CN110923300A - Gene methylation detection method and application - Google Patents
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Abstract
The invention discloses a gene methylation detection method, which comprises the steps of digesting a sample to be detected containing a target gene by using restriction enzyme BstUI, carrying out PCR amplification on an enzyme digestion product, and judging the methylation condition of the target gene according to the PCR amplification result. The method greatly improves the sensitivity and specificity of the gene methylation detection through the unique design of the PCR primer.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a detection method and application of gene methylation.
Background
DNA methylation refers to the selective addition of methyl to cytosine at two nucleotides of DNA CG under the catalysis of methyltransferase to form 5-methylcytosine, which is commonly found in 5'-CG-3' sequences of genes. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.
When tumors occur, the non-methylation degree of CpG sequences except the CpG island of the cancer suppressor gene is increased, and the CpG in the CpG island is in a high-methylation state, so that the expression of the cancer suppressor gene is reduced. Therefore, the study of gene methylation is of great importance for early screening of cancer.
Taking colorectal cancer as an example, research shows that methylation of CpG island regions of a plurality of cancer suppressor gene promoters is accompanied in the process of colorectal cancer occurrence, which provides theoretical basis for developing a new colorectal cancer screening technology. The FDA has approved the product Cologuard for colorectal cancer screening in 2014, whose detection markers include BMP3 and NDRG4 gene methylation, which is mainly targeted to populations in north america. In China, two products for carrying out methylation detection on Septin9 gene in free DNA of plasma and SDC2 gene in DNA of feces for auxiliary diagnosis of colorectal cancer are already approved to be on the market, which is beneficial to carrying out early diagnosis and early screening work of colorectal cancer in a large range.
Although the products for detecting gene methylation are already approved for clinical use, the existing products generally have single detection markers and the traditional sulfite conversion and fluorescence quantitative PCR detection methods, so the sensitivity and specificity of the products are still to be improved.
In the traditional sulfite conversion, unmethylated cytosine (C) in nucleic acid is converted into uracil (U) by utilizing sulfite, methylated cytosine is kept unchanged, the methylated CpG island and the unmethylated CpG island have obvious sequence difference after conversion, and the methylation specific primer is designed to carry out fluorescence quantitative PCR so as to detect whether target gene methylation exists in sample nucleic acid, so that the method has extremely high specificity. However, sulfite conversion causes severe fragmentation of DNA and a large amount of DNA is lost after purification, thereby greatly affecting detection sensitivity and causing false negative detection results.
In addition, the plasma free DNA is from the whole body of a human body and has extremely low concentration, and the DNA derived from intestinal tumor cells is trace and has extremely high individual difference; the fecal nucleic acid is mainly intestinal flora DNA, the human DNA contained in the fecal nucleic acid comes from exfoliated cells of the whole digestive tract and DNA released by degradation of the exfoliated cells, the proportion of the human DNA is only less than 1 percent of the total DNA, and the proportion of the DNA derived from tumor cells is lower. Therefore, how to efficiently use the originally trace sample DNA for detection to achieve the purpose of improving the sensitivity is very important.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method which is different from the traditional sulfite conversion method, combines a unique primer design to carry out fluorescence quantitative PCR (polymerase chain reaction) for detection, and greatly improves the detection sensitivity of gene methylation.
The technical scheme adopted by the invention is as follows:
a method for detecting gene methylation comprises the following steps:
(1) digesting a sample to be detected containing a target gene by using a restriction enzyme BstUI to obtain an enzyme digestion product;
(2) performing PCR amplification on the enzyme digestion product obtained in the step (1), wherein a PCR amplification primer group comprises a target gene primer group, and the 3' tail end of an upstream primer and/or a downstream primer of the target gene primer group is ' CGC ', ' CGCG ' or ' CGCG ' and 1 to 2 bases of a template sequence behind the ' CGC ', preferably ' CGC '; in addition, the number of the "CGCG" sites contained in the primer sequence can be selected according to the specific template sequence, and the more the number of the "CGCG" sites is contained, the higher the specificity is, but the GC proportion of the primer needs to be considered;
(3) and judging the methylation condition of the target gene according to the PCR amplification result.
Preferably, the amplicon of the primer set for the target gene comprises at least one "CGCG" site. The number of "CGCG" sites between the upstream and downstream primers can be selected according to the target sequence, and the amplification specificity is higher as the number of "CGCG" sites covered by the preferred amplification region is larger.
Further preferably, the PCR amplification primer set further comprises an internal standard gene primer set, and the internal standard gene primer set and the amplicon thereof do not contain a "CGCG" site.
In the above detection method, the target gene comprises at least one of SFRP2, NDRG4 and SDC2 genes, preferably SFRP2, NDRG4 and SDC2, and the internal standard gene comprises at least one of ACTB gene or other suitable housekeeping genes or conserved gene sequences with stable copy number, such as GAPDH gene, α -tublin gene, preferably ACTB gene.
Further preferably, a GC enhancer is added to the PCR amplification reaction system, wherein the GC enhancer comprises 20-50mM, preferably 35mM of tetramethylammonium chloride and 6% -15% (v/v), preferably 8% of glycerol.
A gene methylation detection kit, comprising: restriction endonuclease BstUI, a target gene amplification primer, wherein the 3' end of an upstream primer and/or a downstream primer of a target gene amplification primer group is ' CGC ', ' CGCG ' or ' CGCG ' and 1 to 2 bases of a template sequence behind the ' CGC ', preferably ' CGC '; in addition, the number of "CGCG" sites contained in the primer sequence can be selected according to the specific template sequence, and the more the number of "CGCG" sites is contained, the higher the specificity is, but the GC ratio of the primer needs to be considered.
Preferably, the amplicon of the primer set for the target gene comprises at least one "CGCG" site. The number of "CGCG" sites between the upstream and downstream primers can be selected according to the target sequence, and the amplification specificity is higher as the number of "CGCG" sites covered by the preferred amplification region is larger.
Further preferably, the kit further comprises an internal standard gene primer group, and the internal standard gene primer group and an amplicon thereof do not contain a CGCG locus.
Further preferably, the kit further comprises a GC enhancer comprising tetramethylammonium chloride 20-50mM, preferably 35mM, and glycerol 6% -15% (v/v), preferably 8%.
Further preferably, the target gene comprises at least one of SFRP2, NDRG4 and SDC2 genes, preferably SFRP2, NDRG4 and SDC2, and the internal standard gene comprises at least one of ACTB gene or other suitable housekeeping genes or conserved gene sequences with stable copy number, such as GAPDH gene and α -tublin gene, preferably ACTB gene.
The invention has the beneficial effects that:
1. according to the method, the 3' end of the PCR primer is designed into 1 to 2 bases of CGC, CGCG or CGCG and the subsequent template sequence, so that the sensitivity and specificity of gene methylation detection are greatly improved.
2. The method is simple and convenient to operate, can be directly used for PCR detection after enzyme digestion, does not need to purify and recover enzyme digestion products, and can effectively improve the utilization rate of nucleic acid samples, thereby improving the detection sensitivity. The whole detection process only needs 2.5 hours, the detection process is greatly simplified compared with the traditional sulfite conversion method, and the detection efficiency is improved.
3. The method can take the excrement DNA as a detection sample, can sample at home, is completely noninvasive, has extremely high privacy and can improve the acceptance of the examinee.
Drawings
FIG. 1 is a schematic diagram of the detection of the method of the present invention.
FIG. 2 is a graph showing the detection of the internal standard gene ACTB.
FIG. 3 is a graph showing the SFRP2 gene detection.
FIG. 4 is a graph showing the NDRG4 gene detection.
Figure 5 is a graph of SDC2 gene testing.
Detailed Description
The present invention will be further described with reference to the following examples, but is not limited thereto.
The PCR primer and probe design used in the following examples was as follows:
fluorescent PCR primers and probes were designed for the target genes SFRP2, NDRG4 and SDC2, and the internal standard gene ACTB. The 3' end of the upstream primer and/or the downstream primer of the target gene primer group is ' CGC ', ' CGCG ' or ' CGCG ' and 1 to 2 bases of the template sequence after the ' CGC ', preferably ' CGC '; in addition, the number of the "CGCG" sites contained in the primer sequence can be selected according to the specific template sequence, and the more the number of the "CGCG" sites is contained, the higher the specificity is, but the GC proportion of the primer needs to be considered; the amplicon of the primer set for the target gene comprises at least one "CGCG" site. The number of "CGCG" sites between the upstream and downstream primers can be selected according to the target sequence, and the amplification specificity is higher as the number of "CGCG" sites covered by the preferred amplification region is larger. The internal standard gene primer group and the amplicon thereof do not contain CGCG locus. The action principle of the primer obtained by the design scheme is shown in figure 1, the specificity of PCR amplification mainly depends on the matching degree of the 3 'end of the primer and a template, the higher the matching degree is, the higher the amplification efficiency is, otherwise, the amplification efficiency is low, as BstUI restriction endonuclease cuts off an unmethylated CGCG locus from the middle, the template is broken, and the methylated CGCG locus is not cut by enzyme, the sequence integrity is guaranteed, the primer designed by the method can only be completely combined with the sequence which is not cut by enzyme, but can not be completely combined with the sequence which is cut by enzyme, the base at the 3' end is suspended, and can not be subjected to extension amplification, so the primer can only detect the methylated sequence, but the unmethylated sequence can not be amplified, and the primer has good specificity.
Specific primer sequences are shown in Table 1, and probe sequences are shown in Table 2.
TABLE 1 PCR amplification primer sequences
| Primer and method for producing the same | Sequences (5 '> 3') | Serial number |
| SFRP2-F | CGGAGCCCCCCGGAGCTGCGC | SEQ ID NO.1 |
| SFRP2-R | TGGCAGCCGGCGGCTGGGGCGC | SEQ ID NO.2 |
| SDC2-F | AGGAGGAGGGGCGCAGCCGC | SEQ ID NO.3 |
| SDC2-R | GCAGAGCGGCGGGAGCGC | SEQ ID NO.4 |
| NDRG4-F | GGGTGTCCCCCAGGCTCCGC | SEQ ID NO.5 |
| NDRG4-R | GTGGCTTCCGCCTTCTGCGC | SEQ ID NO.6 |
| ACTB-F | GATGACCCAGGTGAGTGGCCCGCTACCTC | SEQ ID NO.7 |
| ACTB-R | GAGAGAACCAGTGAGAAAGGACGCAG | SEQ ID NO.8 |
TABLE 2 fluorescent Probe sequences
Example 1: reference detection
First, experiment sample
The reference substances used in this example were composed as follows:
reference product A: 50ng/uL of wild DNA, the methylation ratio of a target gene is 0 percent;
reference product B: 50ng/uL of mixed DNA, wherein the methylation ratio of the target gene is 5%.
The wild type genomic DNA is extracted from healthy Human leukocyte genomic DNA, and the Methylated DNA is Human standardized Methylated DNA (Universal Methylated Human DNA Standard) purchased from ZYMORESEARCH, and the two are prepared according to a certain proportion.
And (3) carrying out sample treatment and detection by adopting an enzyme digestion and transformation method, and comparing the detection efficiency of the enzyme digestion and the detection efficiency of the sample treatment and the detection efficiency of the sample detection.
Second, Experimental methods
1. Transformation of
The EZ DNA Methylation-gold kit (D5042) of Zymo company is adopted for sample transformation and purification recovery, the nucleic acid transformation amount is 500ng, the elution volume is 50uL, and the theoretically recovered sample concentration is 10ng/uL according to 100% transformation rate and recovery efficiency.
2. Enzyme digestion
A10 uL sample of the reference sample was digested with the enzyme system shown in Table 3. The digested nucleic acid was not purified, and thus the concentration of the digested sample was 10 ng/uL.
TABLE 3 BstUI cleavage System
3. Detection of
Respectively diluting the samples recovered by the enzyme digestion and sulfite conversion treatment to a total volume of 250uL, wherein the theoretical concentration of the diluted samples is 2 ng/uL; the samples obtained by the transformation and the enzyme digestion treatment were subjected to fluorescent quantitative PCR detection 10 times according to the following system and procedure.
After the sample was treated by sulfite Conversion, the detection primers were different from the detection primers after the enzyme digestion treatment, and the amplification procedure was the same, and the sample was converted and purified by using the bisufite Conversion kit from ZYMO RESEARCH, and the detection primers are shown in the following table.
TABLE 4 sulfite conversion PCR amplification primer sequences
TABLE 5 fluorescent quantitative PCR amplification procedure
Third, the detection result
Two methods detect reference a (negative reference) and reference B (positive reference containing 5% methylated DNA). As shown in fig. 2, the reference substance a only detects an internal standard signal, and the detection result of the target gene is negative; as shown in FIGS. 3-5, the detection results of the target genes of reference B were positive, but the difference in ct values was significant.
As can be seen from fig. 2 to 5, after the sample is processed by the enzymatic digestion method, the ct value detected by each channel is about 3 times higher than that detected by the conversion method, so that the sample processed by the enzymatic digestion method has higher nucleic acid utilization rate and detection sensitivity.
Example 2: clinical sample detection contrast
Stool samples collected from 300 enteroscopy rooms were examined, 112 pathologically confirmed colorectal cancer patients, 65 adenoma patients, and 123 approximately normal colonoscopies. We used a sample from a colorectal cancer patient as a positive sample, a sample from a roughly normal enteroscopy as a negative sample, and a stool sample from an adenoma patient was analyzed separately.
1. Sample treatment: the method comprises the steps of extracting nucleic acid from a fecal sample by using a Qiagen fecal DNA rapid extraction kit, and then carrying out detection pretreatment on 10uL of nucleic acid sample by using methods of enzyme digestion and sulfite conversion (kit EZ DNA Methylation-gold kit (D5042)).
2. And (3) qPCR detection: and detecting the processed sample by adopting a PCR system of the first implementation.
3. Analysis of results
And (3) comparing the detection success rate: the internal standard detection positive is taken as the successful detection of the sample, the internal standard ct value of the sample processed by the enzyme cutting method is between 21 and 36, and the internal standard detection rate is 100 percent; the internal standard ct value of the sample processed by the sulfite conversion method is between 24 and 41, wherein no internal standard signal is detected in 12 samples, and the internal standard detection rate is 96%.
Detection sensitivity: and determining a positive judgment value on the premise of ensuring the detection specificity of the negative sample. Setting the specificity of the two methods for detecting clinical negative samples to be 93.0%, and determining positive judgment ct values of the two detection processes, wherein the specificity can reach 93.0% when the positive judgment value of the enzyme cutting method is that any one of target genes (SFRP2/SDC2/NDRG4) is detected to be positive and the ct value is less than 39; when the positive judgment value of the transformation method is that any one of target genes (SFRP2/SDC2/NDRG4) detects a ct value less than 37, the specificity can be ensured to reach 93.0 percent, and when the transformation method also adopts the ct value 39 as the positive judgment value, the specificity can only reach 87.0 percent, so that the enzyme digestion method has better detection specificity. Performing sample detection and analysis according to the positive judgment standard, comparing the detection sensitivity of the sample detection and the analysis, and displaying that the sample is processed by an enzyme cutting method, wherein the positive detection rate is 88.4%, the adenoma detection rate is 48.2%, and the accuracy is 90.6% (not including adenoma); and by adopting sulfite conversion to process a sample, the positive detection rate is 79.5 percent, the adenoma detection rate is 35.4 percent, and the accuracy is 86.4 percent (no adenoma is included).
TABLE 6 results of the enzymatic cleavage assay
TABLE 7 results of the transformation method
Sensitivity (%). true positive/(true positive + false negative) × 100%;
the detection rate of adenomas (%) is adenoma positive/number of adenoma samples × 100%;
accuracy (%) ═ true positive + true negative)/number of samples × 100%;
in summary, the methylation detection of the samples treated by the enzymatic cleavage method has higher sensitivity and accuracy than the detection of the samples treated by sulfite.
SEQUENCE LISTING
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Claims (10)
1. A method for detecting gene methylation, comprising the steps of:
(1) digesting a sample to be detected containing a target gene by using a restriction enzyme BstUI to obtain an enzyme digestion product;
(2) performing PCR amplification on the enzyme digestion product obtained in the step (1), wherein a PCR amplification primer group comprises a target gene primer group, and the 3' tail end of an upstream primer and/or a downstream primer of the target gene primer group is provided with ' CGC ', ' CGCG ' or ' CGCG ' and 1 to 2 bases of a template sequence behind the ' CGC ', ' CGCG ' or ' CGCG ';
(3) and judging the methylation condition of the target gene according to the PCR amplification result.
2. The method for detecting gene methylation of claim 1, wherein the amplicon of the primer set for the target gene comprises at least one "CGCG" site.
3. The method for detecting gene methylation according to claim 1, wherein the PCR amplification primer set further comprises an internal standard gene primer set, and the internal standard gene primer set and an amplicon thereof do not contain a "CGCG" site.
4. The method for detecting gene methylation according to any one of claims 1 to 3, wherein the target gene comprises at least one of SFRP2, NDRG4, SDC2 genes; the internal standard gene includes at least one of the ACTB gene or other suitable housekeeping gene or copy number stable conserved gene sequences.
5. The method for detecting gene methylation according to any one of claims 1 to 3, wherein a GC enhancer is added to the PCR amplification reaction system; the GC enhancer comprises 20-50mM tetramethylammonium chloride and 6% -15% (v/v) glycerol.
6. A gene methylation detection kit, comprising: the restriction endonuclease BstUI, a target gene amplification primer, wherein the 3' end of an upstream primer and/or a downstream primer of a target gene amplification primer group is 1 to 2 bases of ' CGC ', ' CGCG ' or ' CGCG ' and a template sequence behind the CGCG.
7. The gene methylation detection kit of claim 6, wherein the amplicon of the primer set for the target gene comprises at least one "CGCG" site.
8. The gene methylation detection kit according to claim 6, wherein the kit further comprises an internal standard gene primer set, and the internal standard gene primer set and an amplicon thereof do not contain a "CGCG" site.
9. The gene methylation detection kit according to claim 6, wherein the kit further comprises a GC enhancer; the GC enhancer comprises 20-50mM tetramethylammonium chloride and 6% -15% (v/v) glycerol.
10. The gene methylation detection kit according to any one of claims 6 to 9, wherein the target gene comprises at least one of SFRP2, NDRG4, SDC2 genes; the internal standard gene includes at least one of the ACTB gene or other suitable housekeeping gene or copy number stable conserved gene sequences.
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| CN114250280A (en) * | 2020-09-25 | 2022-03-29 | 圣湘生物科技股份有限公司 | Composition, kit, application and method for detecting gene methylation |
| WO2024082624A1 (en) * | 2022-10-21 | 2024-04-25 | 南京普济生物医学有限公司 | Primer having stem-loop structures and use thereof |
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