Single base resolution methylation detection system and detection method
By designing a competitive relationship between specific primers and blockers and combining it with fluorescence quantitative PCR technology, the sensitivity and specificity issues of existing DNA methylation detection methods in low CpG density areas were resolved, and highly sensitive and specific single-base methylation quantitative detection was achieved.
Patent Information
- Application Number
- CN202510880660.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-27
- Publication Date
- 2025-09-19
AI Technical Summary
Existing DNA methylation detection methods are difficult to detect low CpG density regions or non-comethylation of adjacent CpG sites with high sensitivity and specificity. In addition, existing methods are labor-intensive, time-consuming and cost-ineffective.
By using the competitive relationship between specific primers and methylation-specific blockers, specific primers and blockers were designed to inhibit the amplification of non-methylated templates. Single-base methylation detection was performed in combination with fluorescent quantitative PCR technology. Blocking modification of MGB probes and blockers was used to improve detection specificity and sensitivity.
It achieves highly sensitive and specific single-base methylation quantitative detection, can accurately distinguish between fully methylated and unmethylated states, and has a detection sensitivity of 0.01% at a low concentration difference, with good quantitative performance.
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Figure CN120666001A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of analysis and detection, specifically, to the field of nucleic acid molecule detection, and more specifically, to a single-base resolution methylation site quantitative detection system and detection method. Background Art
[0002] DNA methylation profiles can provide valuable biological information. 5-Cytosine DNA methylation profiles in cancer specimens are considered a valuable source of biological information with potential clinical applications. In vertebrates, 5-Cytosine DNA methylation primarily occurs in the context of CpG dinucleotides (a cytosine base followed by a guanine base). Hypermethylation of CpG island (CGI) promoters of tumor suppressor genes has been well documented in many human cancers, leading to transcriptional inactivation. However, promoter CGIs comprise only a small fraction of the methylome, while regions of low CpG density within gene bodies are the most conserved targets of DNA methylation in eukaryotes. Given the clinical relevance of genomic methylation analysis, various analytical methods have been developed. Methylation-specific PCR (MSP) is an endpoint analysis technique, while MethyLight (quantitative methylation-specific PCR (qMSP)) was introduced for quantitative analysis. However, most existing DNA methylation detection methods are designed to measure the local average of multiple CpG sites within high CpG density regions (such as CpG islands), which is based on the assumption that all local CpG sites are fully methylated or unmethylated. Therefore, oligonucleotides are designed to cover as many CpG dinucleotides as possible to improve analytical sensitivity and specificity.
[0003] Previous studies have employed various strategies to measure single-base methylation in patient samples using modified MethyLight assays, but these studies lack systematic evidence and optimized methods. Therefore, identifying optimal solutions for regions of low CpG density or when adjacent CpG sites are not comethylated is crucial. Bisulfite pyrosequencing or targeted bisulfite sequencing can be used in this context, but these techniques are labor-intensive, time-consuming, and not cost-effective for cohort studies or clinical practice. The MethyLight assay, based on an ALU repeat input control reaction, has been widely used to measure CpG island methylation in bulk samples. Another limitation of the MethyLight assay is that an input control and fully methylated DNA are required as reference controls for calculating percent methylation. However, input controls can introduce random errors, and fully methylated DNA is not readily available as quality control material. To investigate the methylation status of individual CpG sites, we developed a single-base-resolution quantitative methylation assay. Summary of the Invention
[0004] The present invention aims to overcome the shortcomings of the aforementioned existing methods and provide a highly specific and sensitive single-base methylation quantitative detection system and method. This method utilizes specific primers and a methylation-specific blocker to compete with the unmethylated template, thereby inhibiting amplification of the unmethylated template and achieving the desired detection. Furthermore, the method offers enhanced specificity and ensures detection sensitivity.
[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:
[0006] A single-base methylation detection system, the detection system is a fluorescent quantitative PCR amplification system, the detection system includes a forward primer, a reverse primer, a probe and a blocker;
[0007] The forward primer is 20-30 bp in length. If the complementary template sequence contains a CpG site, the bases therein are designed to be degenerate bases.
[0008] The reverse primer is 20-30 bp in length. If the complementary template sequence contains a CpG site, the base at that site is designed to be a degenerate base. The last base at the 3' end of the reverse primer is a complementary base to the single methylation site to be detected and is not a degenerate base.
[0009] The 3' end of the blocker contains a blocking modification to hinder its extension; if the blocker complementary template sequence contains a CpG site, the base at this site is designed as a degenerate base.
[0010] Furthermore, the base 0-8 bases away from the 5' end of the Blocker sequence is a non-methylated blocking site corresponding to the single-base methylated site, and this site has the above-mentioned competitive relationship with the last base at the 3' end of the reverse primer.
[0011] Furthermore, the 5' end of the probe contains a fluorescent group and the 3' end contains a quenching group, the fluorescent group is selected from one of FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 or JOE, and the quenching group is selected from one of MGB, BHQ1 or BHQ2; if the probe complementary template sequence contains a CpG site, the base there is designed as a degenerate base.
[0012] Furthermore, the probe is an MGB probe.
[0013] Furthermore, the misalignment distance between the 5' end of the blocker and the detection site is greater than 3 nt.
[0014] Furthermore, the forward primer is shown as SEQ ID No: 1, the sequence of the reverse primer is shown as SEQ ID No: 2, the sequence of the probe is shown as SEQ ID No: 12, and the sequence of the blocker is shown as SEQ ID No: 4.
[0015] Furthermore, the detection system also includes PCR buffer, Mg 2+ ions, dNTPs, and hot-start DNA polymerase.
[0016] The present invention also provides a detection kit comprising the above-mentioned single-base methylation detection system.
[0017] The present invention also provides a single-base resolution methylation detection method, which uses the above-mentioned single-base methylation detection system to perform quantitative PCR detection and uses Taqman probes, molecular beacons or other luminescent probes to obtain signals.
[0018] Furthermore, the detection method includes: enzyme activation at 95°C for 10 minutes, denaturation at 95°C for 10 seconds, annealing and extension at 52-53°C for 30 seconds, and 45-50 cycles.
[0019] The 3' end of the blocker contains a blocking modification that hinders its extension. Blocking groups include but are not limited to MGB modifications. The bases 0-8 bases away from the 5' end of the blocker sequence are non-methylated blocking sites corresponding to the single-base methylation site, and this site has the aforementioned competitive relationship with the last base at the 3' end of the reverse primer. If the complementary template sequence contains CpG sites at other positions in the blocker sequence, the bases at these positions are designed as degenerate bases.
[0020] The competition between the blocker sequence and the reverse primer works as follows: If the detection site is methylated, the 3' end of the reverse primer binds to the template and extends. However, since the blocker is unmethylated at this site, its binding ability is weaker and it cannot compete with the reverse primer. If the detection site is unmethylated, the blocker binds to the template, preventing primer extension.
[0021] The probe contains a fluorescent group at the 5' end and a quenching group at the 3' end. The fluorescent group is selected from one of FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 or JOE. The quenching group is selected from one of MGB, BHQ1 or BHQ2. The MGB probe is preferred. If the complementary template sequence contains a CpG site, the base there is designed to be a degenerate base.
[0022] The detection system uses bisulfite-converted DNA as the template, specifically converting unmethylated cytosine (C) to uracil (U), while methylated cytosine remains unchanged. The detection system also includes primers, probes, blockers, PCR buffer, Mg2+ ions, dNTPs, and a hot-start DNA polymerase. The concentrations of primers, probes, and blockers range from 0.1 to 1 μM.
[0023] During the test, a PCR system is composed of primers, probes, blockers and other raw materials required for PCR at a certain concentration, and the sample is subjected to quantitative PCR detection.
[0024] In the quantitative PCR detection, Taqman probes, molecular beacons or other luminescent probes are used to obtain signals.
[0025] Beneficial effects
[0026] The present invention provides a highly specific and sensitive method for quantitatively detecting single-base methylation. This method utilizes specific primers and a methylation-specific blocker to compete with the unmethylated template, thereby inhibiting amplification of the unmethylated template and achieving the purpose of detection. Furthermore, this method has higher specificity and ensures both sensitivity and quantitative performance. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 Schematic diagram of the amplification principle of four synthetic templates;
[0028] Figure 2 The △Ct value histogram of the positive and negative templates with and without blocker;
[0029] Figure 3 A bar graph showing the △Ct values of primers indicating whether degenerate base yin-yang templates are used;
[0030] Figure 4 The △Ct value histogram of the long and short reverse primers and the positive and negative templates;
[0031] Figure 5 It is the bar graph of △Ct values of positive and negative templates of long and short forward primers;
[0032] Figure 6 The △Ct value histogram of the misalignment distance between the 5' end of the blocker and the detection site for the positive and negative templates;
[0033] Figure 7 It is the amplification curve diagram of four positive and negative templates;
[0034] Figure 8 The following are histograms of Ct values of four yin-yang templates;
[0035] Figure 9 It is the amplification curve diagram of the positive and negative templates incorporating in proportion;
[0036] Figure 10 The Ct value histogram is added proportionally to the positive and negative templates;
[0037] Figure 11 Linear regression analysis of template concentration gradient. DETAILED DESCRIPTION
[0038] The present invention is further described in detail below with reference to the embodiments so that those skilled in the art can implement the invention with reference to the description.
[0039] Example 1
[0040] 1. Design primers, probes, and blockers
[0041] Based on the synthetic template, primers, probes, and blockers were designed (Table 1). The synthetic template was detected using this method, and the sequence was as follows:
[0042] Table 1 Primers, probes, and blockers used in Example 1
[0043]
[0044] Y represents the base C or T; R represents the base A or G.
[0045] 2. Effect of blocker on test specificity
[0046] The total reaction volume was 30 μl, containing 3 mM MgCl2, 0.2 mM dNTPs, 0.2 μM forward primer, 0.1 μM reverse primer, 0.1 μM probe, 0.2 μM blocker, 0.05 U / μl hot start polymerase, and 15 μl template. The reaction was activated at 95°C for 10 min, followed by denaturation at 95°C for 10 s and annealing and extension at 53°C for 30 s for 50 cycles. PCR and signal acquisition were performed using the Macrostone SLAN96s system. Figure 2 It can be seen that the blocker can increase the △Ct value of the positive and negative templates, indicating that the blocker improves the detection specificity by blocking primer extension.
[0047] Example 2
[0048] 1. Design primers, probes, and blockers
[0049] Based on the synthetic template, primers, probes, and blockers were designed (Table 2). The synthetic template was detected using this method, and the sequence was as follows:
[0050] Table 2 Primers, probes, and blockers used in Example 2
[0051]
[0052] aacc indicates bases that are not complementary to the template.
[0053] 2. Whether the detection primer contains degenerate bases affects the specificity and sensitivity of the detection
[0054] The total reaction volume was 30 μl, containing 3 mM MgCl2, 0.2 mM dNTPs, 0.2 μM forward primer, 0.1 μM reverse primer, 0.1 μM probe, 0.4 μM blocker, 0.05 U / μl hot start polymerase, and 15 μl template. The reaction was activated at 95°C for 10 min, followed by denaturation at 95°C for 10 s and annealing and extension at 55°C for 30 s for 50 cycles. PCR and signal acquisition were performed using the Macrostone SLAN96s system. Figure 3 It can be seen that the use of degenerate bases can increase the △Ct value of the positive and negative templates and improve the detection specificity.
[0055] Example 3
[0056] 1. Design primers, probes, and blockers
[0057] In this example, the forward primer and the reverse primer of SEQ ID No: 7 in Example 2 were used as short reverse primers, and the probe, blocker, and reverse primer in Example 1 were used as long reverse primers.
[0058] 2. Detection of the effect of reverse primers of different lengths on detection specificity
[0059] This method was used to detect the synthetic template. The total reaction volume was 30ul, containing 3 mM MgCl2, 0.2mM dNTPs, 0.2μM forward primer, 0.1μM reverse primer, 0.1μM probe, 0.4μM blocker, 0.05 U / ul hot start polymerase, and 15ul template. The reaction was activated at 95℃ for 10min, followed by denaturation at 95℃ for 10s, annealing and extension at 53℃ for 30s, and 50 cycles. PCR and signal acquisition were performed using the Macrostone SLAN96s system. Figure 4 It can be seen that extending the reverse primer by 4-5 nt bases (long reverse primer) relative to the original reverse primer can increase the △Ct value of the positive and negative templates and improve the detection specificity.
[0060] Example 4
[0061] 1. Design primers, probes, and blockers
[0062] In this example, the forward primer in Example 2 is used as a short forward primer, and the probe, blocker, reverse primer, and forward primer in Example 1 are used as a long forward primer.
[0063] 2. Detection of the effect of different lengths of forward primers on detection specificity
[0064] This method was used to detect the synthetic template. The total reaction volume was 30ul, containing 3 mM MgCl2, 0.2mM dNTPs, 0.2μM forward primer, 0.1μM reverse primer, 0.1μM probe, 0.2μM blocker, 0.05 U / ul hot start polymerase, and 15ul template. The reaction was activated at 95℃ for 10min, followed by denaturation at 95℃ for 10s, annealing and extension at 53℃ for 30s, and 50 cycles. PCR and signal acquisition were performed using the Macrostone SLAN96s system. Figure 5 It can be seen that the long forward primer can increase the △Ct value of the positive and negative templates and improve the detection specificity.
[0065] Example 5
[0066] 1. Primer, probe, and blocker design
[0067] This example uses the primers, probes, and blockers in Example 1 as far-distance blockers, and designs near-distance blockers based on the synthetic template (Table 3). The synthetic template is detected using this method, and the sequence is as follows:
[0068] Table 3 Blocker used in Example 3
[0069]
[0070] 2. The effect of the misalignment distance between the 5' end of the detection blocker and the detection site on the detection specificity
[0071] This method was used to detect the synthetic template. The total reaction volume was 30ul, containing 3 mM MgCl2, 0.2mM dNTPs, 0.2μM forward primer, 0.1μM reverse primer, 0.1μM probe, 0.2μM blocker, 0.05 U / ul hot start polymerase, and 15ul of each of the four templates. The reaction was activated at 95℃ for 10min, followed by denaturation at 95℃ for 10s, annealing and extension at 53℃ for 30s, and 50 cycles. PCR and signal acquisition were performed using the Macrostone SLAN96s system. Figure 6 It can be seen that the blocker with a long misalignment distance (5nt) can better block the binding and extension of the primer, increase the △Ct value of the positive and negative templates, and improve the detection specificity.
[0072] Example 6
[0073] 1. Primer, probe, and blocker design
[0074] This example uses the primers and blocker in Example 1, and the probe is the MGB probe (SEQ ID No: 12) designed from the probe in Example 1.
[0075]
[0076] 2. Specificity of the detection method
[0077] This method was used to detect the synthetic template. The total reaction volume was 30ul, containing 2 mM MgCl2, 0.2mM dNTPs, 0.4μM forward primer, 0.1μM reverse primer, 0.1μM probe, 0.1μM blocker, 0.05 U / ul hot start polymerase, and 15ul template. The reaction was activated at 95°C for 10min, followed by denaturation at 95°C for 10s, annealing at 53°C for 30s, and extension at 72°C for 15s, for 50 cycles. PCR and signal acquisition were performed using the Macrostone SLAN96s system. Figure 7 It can be seen that the fluorescence quantitative PCR amplification curve is a standard S-shaped curve. Figure 8 It can be seen that the Ct values of the positive and negative templates are significantly different, and the two positive templates can be clearly distinguished from the two negative templates, proving that the method of the present invention has good specificity.
[0078] Example 7
[0079] 1. Primer, probe, and blocker design
[0080] This example uses the primers, blocker, and probe in Example 6.
[0081] 2. Sensitivity of the detection method
[0082] The reaction system and PCR procedure of Example 6 were used to mix the positive template into the negative template at different ratios as the target template. The target template mixing ratios were 50%, 10%, 1%, 0.1%, 0.01% and 0%. Figure 9 It can be seen that the fluorescence quantitative PCR amplification curves in the four cases are standard S-shaped curves. Figure 10 It can be seen that the three situations of full methylation and full non-methylation, full methylation and single-base non-methylation, and single-base methylation and full non-methylation can still be detected when the incorporation ratio is as low as 0.01%. The lowest incorporation ratio that can be detected for single-base methylation and single-methyl non-methylation is 1%, indicating that the method of the present invention has high sensitivity.
[0083] Example 8
[0084] 1. Primer, probe, and blocker design
[0085] This example uses the primers, blocker, and probe in Example 6.
[0086] 2. Detection of concentration gradient
[0087] The reaction system and PCR program of Example 6 were used, and HCT116 was used as the target template. The target template concentration gradient was detected, and the concentration gradient was: 10000 copies / reaction, 1000 copies / reaction, 10 copies / reaction, and 10 copies / reaction. The logarithmic value of the template concentration and the Ct value were subjected to linear regression analysis. The results are as follows Figure 11 As shown, the correlation coefficient r = -0.99958, indicating that the method of the present invention has good quantitative performance.
Claims
1. A single base methylation detection system, characterized in that: The detection system is a fluorescent quantitative PCR amplification system, which includes a forward primer, a reverse primer, a probe and a blocker; The forward primer is 20-30 bp in length. If the complementary template sequence contains a CpG site, the bases therein are designed to be degenerate bases. The reverse primer is 20-30 bp in length. If the complementary template sequence contains a CpG site, the base at that site is designed to be a degenerate base. The last base at the 3' end of the reverse primer is a complementary base to the single methylation site to be detected and is not a degenerate base. The 3' end of the blocker contains a blocking modification to hinder its extension; if the blocker complementary template sequence contains a CpG site, the base at this site is designed as a degenerate base.
2. The single-base methylation detection system according to claim 1, characterized in that The base 0-8 bases away from the 5' end of the Blocker sequence is a non-methylated blocking site corresponding to the single-base methylated site, and this site has the above-mentioned competitive relationship with the last base at the 3' end of the reverse primer.
3. The single base methylation detection system according to claim 1, wherein The 5' end of the probe contains a fluorescent group, and the 3' end contains a quenching group, the fluorescent group is selected from one of FAM, HEX, ROX, VIC, CY5, 5-TAMRA, TET, CY3 or JOE, and the quenching group is selected from one of MGB, BHQ1 or BHQ2; if the probe complementary template sequence contains a CpG site, the base there is designed as a degenerate base.
4. The single base methylation detection system according to claim 1, wherein The probe is an MGB probe.
5. The single base methylation detection system according to claim 1, wherein The misalignment distance between the 5' end of the blocker and the detection site is greater than 3 nt.
6. The single-base methylation detection system according to claim 1, characterized in that The forward primer is shown as SEQ ID No: 1, the reverse primer sequence is shown as SEQ ID No: 2, the probe sequence is shown as SEQ ID No: 12, and the blocker sequence is shown as SEQ ID No:
4.
7. The single-base methylation detection system according to claim 1, characterized in that The detection system also includes PCR buffer, Mg 2+ ions, dNTPs, and hot-start DNA polymerase.
8. A detection kit, characterized in that: Comprising the single-base methylation detection system according to any one of claims 1 to 7.
9. A single-base resolution methylation detection method, characterized in that: The single-base methylation detection system according to any one of claims 1 to 7 is used for quantitative PCR detection, and a Taqman probe, a molecular beacon or other luminescent probe is used to obtain a signal.
10. The single-base resolution methylation detection method according to claim 9, characterized in that: The detection method comprises: enzyme activation at 95° C. for 10 minutes, denaturation at 95° C. for 10 seconds, annealing and extension at 52-53° C. for 30 seconds, and 45-50 cycles.