Detection method based on Cas protein

Abstract

The invention relates to a detection method based on Cas protein. The detection method comprises the following steps: S1, designing and synthesizing a PCR amplification primer according to a target gene; synthesizing crRNA according to the PCR amplification primer; and S2, performing PCR amplification on the target gene by adopting a PCR amplification primer to obtain an amplification product; detecting an amplification product by using a Cas protein, crRNA and a fluorescent probe system; wherein the sequence of the primer comprises a PAM sequence and a crRNA recognition region sequence. The invention breaks through the limitation of the PAM sequence and the crRNA, and even if no PAM sequence exists near the mutation site to be detected or proper crRNA cannot be designed, the PAM sequence and the crRNA can be manually introduced through the primer for detection. The primer-mediated Cas12a qualitative detection can detect a plurality of variation sites in one reaction system, so that the capability of multiple detection is reflected, the operation steps are reduced, and the detection cost is reduced.

Description

technical field [0001] The invention relates to a method for detecting nucleic acid molecules based on Cas protein. Background technique [0002] Clustered regularly interspaced short palindrome repeats (CRISPR) and CRISPR-associated nucleases (CRISPR associated proteins, Cas), which are ubiquitous in bacteria and archaea, are the defense of bacteria against foreign plasmid or phage infection Scientists have successively discovered a variety of Cas proteins and their working mechanisms. Cas12a (also known as Cpf1) specifically recognizes the target double-stranded DNA (double stranded DNA, dsDNA) under the guidance of crRNA and is activated. The activated Cas12a can not only specifically cut the target dsDNA molecule, but also non-specifically cut irrelevant single-stranded DNA (single stranded DNA, ssDNA), that is, with cleavage activity. [0003] Using the incidental cleavage activity of Cas12a to detect target dsDNA molecules, the principle of the existing technical sch...

AI Technical Summary

Problems solved by technology

If there is no PAM sequence in the target sequence, crRNA cannot be designed, so Cas12a cannot be used for detection

PAM sequences greatly limit the choice of target sequences and the flexibility of crRNA design

[0008] 2. crRNA restriction

[0010] 3. Limited to qualitative detection

[0011] Most of the existing Cas12a nucleic acid detection technologies are qualitative detection. Qualitative detection can only determine the presence or absence of target nucleic acid molecules, but cannot determine the number of copies of target nucleic acid molecules

Only a few studies have conceptually achieved quantitative detection [3,4] , but in practical applications, there are often concentration differences between samples. In order to compare the differences in the target nucleic acid copy number between samples, it is also necessary to set up internal references to eliminate the differences in quantitative detection results caused by sample concentration differences.

[0012] 4. Limited to pathogen detection

[0017] Because the mutation type of β-thalassemia is mainly point mutation, the mutation is limited to one or several bases, and there is often a lack of PAM sequence near it or it is difficult to design a suitable crRNA, so it is difficult to use the existing Cas12a nucleic acid detection technology to detect detection

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