CRISPR/Cas12a one-step nucleic acid detection method

Abstract

The invention provides a CRISPR/Cas12a one-step nucleic acid detection method. The method comprises the following steps of 1, selecting a target sequence of bacteria to be detected, designing upstream and downstream primers of RPA, performing specific screening, amplifying the target sequence to obtain a primer amplicon sequence, and performing specific verification on the primer amplicon sequence by utilizing NCBI BLAST; 2, designing specific crRNA; 3, designing a single-stranded DNA reporter molecule; 4, mixing the single-stranded DNA reporter molecule, the RPA upstream and downstream primers, freeze-dried RPA reaction particles, magnesium acetate, an RPA hydration buffer solution, NEB buffer 2.1, RNase Inhibitor and a target to be detected to obtain a reaction mixture; 5, constructing a one-step detection kit comprising a reactant placing tube and an injector, placing the reaction mixture at the bottom of the reactant placing tube, and preloading crRNA and Cas12a protein into the injector; and 6, after the reaction mixture reacts, pushing a piston of the injector to enable the crRNA and the Cas12a protein to be mixed with the reaction mixture for a reaction, and performing blue light excitation to obtain a detection result.

Description

technical field [0001] The invention belongs to the technical field of gene detection, in particular to a CRISPR / Cas12a one-step nucleic acid detection method. Background technique [0002] Listeria monocytogenes (Listeria monocytogenes, LM) is an important class of food-borne pathogens, which can penetrate the intestinal barrier, placental barrier and blood-brain barrier, with a mortality rate of 20% to 30%. Human safety and health. The pathogenicity of Listeria monocytogenes is closely related to virulence factors, and rapid and accurate identification of Listeria monocytogenes-related virulence genes can provide an important reference for the study of its pathogenicity. [0003] At present, the main method of gene level detection is to amplify the target gene by polymerase chain reaction, and the amplified product is visualized by agarose gel electrophoresis. Quantitative PCR or fluorescent real-time PCR uses TaqMan probes and fluorescent dyes for real-time detection on...

AI Technical Summary

Problems solved by technology

Although this method has high sensitivity, it prolongs the detection time and still needs to open the cover, which is easy to cause aerosol pollution and false positives, which is not conducive to practical application

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