15/74C12Q1/6876C12N2310/10
Inventor 陈侃罗镇华
Owner GUANGZHOU PLUSLIFE TECH CO LTD
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