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One-step nucleic acid test method based on CRISPR/Cas and isothermal amplification and kit

A detection method, constant temperature amplification technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of long detection time, expensive price, complicated operation, etc., to ensure accuracy, convenient and rapid detection , The effect of improving the ease of operation

Inactive Publication Date: 2019-08-30
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Although these methods can effectively and qualitatively detect specific nucleic acid sequences, most of them require special instruments, are expensive, or require more complicated operations and longer detection times
In order to solve this problem, constant temperature amplification technology has been developed, but the simple constant temperature amplification technology usually faces the problems of poor specificity and inconvenient display of test results

Method used

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  • One-step nucleic acid test method based on CRISPR/Cas and isothermal amplification and kit

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Embodiment 1

[0041] In the embodiment of the present invention, based on CRISPR / Cas12a technology, on-site rapid lateral flow immunochromatography test paper detects nucleic acid. This method is used for non-diagnostic or therapeutic purposes, and includes the following steps:

[0042] 1) Design and transcribe crRNA: find the crRNA target sequence (such as SEQ ID NO: 1) that conforms to the TTTN 18N principle in the target nucleic acid sequence to be tested, and determine the crRNA sequence from it. RPA upstream and downstream amplification primers (such as SEQ ID NO: 2-3) were designed within 200 bp of both ends of the target sequence.

[0043] The method of crRNA in vitro transcription is as follows: react at 37°C for 16 hours in the following system: add Nuclease-free water to 20 μL, NTP 1.5 μL each, 10×reaction buffer 1.5 μL, Template DNA 1 μg, T7RNA Polymerase Mix 1.5 μL.

[0044] CrRNA purification method: Mix 20 μL of in vitro transcription product with 160 μL nuclease-free water an...

Embodiment 2

[0052] In the embodiment of the present invention, based on CRISPR / Cas12a technology, on-site rapid fluorescent detection of nucleic acid, the method is used for non-diagnostic or therapeutic purposes, including the following steps:

[0053] 1) Design and transcribe crRNA: find the crRNA target sequence (such as SEQ ID NO: 1) that conforms to the TTTN 18N principle in the target nucleic acid sequence to be tested, and determine the crRNA sequence from it. RPA upstream and downstream amplification primers (such as SEQ ID NO: 2-3) were designed within 200 bp of both ends of the target sequence.

[0054] The method of in vitro transcription of crRNA is as follows: react at 37°C for 16 hours in the following system: add Nuclease-free water to 20 μL, NTP 1.5 μL each, 10×reaction buffer 1.5 μL, Template DNA 1 μg, T7 RNA Polymerase Mix 1.5 μL.

[0055] CrRNA purification method: Mix 20 μL of in vitro transcription product with 160 μL nuclease-free water and 20 μL 3M ammonium acetate,...

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Abstract

The invention discloses a one-step nucleic acid test method based on CRISPR / Cas (clustered regularly interspaced short palindromicrepeats / CRISPR-associated protein) and isothermal amplification and akit. The method is a quick field test method used for testing nucleic acid and displaying visual test results through fluorescent light or lateral side immunochromatographic test paper. The test accuracy is effectively ensured through specificity of crRNA (RISPR-derived ribonucleic acid); at a room temperature, whether a test sample contains test nucleic acid can be determined through the test paper or the fluorescent light within 1-2h; and compared with the traditional test method, the precision, simplicity and convenience in operation of the test method are improved to some extent. The kit with purified freeze-drying LbaCas12a, crRNA and RPA (recombinase polymerase amplification) can be formed; a nucleic acid segment of a specific sequence can be conveniently and quickly tested on site;a complicated temperature control instrument such as a PCR (polymerase chain reaction) instrument and other electrophoresis and centrifugation equipment are not required; and nucleic acid of the specific sequence can be tested quickly and sensitively only by a thermostatic device and fluorescence detection equipment and even visual observation.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a CRISPR / cas system and an RPA constant temperature amplification system, in particular to a one-step nucleic acid detection method and kit based on CRISPR / Cas12a and constant temperature amplification. Background technique [0002] Nucleic acid detection is of great significance in the fields of disease diagnosis and pathogen detection. Among them, in the field of pathogen detection, because nucleic acid fragments are more stable than protein antigens, and are not as susceptible to sample background as RNA, pathogen nucleic acid detection technology using pathogen nucleic acid-specific sequences is of great significance. [0003] Current nucleic acid detection methods include RT-PCR, Real-Time qPCR, gene chips, LAMP, NASBA, HRM, PCR, RPA, and Oxford Nanopore sequencing technologies. When the RT-PCR method is used for nucleic acid detection, reverse transcription is first performed, RN...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6844
CPCC12Q1/6844C12Q2563/107C12Q2521/327C12Q2565/625
Inventor 黄黎珍白静林浩斯周阳汪凯婷李浩健刘俊杉武鹭婷杜红丽
Owner SOUTH CHINA UNIV OF TECH
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