Nucleic acid detection method based on PCR amplification and CRISPR-Cas12a and application

An amplification reaction, nucleic acid technology, applied in the fields of molecular biology and microbiology, can solve the problems of long time required for isolation and culture, misdiagnosis, delayed treatment and other problems

Active Publication Date: 2021-09-14
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Nocardia farcinica (N. farcinica) is one of the most common pathogens causing nocardiosis. It is very similar to mycobacteria in morphology and is easily confused, leading to misdiagnosis
For many nocardiosis patients, misdiagnosis can le

Method used

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  • Nucleic acid detection method based on PCR amplification and CRISPR-Cas12a and application
  • Nucleic acid detection method based on PCR amplification and CRISPR-Cas12a and application
  • Nucleic acid detection method based on PCR amplification and CRISPR-Cas12a and application

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1. Construction of CRISPR-Pa method

[0032] The workflow of CRISPR-Pa disclosed by the present invention:

[0033] Such as figure 1 As shown, the CRISPR-Pa platform includes the following three steps: 1. Sample pretreatment and DNA extraction, about 20 minutes; 2. PCR amplification, 60 minutes; 3. CRISPR-based fluorescence detection, 10 minutes. The entire CRISPR-Pa detection process can be completed within 90 minutes.

[0034] The detection principle of CRISPR-Pa disclosed in the present invention:

[0035] Such as figure 2 As shown, the detection process after DNA extraction is divided into two steps: 1. The target sequence is exponentially amplified by PCR. In the annealing stage of PCR, the upstream and downstream primers modified with the PAM site: ATTTA (SEQ ID NO: 1) bind to the template DNA strand, and are copied together with the template DNA strand in the amplification stage. After PCR amplification, the upstream of the target amplicon carrie...

Embodiment 2

[0062] Example 2. Sensitivity evaluation of Nocardia melioides CRISPR-Pa detection system

[0063] CRISPR-Pa was performed using the genomic DNA of the standard strain IFM 10152 of Nocardia melioli, which was serially diluted, and the results were as follows: Figure 5 show, Figure 5 Among them, 1-9 are 10ng, 1ng, 100pg, 10pg, 1pg, 100fg, 10fg, 1fg template DNA per reaction and blank control respectively. The lower limit of detection of Nocardia mallei CRISPR-Pa detection system for pure culture bacteria is 1 pg of template DNA.

Embodiment 3

[0064] Example 3. Optimization of the reaction conditions of the Nocardia melioides CRISPR-Pa detection system

[0065] Under standard reaction system conditions, the PCR amplification annealing temperature is carried out at 61-73° C. (2° C. increment). After PCR amplification, 2% agarose gel electrophoresis was used to detect and evaluate the amount of PCR amplification products at different annealing temperatures, and CRISPR detection was used to further evaluate the detection results. Such as Image 6 As shown, positive amplification can occur when the standard reaction system is annealed at 61-73°C, and 69-73°C is the best. In CRISPR assays, such as Figure 7 As shown, all positive results appeared within 10 minutes of real-time fluorescence signal detection, and the fluorescence value of the PCR product annealed at 69°C was the highest. Therefore, we believe that the optimal reaction time for N. mallei CRISPR-Pa detection at 37°C is 10 minutes, and the optimal annealin...

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Abstract

The invention discloses a nucleic acid detection method based on CRISPR-Cas12a and an application. According to the method, the 5'end of a PCR upstream primer or the 3 'end of a PCR downstream primer is modified with the PAM sequence so as to detect any nucleic acid fragment by using the CRISPR-Cas12a-based method, and even if the target fragment does not contain the PAM sequence, a nucleic acid detection platform can be used for identifying the nocardia gangrene, and has characteristics of simple and convenient detection and high specificity.

Description

technical field [0001] The invention discloses a nucleic acid detection method based on PCR amplification and CRISPR-Cas12a, belonging to the fields of molecular biology and microbiology. Background technique [0002] The CRISPR-Cas system composed of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas protein) is considered to be the adaptive immunity of archaea and bacteria. system. The CRISPR-Cas system is not only applied to gene editing, but also becomes an attractive tool for nucleic acid detection, showing the great potential of next-generation molecular diagnostic methods. In 2018, Chen et al. reported their discovery that when the Cas12a protein cleaves double-stranded DNA (dsDNA) in a sequence-specific manner, it induces robust nonspecific single-stranded DNA (ssDNA) cleavage in trans. The researchers used the trans-cleavage activity of the Cas12a protein to establish the DETECTR (DNA Endonuclease-Targeted CRISPR ...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/686C12Q1/04C12R1/365
CPCC12Q1/689C12Q1/686C12Q2521/327C12Q2525/161C12Q2525/151C12Q2525/131Y02A50/30
Inventor 李振军邱小彤徐帅
Owner ICDC CHINA CDC
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