MGMT gene methylation detection primer probe system and kit thereof

Abstract

The invention discloses an MGMT gene methylation detection primer probe system and a kit thereof.The MGMT gene methylation detection primer probe system comprises a primer probe set A used for detecting methylated genes and a primer probe set B used for detecting unmethylated genes, the nucleotide sequence of an upstream primer a of the primer probe set A is shown as SEQ ID NO.1, the nucleotide sequence of a downstream primer a of the primer probe set A is shown as SEQ ID NO.2, and the nucleotide sequence of the downstream primer a of the primer probe set B is shown as SEQ ID NO.2; the nucleotide sequence of the probe a is as shown in SEQ ID NO.3; a nucleotide sequence of an upstream primer b on the primer probe group B is shown as SEQ ID NO.4, a nucleotide sequence of a downstream primer b on the primer probe group B is shown as SEQ ID NO.5, and a nucleotide sequence of a probe b on the primer probe group B is shown as SEQ ID NO.6; different reporter fluorophores are arranged at the 5'ends of the probe a and the probe b. By adopting the primer probe system, the methylation level of the human genome MGMT gene can be rapidly and accurately determined.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer probe system for detecting methylation of an MGMT gene and a kit thereof. Background Art

[0002] DNA methylation refers to the covalent bonding of a methyl group to the 5th carbon position of cytosine in the CpG dinucleotide of the genome under the action of DNA methyltransferase. Methylation is an enzymatic catalytic reaction that is ubiquitous in the human body, and abnormal methylation levels are generally associated with many diseases. The frequency of CpG sequences in the mammalian genome is only about 1%, which is much lower than other dinucleotide sequences in the genome. However, in some regions, the density of CpG sequences is very high, which can reach more than 5 times the average, becoming a cytosine and guanine-rich area, forming the so-called CpG island. CpG islands are usually located in the promoter region or the first exon region of a gene. In the genome of a healthy person, CpG sites in CpG islands are usually in a non-methylated state, while CpG sites outside CpG islands are usually methylated. Determining the degree of methylation by testing circulating tumor cells or free DNA (cfDNA) in blood or other body fluids has important clinical significance for early detection and screening of tumors.

[0003] MGMT, O6-methylguanine-DNAmethyltransferase, O6-methylguanine-DNA methyltransferase. It is a DNA repair enzyme. It is located on the long arm of human chromosome 10. Its basic function is to repair damaged guanine nucleotides by transferring the methyl group at the guanine O6 site to the cysteine ​​residue, thereby avoiding gene mutations, cell death and tumorigenesis caused by alkylating agents. Therefore, the MGMT gene is a factor causing genetic chemotherapy resistance. MGMT is mainly regulated by epigenetics of the methylation state of the MGMT promoter. DNA methylation is the most widely studied epigenetic modification. It plays a vital role in gene regulation by affecting chromatin accessibility and thus gene transcription. Methylation of CpG dinucleotides (CpGs) in the promoter region usually leads to gene transcriptional inactivation. As an early discovered chemotherapy sensitivity-related gene, MGMT plays an important role in the treatment of gliomas and has a high frequency of promoter region methylation. The MGMT promoter contains a CpG island consisting of 98 CpGs and extends into noncoding exon 1 (CpGs 74–83; NCBI reference sequence: NG_052673.1) [within the CpG island, a minimal promoter (7 bp, CpGs 88–50; −173--86 according to NCBI exon 62 coordinates) and an enhancer region (1 bp, CpGs 59–82; +87 to +40) have been identified. Hypermethylation of the MGMT promoter results in transcriptional silencing and is associated with a survival benefit from treatment with the alkylating agent temozolomide (TMZ). Since clinical trials have confirmed this finding, MGMT promoter methylation status has become a predictive biomarker for TMZ response, particularly in an elderly patient population with newly diagnosed glioblastoma (GBM). In addition, multiple studies have confirmed that MGMT promoter methylation can serve as a prognostic biomarker for GBM.

[0004] Among the commonly used methylation detection methods, the pyrophosphate sequencing method is cumbersome and time-consuming, and the interpretation of the results requires a certain technical foundation. The fluorescent probe method requires the establishment of a fluorescent quantitative PCR platform, and the sensitivity and specificity are closely related to the primer design. The BSP method can only target known CpG site regions, and the experimental results are related to the number of selected clones. The more clones are selected, the more accurate the results. The existing QPCR and NGS methods for detecting MGMT methylation have low sensitivity and are all relative quantitative, and cannot accurately measure the actual level of methylation. Summary of the invention

[0005] Technical problems solved: In response to the above technical problems, the present invention provides a primer probe system and a kit for detecting methylation of the MGMT gene, which can effectively solve the shortcomings of existing QPCR or NGS methods for detecting MGMT methylation, such as low sensitivity, relative quantification, and inability to accurately determine the actual level of methylation.

[0006] Technical solution: In a first aspect, the present invention provides a primer probe system for detecting methylation of the MGMT gene, comprising a primer probe set A for detecting methylated genes and a primer probe set B for detecting unmethylated genes. The primer probe set A comprises an upstream primer a, a downstream primer a and a probe a, the nucleotide sequence of the upstream primer a is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer a is shown in SEQ ID NO.2, and the nucleotide sequence of the probe a is shown in SEQ ID NO.3; The primer probe set B comprises an upstream primer b, a downstream primer b and a probe b, wherein the nucleotide sequence of the upstream primer b is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer b is shown in SEQ ID NO.5, and the nucleotide sequence of the probe b is shown in SEQ ID NO.6; The 5' ends of the probe a and the probe b are provided with different reporter fluorescent groups.

[0007] Preferably, the final concentration of each primer used in the PCR reaction is 900 nM, and the final concentration of each probe used in the PCR reaction is 250 nM.

[0008] Preferably, the reporter fluorescent group is FAM or VIC.

[0009] Preferably, the probability of MGMT gene methylation = the number of copies of the fluorescence carried by reporter probe a ÷ (the number of copies of the fluorescence carried by reporter probe a + the number of copies of the fluorescence carried by reporter probe b).

[0010] In a second aspect, the present invention provides a MGMT gene methylation detection kit comprising the primer-probe system described in the first aspect.

[0011] Beneficial effects: The primer-probe system of the present invention can be used to rapidly and accurately determine the methylation level of the MGMT gene in the human genome, and has the advantages of absolute quantification and high sensitivity, which is of great significance for the development of methylation diagnostic kits and cross-validation of other MGMT methylation detection methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a schematic diagram of the test results of sample 1 in Example 1 of the present invention; Figure 2It is a schematic diagram of the detection results of sample 1 in Example 2 of the present invention. DETAILED DESCRIPTION

[0013] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments: Example 1

[0014] The steps of using the primer-probe system of the present invention to detect the methylation probability of the sample to be tested (the sample verified by pyrophosphate sequencing and BSP sequencing) are as follows: 1. Pretreatment of the sample to be tested: Use the DNA methylation modification kit (EZ DNA Methylation-Lightning, KitZYMO RESEARCH) to treat the DNA of the sample to be tested with sulfite to obtain a sulfite-treated sample; 2. Synthesize primers and probes: According to the sequence in Table 1 below, entrust Nanjing Kebai Biotechnology Co., Ltd. to complete the synthesis.

[0015] Table 1 Primer-probe system of the present invention

[0016] Note: The 5' end of the probe MGMT-MP is modified with FAM, and the 5' end of the probe MGMT-UM-P is modified with VIC.

[0017] 3. Test the samples after sulfite treatment using Bio-Rad's ddPCR supermix for probes: 3.1. Prepare the PCR reaction system as shown in Table 2 below: Table 2 PCR reaction system

[0018] Note: The concentration of DNA after sulfite treatment is 50 ng / μL, the concentrations of upstream primer a, downstream primer a and probe a in primer probe set A are 450nM, 450nM and 250nM respectively, and the concentrations of upstream primer b, downstream primer b and probe b in primer probe set B are 450nM, 450nM and 250nM respectively.

[0019] 3.2. The setting of the reaction program is shown in Table 3 below: Table 3 PCR reaction procedure

[0020] Note: The heating rate is 2°C / sec., the cover temperature is set to 105°C, and the sample volume is set to 40 μL.

[0021] 3.3 Results and Analysis The experimental results are as follows Figure 1-Figure 2The corresponding data are shown in Table 4 below: Table 4 Experimental results

[0022] The results show that the results of the primer-probe system of the present invention are consistent with the known sample types, and can detect whether the sample is methylated or not. The method of the present invention can directly quantify the methylation probability, with higher efficiency and more intuitive data. Compared with other sequencing methods, the method of the present invention takes less time and is more efficient. Example 2

[0023] The positive samples and negative samples were mixed in different proportions, and the primer-probe system of the present invention was used for detection according to Example 1. The results are shown in Table 5 below: Table 5 Sample combinations with different proportions and test results

[0024] The results showed that the detection process showed high specificity and appropriate sensitivity, and the target substances could be detected in positive samples. Example 3

[0025] Low concentration samples were prepared, numbered 6-8, and the primer-probe system of the present invention was used for detection according to Example 1. The results are shown in Table 6 below: Table 6 Low concentration samples and their test results

[0026] The results show that the accuracy and sensitivity of detection can be guaranteed even for low-copy and low-concentration samples.

[0027] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer-probe system for detecting methylation of the MGMT gene, characterized in that: The method comprises a primer probe set A for detecting methylated genes and a primer probe set B for detecting unmethylated genes. The primer probe set A comprises an upstream primer a, a downstream primer a and a probe a, the nucleotide sequence of the upstream primer a is shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer a is shown in SEQ ID NO.2, and the nucleotide sequence of the probe a is shown in SEQ ID NO.3; The primer probe set B comprises an upstream primer b, a downstream primer b and a probe b, wherein the nucleotide sequence of the upstream primer b is shown in SEQ ID NO.4, the nucleotide sequence of the downstream primer b is shown in SEQ ID NO.5, and the nucleotide sequence of the probe b is shown in SEQ ID NO.6; The 5' ends of the probe a and the probe b are provided with different reporter fluorescent groups.

2. The MGMT gene methylation detection primer-probe system according to claim 1, characterized in that: The final concentration of each primer used in the PCR reaction was 900 nM, and the final concentration of each probe used in the PCR reaction was 250 nM.

3. The MGMT gene methylation detection primer-probe system according to claim 1, characterized in that: The reporter fluorescent group is FAM or VIC.

4. The MGMT gene methylation detection primer-probe system according to claim 1, characterized in that: The probability of MGMT gene methylation = the number of copies of the fluorescence carried by reporter probe a ÷ (the number of copies of the fluorescence carried by reporter probe a + the number of copies of the fluorescence carried by reporter probe b).

5. A MGMT gene methylation detection kit comprising the primer-probe system as claimed in claim 1.

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