CN-121992081-A - Detection method for isothermal one-pot DNA methylation based on MSRE-RPA-CRISPR

Abstract

The invention provides a detection method of isothermal one-pot DNA methylation based on MSRE-RPA-CRISPR, and relates to the technical field of gene detection. The invention provides a buffer solution system and application of an RPA upstream mutation primer in MSRE-RPA-CRISPR-based isothermal one-pot DNA methylation detection, wherein the buffer solution system comprises a Twist buffer solution, and the nucleotide sequence of the RPA upstream mutation primer is a mutant inserted into an MSRE recognition site at the 3' -end of the RPA upstream primer. The buffer solution system and the RPA upstream mutation primer of the invention can realize constant temperature and high sensitivity detection of one-pot DNA methylation without step separation.

Inventors

• LIU RUOCHEN
• CUI HONGJUAN
• SONG WENJIA

Assignees

• 西南大学

Dates

Publication Date

20260508

Application Date

20260304

Claims (10)

1. 1. The application of the buffer solution system and the RPA upstream mutation primer in the isothermal one-pot method DNA methylation detection based on MSRE-RPA-CRISPR is characterized in that the buffer solution system comprises a Twist buffer solution, and the nucleotide sequence of the RPA upstream mutation primer is a mutant inserted into an MSRE recognition site at the 3' -end of the RPA upstream primer.
2. 2. The use of claim 1, wherein the MSRE recognition site comprises GCGC, CCGG, CGCG or CCGC.
3. 3. The use according to claim 1 or 2, characterized in that the mutant is a mutation of any one or several bases of the MSRE recognition site.
4. 4. The use according to claim 1, wherein the nucleotide sequence of the RPA upstream mutation primer is shown in SEQ ID No. 1.
5. 5. A composition for detecting DNA methylation by an isothermal one-pot method based on MSRE-RPA-CRISPR, which is characterized by comprising a buffer system, an RPA upstream mutation primer and crRNA; The buffer solution system comprises a Twist buffer solution, the nucleotide sequence of the RPA upstream mutation primer is a mutant inserted into an MSRE recognition site at the 3' end of the RPA upstream primer, and the nucleotide sequence of the crRNA is designed according to 0-2 bases at the downstream interval of the 3' end of the RPA upstream primer and the downstream interval of the 3' end of the RPA downstream primer.
6. 6. The composition of claim 5, wherein the nucleotide sequence of the RPA upstream mutation primer is set forth in SEQ ID No. 1.
7. 7. The composition of claim 5, wherein the crRNA comprises crRNA-1 and crRNA-2, the nucleotide sequence of crRNA-1 is shown in SEQ ID No.3, and the nucleotide sequence of crRNA-2 is shown in SEQ ID No. 4.
8. 8. The composition of claim 5, further comprising a fluorescent probe, an RPA downstream primer, and an MSRE.
9. 9. The composition of claim 8, wherein the MSRE is selected from any one of HhaI, aciI, hpaII and BstUI.
10. 10. A method for detecting DNA methylation by an isothermal one-pot method based on MSRE-RPA-CRISPR is characterized by comprising the following steps of reacting a sample DNA to be detected with the composition according to any one of claims 5-9 for 30-70 min at 37-42 ℃ and then observing and detecting.

Description

Detection method for isothermal one-pot DNA methylation based on MSRE-RPA-CRISPR Technical Field The invention belongs to the technical field of gene detection, and particularly relates to a detection method for isothermal one-pot DNA methylation based on MSRE-RPA-CRISPR. Background Methylation of genomic DNA is an evolutionarily conserved epigenetic regulatory mechanism, primarily referring to the covalent bonding of a methyl group at the 5 th carbon atom of cytosine (Cytosine; C) to a process of 5-methylcytosine (5-mC). DNA methylation has profound effects on gene expression and is thus widely involved in animal development and disease progression. Methylation modification disorder is closely related to various diseases of human beings, such as cancers, diabetes, cardiovascular and cerebrovascular diseases and the like, so that DNA methylation becomes a biomarker which is widely researched and used, and the accurate detection of the biomarker is of great significance to diagnosis and treatment of the diseases. The core step in DNA methylation detection is the transformation extraction of methylation information encoded on DNA sequences, i.e. the recognition of methylated and unmethylated cytosines. The most commonly used and gold standard method at present is the bisulfite conversion method, after treatment the unmethylated cytosines are converted to uracil (U), while the methylated cytosines remain unchanged, and then are subjected to downstream molecular detection, such as clone sequencing, methylation-specific PCR, etc. However, the method is complex in operation and depends on professionals and professional equipment, and the reaction needs to be carried out under severe conditions such as high temperature, alternating strong acid/strong alkali, long-time reaction and the like, so that DNA degradation and detection failure are extremely easy to cause. In contrast, the DNA methylation detection method based on the Methylation Sensitive Restriction Enzyme (MSRE) digestion method utilizes the recognition of the MSRE on the methylated and unmethylated sequences to convert and extract the methylation information encoded on the DNA sequence, i.e., the cleavage site containing unmethylated cytosine is recognized and cleaved, while the site containing methylated cytosine can resist cleavage and remain intact. And after digestion, the downstream is connected with nucleic acid amplification detection methods such as Real-time PCR, RPA-CRISPR and the like to realize qualitative or quantitative detection of methylation level. The MSRE-RPA-CRISPR method has mild conditions and avoids the degradation of DNA. However, the operation is still cumbersome, and the professional personnel and professional equipment are required. The method involves multi-step pipetting and multitube operations, i.e. the aspiration of small amounts of the products of the previous reaction in the subsequent reaction to minimize the impact on the next reaction system. However, such an operation not only increases the complexity of the operation, but also fails to make full use of the product of the previous reaction, especially in the field of methylation analysis, because the target DNA content in the sample is low (such as cell-free DNA), thereby decreasing the detection sensitivity. In addition, the current method requires temperature change treatment to inactivate MSRE to avoid interference to subsequent RPA and CRISPR reactions, which not only increases operation complexity and prolongs detection time, but also fails to realize isothermal detection. Thus, currently, single tube, isothermal, rapid, simple DNA methylation detection is not possible with either bisulfite conversion or MSRE-RPA-CRISPR based methods. Therefore, it is highly desirable to provide a simple, rapid and sensitive method for detecting DNA methylation in a single tube. Disclosure of Invention In view of the above technical problems, a first object of the present invention is to provide a buffer system and an application of an RPA upstream mutation primer in an isothermal one-pot DNA methylation detection based on MSRE-RPA-CRISPR. The buffer solution system can be compatible with a restriction endonuclease digestion reaction, an RPA isothermal amplification reaction and a CRISPR-Cas12a cutting reaction, and the RPA upstream mutation primer not only maintains the RPA amplification efficiency, but also ensures that the amplicon is not recognized and cut by MSRE any more, and the constant-temperature and high-sensitivity detection of one-pot DNA methylation is realized without step by step. A second object of the present invention is to provide a composition for detecting DNA methylation based on the MSRE-RPA-CRISPR isothermal one-pot method. The third object of the invention is to provide a detection method of isothermal one-pot DNA methylation based on MSRE-RPA-CRISPR. In order to achieve the above object, the present invention provides the following technical solution