Kit for detecting colorectal cancer related gene methylation and application thereof

Abstract

The invention relates to the technical field of biological detection, in particular to a kit for detecting methylation of colorectal cancer related genes and application of the kit. According to the kit provided by the invention, by utilizing the characteristic that methylation sensitive restriction enzyme (MSRE) can specifically recognize and cut unmethylated DNA sequences but cannot cut methylated (such as 5 '-methylcytosine, 5mC) sites, a sample to be detected is subjected to enzyme digestion reaction in combination with a specific primer and a probe designed for a target gene, so that the methylated DNA sequences can be specifically recognized and cut, and the methylated (such as 5'-methylcytosine, 5mC) sites can be detected. The Ct difference value (namely delta Ct) between the target gene and the reference gene is calculated through qPCR reaction, so that the methylation occurrence state of the sample to be detected can be determined.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a kit for detecting methylation of colorectal cancer-related genes and its application. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors, ranking among the top cancers in both incidence and mortality. CRC largely follows an adenoma-carcinoma progression, with a disease development cycle of 5-10 years. Early detection of precancerous lesions and early screening are the most effective means of reducing CRC incidence and mortality; therefore, CRC is the most suitable malignant tumor for screening and offers significant benefits. Abnormal DNA methylation modification is an early molecular event in tumorigenesis, and abnormal methylation of specific genes has been shown to have high sensitivity in the diagnosis of colorectal cancer. Therefore, detection of methylation markers associated with colorectal cancer development can serve as a non-invasive and effective means of early screening.

[0003] Current technologies for gene methylation detection include: 1) High-throughput sequencing, the mainstream method of which involves treating DNA with sulfite to convert unmethylated cytosine (C) into uracil (U), while methylated cytosine (5mC) remains unchanged. After sequencing, uracil is read as thymine (T), and methylation sites (unconverted C is the methylation site) can be identified by comparing with the original genome sequence; 2) Methylation-specific PCR, where specific primers and probes are designed to match only the methylated sequence after DNA sulfite treatment (methylated C remains C, unmethylated C becomes T). The amplification products are detected by real-time quantitative PCR (qPCR), and the fluorescence signal intensity is proportional to the methylated DNA content; 3) High-resolution melting curve analysis, where the difference in GC content between methylated and unmethylated DNA after sulfite treatment leads to different melting temperatures (Tm). By accurately monitoring the fluorescence changes during PCR product melting (such as the release of SYBR Green dye), the methylation state can be distinguished based on the shape of the melting curve and the difference in Tm value.

[0004] However, existing technologies generally require sulfite treatment of the nucleic acids to be tested, which can damage the nucleic acids to some extent, thus requiring a large amount of nucleic acid input. In addition, the cost of instruments, reagents, and data analysis required for high-throughput sequencing technology to detect gene methylation is high, especially whole-genome methylation sequencing, which has high requirements for sample size and sequencing depth; it also requires the support of bioinformatics professionals, and the raw data volume is large and the processing procedure is cumbersome.

[0005] Methylation-specific PCR technology requires primers to be designed for the sulfite-converted sequence. Due to the high T base content of the converted sequence, primer specificity is difficult to guarantee, and primer and probe design is challenging. In addition, the detection capability is insufficient for samples with low methylation levels below 1%.

[0006] High-resolution melting curve analysis requires high-resolution melting equipment, which ordinary qPCR instruments cannot meet; the differences in melting curve morphology are subtle, relying on the operator's experience and judgment, which can easily introduce human error; and it is difficult to accommodate multiple targets for multiple detection in a single reaction. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a kit for detecting methylation of colorectal cancer-related genes and its application. The kit provided by this invention does not require sulfite conversion and can detect the methylation level of colorectal cancer-related genes through enzymatic digestion combined with fluorescent probes. It achieves methylation detection with a nucleic acid input of only 0.1 ng, offering advantages such as low cost, simple operation, and short processing time; results of methylation levels of related genes can be obtained in just 2.5 hours.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] This invention provides a kit for detecting methylation of colorectal cancer-related genes, comprising: a methylation-sensitive restriction endonuclease, primers and probes for detecting the genes; the genes include a target gene and an internal reference gene; both the probes for detecting the target gene and the internal reference gene are modified with fluorescent groups and quenching groups; the fluorescent groups modified on the probes for detecting the target gene are different from those modified on the probes for detecting the internal reference gene.

[0010] The target genes include one or more of the following: BMP3 gene, SDC2 gene, Septin 9 gene, and SPG20 gene.

[0011] The nucleotide sequences of the primers and probes for detecting the BMP3 gene are shown in SEQ ID NO. 23-25;

[0012] The nucleotide sequences of the primers and probes for detecting the SDC2 gene are shown in SEQ ID NO.32-34;

[0013] The nucleotide sequences of the primers and probes for detecting the Septin 9 gene are shown in SEQ ID NO. 35–37;

[0014] The nucleotide sequences of the primers and probes for detecting the SPG20 gene are shown in SEQ ID NO.41-43.

[0015] Preferably, the internal reference gene includes the ACTB gene.

[0016] Preferably, the nucleotide sequences of the primers and probes corresponding to the ACTB gene are shown in SEQ ID NO.11-13.

[0017] Preferably, the methylation-sensitive restriction endonuclease includes one or more of AciI, HpaII, and HhaI.

[0018] Preferably, the fluorescent group includes a TAMRA group, a VIC group, or a FAM group; the quenching group includes a BHQ1 quenching group or a BHQ2 quenching group.

[0019] Preferably, the kit further includes one or more of Probe qPCRMixMultiPlus, dimethyl sulfoxide (DMSO), and betaine.

[0020] This invention provides the application of the kit described in the above technical solution in detecting the methylation level of colorectal cancer-related genes, the direct purpose of which is non-diagnostic and non-therapeutic.

[0021] This invention provides a method for detecting the methylation level of rectal cancer-related genes using the kit described above. The direct purpose of this method is non-diagnostic and non-therapeutic, and it includes the following steps:

[0022] The DNA of the sample to be tested was digested using a methylation-sensitive restriction endonuclease to obtain the reaction product;

[0023] The reaction product was subjected to qPCR with primers and probes to obtain the Ct values ​​of the target gene and internal reference gene.

[0024] Calculate the value of ΔCt according to Equation I:

[0025] ΔCt = Target gene Ct value - Internal reference gene Ct value (Equation I);

[0026] The smaller the ΔCt value, the higher the methylation level of the sample being tested.

[0027] Preferably, when performing enzyme digestion, the amount of DNA used in the sample to be tested is ≥1 ng.

[0028] Preferably, in addition to the reaction products, primers, and probes, the qPCR reaction system further includes: 25 μL ProbeqPCR Mix MultiPlus, 1 μL ROX Reference Dye, 5 μL GC Enhancer, and nuclease-free water to a final volume of 50 μL; the concentration of the primers in the reaction system is 20 μM; and the concentration of the probes in the reaction system is 10 μM.

[0029] The qPCR reaction program was as follows: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, for 45 cycles.

[0030] Beneficial effects:

[0031] The kit provided by this invention utilizes the characteristic that methylation-sensitive restriction endonucleases (MSREs) can specifically recognize and cleave unmethylated DNA sequences, but cannot cleave methylated sites (such as 5'-methylcytosine, 5mC). Combined with specific primers and probes designed for the target gene, after the sample to be tested is digested with enzymes, the Ct difference (i.e., ΔCt) between the target gene and the internal reference gene is calculated by qPCR, thereby determining the methylation status of the sample to be tested. That is, the higher the methylation level, the smaller the ΔCt.

[0032] The kit provided by this invention has the following advantages in detecting the methylation level of colorectal cancer-related genes:

[0033] 1) No sulfite treatment required: preserves DNA integrity and is suitable for micro-samples (such as liquid biopsy).

[0034] 2) High sensitivity and specificity: Directly detects sample DNA, avoiding interference from sequence complexity, with a sensitivity of up to 0.4% methylation level.

[0035] 3) Rapid and efficient: Enzyme digestion and qPCR integration can be completed within 2-3 hours, making it suitable for rapid clinical testing.

[0036] 4) Cost controllable: No expensive sequencing or chip platform is required; conventional qPCR instruments can be used.

[0037] 5) Multiplex detection potential: By using multiple enzymes in combination or designing multi-site primers, multiple methylation targets can be analyzed simultaneously. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0039] Figure 1 The results show the performance of templates with different methylation levels in three fluorescence channels;

[0040] Figure 2 A model fitting ΔCt1 to methylation level;

[0041] Figure 3 A model fitting ΔCt2 to methylation level;

[0042] Figure 4 This is the result of a 10ng clinical sample test;

[0043] Figure 5 This is the result of a 1ng clinical sample test;

[0044] Figure 6 The results of comparing Septin 9 methylation levels in 20 clinical samples measured by three methods are presented. Detailed Implementation

[0045] This invention provides a kit for detecting methylation of colorectal cancer-related genes, comprising: a methylation-sensitive restriction endonuclease, primers and probes for detecting the genes; the genes include a target gene and an internal reference gene; both the probes for detecting the target gene and the internal reference gene are modified with fluorescent groups and quenching groups; the fluorescent groups modified on the probes for detecting the target gene are different from those modified on the probes for detecting the internal reference gene.

[0046] The target genes include one or more of the following: BMP3 gene, SDC2 gene, Septin 9 gene, and SPG20 gene.

[0047] The nucleotide sequences of the primers and probes for detecting the BMP3 gene are shown in SEQ ID NO. 23-25;

[0048] The nucleotide sequences of the primers and probes for detecting the SDC2 gene are shown in SEQ ID NO.32-34;

[0049] The nucleotide sequences of the primers and probes for detecting the Septin 9 gene are shown in SEQ ID NO. 35–37;

[0050] The nucleotide sequences of the primers and probes for detecting the SPG20 gene are shown in SEQ ID NO.41-43.

[0051] The completeness of the enzyme digestion step is crucial to the detection results. Incomplete digestion can lead to an overestimation of the methylation level of related genes in the test sample, resulting in false positives. The primers provided in this invention are mainly designed in the promoter region of the gene, amplifying the target region with 3 or more enzyme digestion sites, which can improve the efficiency of enzyme digestion and further improve the accuracy of the detection results.

[0052] The kit provided by this invention is a kit that can perform enzyme digestion and qPCR reactions in one tube without the need for purification and replacement of reaction tubes in between. It can improve the sensitivity of colorectal cancer-related gene methylation level detection and has the advantages of simple operation and minimal DNA loss.

[0053] In one implementation, the internal reference gene includes the ACTB gene.

[0054] As one implementation, the nucleotide sequences of the primers and probes corresponding to the ACTB gene are shown in SEQ ID NO. 11-13.

[0055] In one embodiment, the methylation-sensitive restriction endonuclease includes one or more of AciI, HpaⅡ, and HhaI.

[0056] In one embodiment, the fluorescent group includes a TAMRA group, a VIC group, or a FAM group; the quenching group includes a BHQ1 quenching group or a BHQ2 quenching group.

[0057] In one embodiment, the kit further includes one or more of Probe qPCRMix MultiPlus, dimethyl sulfoxide, and betaine. The dimethyl sulfoxide and / or betaine provided by this invention can enhance the amplification capability for high-GC templates.

[0058] This invention provides the application of the kit described above in detecting the methylation level of colorectal cancer-related genes, where the direct purpose of the application is non-diagnostic and non-therapeutic. When detecting the methylation level of colorectal cancer-related genes for non-diagnostic and non-therapeutic purposes, the kit provided by this invention is only intended to obtain an intermediate value of the methylation level of colorectal cancer-related genes.

[0059] This invention provides a method for detecting the methylation level of rectal cancer-related genes using the kit described above. The direct purpose of this method is non-diagnostic and non-therapeutic, and it includes the following steps:

[0060] The DNA of the sample to be tested was digested using a methylation-sensitive restriction endonuclease to obtain the reaction product;

[0061] The reaction product was subjected to qPCR with primers and probes to obtain the Ct values ​​of the target gene and internal reference gene.

[0062] Calculate the value of ΔCt according to Equation I:

[0063] ΔCt = Target gene Ct value - Internal reference gene Ct value (Equation I);

[0064] The smaller the ΔCt value, the higher the methylation level of the sample being tested.

[0065] As one implementation method, when performing enzyme digestion, the amount of DNA used in the sample to be tested is ≥1 ng.

[0066] As one implementation method, the reaction conditions for the enzymatic digestion treatment are 37℃ for 60 min and 80℃ for 20 min.

[0067] In one embodiment, the amount of methylation-sensitive restriction endonuclease used in the enzyme digestion process is 5-20 U; in another embodiment, the amount of methylation-sensitive restriction endonuclease used in the enzyme digestion process is 10-15 U. The reaction product obtained after enzyme digestion in this invention can be directly used for qPCR without purification.

[0068] In one implementation, in addition to the reaction products, primers, and probes, the qPCR reaction system also includes: 25 μL Probe qPCR Mix MultiPlus, 1 μL ROX Reference Dye, 5 μL GC Enhancer, and nuclease-free water to a final volume of 50 μL; the concentration of the primers in the reaction system is 20 μM; and the concentration of the probes in the reaction system is 10 μM.

[0069] As one implementation method, the qPCR reaction program is: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, for 45 cycles.

[0070] In one implementation method, the present invention uses ΔCt as the independent variable and methylation level as the dependent variable to fit an exponential model. For the detection of colorectal cancer samples, the cut-off value of ΔCt is 10.0, that is, ΔCt less than 10.0 is defined as positive (the sample to be tested is colorectal cancer), and ΔCt greater than 10.0 is defined as negative (the sample to be tested is not colorectal cancer).

[0071] The method provided by this invention can perform the enzyme digestion reaction and qPCR reaction in one tube without the need for purification and replacement of reaction tubes in between. This can improve the sensitivity of detecting the methylation level of colorectal cancer-related genes and has the advantages of simple operation and minimal DNA loss.

[0072] To further illustrate the present invention, the kit for detecting methylation of colorectal cancer-related genes and its application are described in detail below with reference to embodiments and accompanying drawings, but these should not be construed as limiting the scope of protection of the present invention.

[0073] Example 1: Selection of the number of enzyme cleavage sites based on fluorescent dye method

[0074] Considering that the number of restriction enzyme sites affects the detection signal of methylation levels, too many restriction enzyme sites on the gene detection target region may result in the loss of signals from individual sites with high methylation levels, leading to missed detections. This invention first screens the number of restriction enzyme sites in the target region, and the operation steps are as follows:

[0075] 1. Enzyme digestion reaction

[0076] A 20 μl enzyme digestion reaction system was prepared using HhaI from Thermo Fisher Scientific, as follows: X μL of DNA sample to be tested, 2 μL of 10× Buffer Tango, 1 μL of HhaI, and nuclease-free water to a final volume of 20 μL.

[0077] The reaction conditions were: 37℃, 1h; 80℃, 20min; 4℃, Hold.

[0078] 2. Purification of enzyme digestion products

[0079] 1) Advance delivery of the materials from Nanoda (Nanjing) Biotechnology Co., Ltd. Remove SP Beads, vortex mix thoroughly, and allow to equilibrate at room temperature for 30 minutes before use.

[0080] 2) Add 30 μL of Nuclease Free Water to the enzyme digestion product, vortex to mix, and then add 90 μL of Nuclease Free Water. Mix SP Beads thoroughly and incubate at 25°C for 5 minutes.

[0081] 3) After briefly centrifuging the PCR tube, place it on a magnetic rack for 5 minutes until the liquid is completely clear. Use a pipette to remove and discard the supernatant.

[0082] 4) Slowly add 200 μL of 80% ethanol along the side wall of the PCR tube, being careful not to disturb the magnetic beads. Let it stand for 30 seconds, then use a pipette to remove and discard the supernatant.

[0083] 5) Repeat step 4) once.

[0084] 6) After briefly centrifuging the PCR tube, place it on a magnetic rack and use a 10μL pipette tip to remove any remaining ethanol.

[0085] 7) Open the PCR tube cap and let it stand at room temperature for 5 minutes until the ethanol has completely evaporated.

[0086] 8) Remove the PCR tube, add 10 μL of Nuclease Free Water to the PCR tube, mix well, incubate at room temperature for 5 min, and take 9.2 μL into a new PCR tube for subsequent qPCR reactions.

[0087] 3. qPCR reaction

[0088] The qPCR reaction used TB Green Premix Ex Taq II reagent from TAKARA, and the qPCR instrument used was the Stepone Plus instrument from ABI.

[0089] Prepare the reaction system in the qPCR reaction tubes according to Table 1.

[0090] Table 1 qPCR reaction system

[0091] The product from the previous enzymatic digestion purification step 9.2μL TB Green Premix Ex Taq II 10μL ROX Reference Dye (50X) 0.4μL PCR Forward Primer (20 μM) 0.2μL PCR Reverse Primer (20μM) 0.2μL Total 20μL

[0092] Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the qPCR reaction tube.

[0093] Start the following reaction program on the Stepone Plus qPCR instrument: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, 45 cycles.

[0094] Fluorescence signal acquisition was set at 60℃ with a 30s extension step.

[0095] First, the GDNF gene was selected to test the detection effect when the amplified target region contained only one restriction site. The primer sequences are shown in Table 2.

[0096] Table 2 Primer sequence list

[0097]

[0098] The initial template was 10 ng of human gDNA standard. Following the above operating steps, the results are shown in Table 3.

[0099] Table 3 Results of qPCR reaction of enzyme digestion products

[0100]

[0101] Note: No methylation-sensitive restriction endonucleases were added to the non-enzyme digestion group; only a simulated enzymatic digestion reaction was performed. The same applies below.

[0102] The results showed that incomplete digestion occurred when the target region contained only a single restriction enzyme site. To ensure complete digestion, PCR primers with the minimum required number of restriction enzyme sites were designed based on the SDC2 gene to amplify the target region containing 3, 4, 5, and 6 restriction enzyme sites (see Table 2).

[0103] The initial template used 10 ng of standards with different methylation levels. The standards for each methylation level were obtained by mixing fully methylated and non-methylated standards from ZYMO in a certain proportion to obtain samples with different mass percentages of methylation level. Following the above operating steps, the results are shown in Table 4.

[0104] Table 4. qPCR reaction results of different samples using different primers

[0105]

[0106] Note: In Table 4, ΔCt = Ct(enzyme digestion group) - Ct(non-enzyme digestion group).

[0107] The test results did not follow the theory that higher methylation levels correspond to smaller ΔCt values, and the number of restriction sites was not directly related to the completeness of restriction digestion. This is likely because the use of fluorescent dyes in qPCR assays resulted in non-specific amplification signals that significantly interfered with the remaining small amount of template signal after restriction digestion.

[0108] Example 2: Selection of the number of restriction sites based on fluorescent probe method

[0109] Example 1 could not meet the detection requirements of high sensitivity, specificity, and multiplex PCR. Therefore, this invention uses a fluorescent probe method for subsequent experiments. The fluorescent probe is a Taqman probe. The restriction enzyme digestion effect was detected when the target region contained 1, 2, and 3 restriction enzyme sites, respectively. Meanwhile, ACTB was used as an internal reference gene, and the amplified fragment of the internal reference gene did not contain the required restriction enzyme sites. Primer and probe information is shown in Table 5.

[0110] Table 5 Primers and probes for different genes

[0111]

[0112]

[0113] Note: Probe 1 has a TAMRA group at its 5' end and a BHQ2 quencher group at its 3' end; Probe 2 has a FAM group at its 5' end and a BHQ1 quencher group at its 3' end; Probe 3 has a FAM group at its 5' end and a BHQ1 quencher group at its 3' end; Probe 4 has a FAM group at its 5' end and a BHQ1 quencher group at its 3' end.

[0114] The enzyme digestion reaction and enzyme digestion product purification process are the same as in Example 1.

[0115] qPCR reaction

[0116] The qPCR reaction used TAKARA's Probe qPCR Mix MultiPlus reagent, and the qPCR instrument used was ABI's Stepone Plus instrument.

[0117] Prepare the reaction system in the qPCR reaction tubes according to Table 6.

[0118] Table 6. qPCR reaction system

[0119] The product from the previous enzymatic digestion purification step 9.2μL Probe qPCR Mix MultiPlus 12.5μL ROX Reference Dye (50X) 0.5μL PCR Forward Primer (20 μM) 0.25μL PCR Reverse Primer (20μM) 0.25μL Probe (10μM) 0.5μL Nuclease Free Water To 25μL

[0120] Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the qPCR reaction tube.

[0121] Start the following reaction program on the Stepone Plus qPCR instrument: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, 45 cycles.

[0122] Fluorescence signal acquisition was set at 60℃ with a 30s extension step.

[0123] Using 10 ng of standards with different methylation levels (prepared in the same way as in Example 1) as the initial input template, the results are shown in Table 7.

[0124] Table 7. qPCR reaction results of 10 ng standards with different methylation levels

[0125]

[0126] Note: In label 7, ΔCt = Site[Ct(enzyme-digested group) - Ct(non-enzyme-digested group)] - ACTB[Ct(enzyme-digested group) - Ct(non-enzyme-digested group)].

[0127] The results showed that a higher number of cleavage sites resulted in a larger ΔCt, indicating more thorough cleavage of unmethylated samples. Regardless of the number of cleavage sites, 100% methylated samples were not cleaved. To ensure clear differentiation between different methylation levels, a larger ΔCt is preferable when using unmethylated samples. Furthermore, based on the commercially available ZYMO OneStepPLUS qMethyl... TM The equation for calculating methylation levels in a PCR kit is: methylation level percentage = 100 × 2 -ΔCt (ΔCt = target gene Ct - reference gene Ct). Substituting ΔCt into the table, the number of restriction enzyme sites is close to the theoretical value when it is 3. That is, primer design should ensure that the number of restriction enzyme sites is greater than or equal to 3.

[0128] Example 3 One-tube MSRE-qPCR reaction

[0129] To minimize sample loss during purification after enzyme digestion and ensure detection sensitivity, the enzyme digestion reaction and qPCR reaction were performed in a single tube, eliminating the need for purification and tube replacement. The primers used were Site-3 from Example 2, and the procedure was as follows:

[0130] Enzyme digestion reaction

[0131] A 10 μL enzyme digestion reaction system was established using HhaI from Thermo Fisher Scientific, as follows: X μL of DNA sample to be tested, 1 μL of 10× Buffer Tango, 1 μL of HhaI, and nuclease-free water was added to bring the total volume to 10 μL.

[0132] The reaction conditions were 37℃ for 1 h; 80℃ for 20 min; and 4℃, held.

[0133] qPCR reaction

[0134] The qPCR reaction used TAKARA's Probe qPCR Mix MultiPlus reagent, and the qPCR instrument used was ABI's Stepone Plus instrument.

[0135] Prepare the reaction system in the qPCR reaction tubes according to Table 8.

[0136] Table 8 qPCR reaction system

[0137] The product from the previous enzymatic digestion purification step 10μL Probe qPCR Mix MultiPlus 25μL ROX Reference Dye (50X) 1μL PCR Forward Primer (20 μM) 0.5μL PCR Reverse Primer (20μM) 0.5μL Probe (10μM) 0.5μL Nuclease Free Water To 50μL

[0138] Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the qPCR reaction tube.

[0139] The qPCR reaction conditions were the same as in Example 2.

[0140] The initial template was 10 ng male gDNA standard. Following the above operating steps, the results are shown in Table 9.

[0141] Table 9 qPCR reaction results

[0142] Group Ct Enzyme digestion group 41.979 Non-enzyme digestion group 31.027

[0143] The results showed that a single-tube MSRE-qPCR reaction could effectively distinguish between the enzyme-digested group and the non-enzyme-digested group, and could be used for subsequent testing.

[0144] Example 4: Composition of the Colorectal Cancer Related Gene Detection Kit

[0145] Based on the detection system established in Examples 1-3, the detection of colorectal cancer-related genes was specifically optimized. The detection kit consists of the following components:

[0146] 1. Primer and probe sequences for methylation markers associated with colorectal cancer

[0147] Although there are numerous methylation biomarkers for colorectal cancer, many targets involve high-GC regions, making primer and probe design challenging. To ensure amplification efficiency while also considering the number of restriction enzyme sites, this invention selected several genes already reported to be associated with colorectal cancer and designed primers and probes. Specific information is shown in Table 10.

[0148] Table 10 Primers and probes used for colorectal cancer-related genes.

[0149]

[0150]

[0151] 2. Test reagents:

[0152] The selection of methylation-sensitive restriction endonucleases has a significant impact on the efficiency of the enzyme digestion reaction. The results of Example 2 show that primer amplification regions containing three or more restriction sites are optimal. To ensure sufficient restriction sites in the primer amplification region, a methylation-sensitive restriction endonuclease mix (Mix) consisting of ACiI, HpaⅡ, and HhaⅠ was selected, with equal amounts of the three enzymes used, and a total mixed volume of 1 μL.

[0153] The amplification reagents include: Probe qPCRMixMultiPlus, primers, probes, GC enhancer (a mixture of betaine and DMSO), and Nuclease-Free Water.

[0154] 3. Reference materials:

[0155] Positive and negative reference materials for colorectal cancer.

[0156] Example 5: Detection Reaction System and Process

[0157] The detection reaction system and process established in this embodiment are applied to Examples 6-10.

[0158] MSRE reaction

[0159] 1. Take out ACII, HpaⅡ, HhaI and mix them evenly to obtain Enzyme Mix, centrifuge briefly, and place on ice for later use.

[0160] 2. Take out 10×MSRE buffer and place it on ice to thaw naturally. Mix well and centrifuge briefly for later use.

[0161] 3. Prepare the reaction system in the qPCR reaction tube according to the table below: X μL of DNA sample to be tested, 1 μL of 10×MSREBuffer, 1 μL of Enzyme Mix, and Nuclease Free Water to a final volume of 10 μL.

[0162] 4. Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the qPCR reaction tube.

[0163] 5. Start the following reaction program on the PCR instrument. Once the temperature stabilizes at 37℃, place the reaction tubes into the PCR instrument: 37℃ for 60 min, 80℃ for 20 min, 4℃ Hold. Set the hot lid temperature to 90℃ during program execution.

[0164] qPCR reaction

[0165] 1. Take out Probe qPCRMix MultiPlus and ROX Reference Dye (50×conc.), place the primers and probes on ice to thaw naturally, mix well, and centrifuge briefly for later use.

[0166] 2. Prepare the reaction system in the qPCR reaction tubes according to Table 11.

[0167] Table 11 qPCR reaction system

[0168] The product of the previous MSRE reaction 10μL ProbeqPCRMixMultiPlus 25μL ROXReferenceDye(50×conc.) 1μL GCEnhancer 5μL Primers (20 μM) 0.5 μL of each primer Probe (10μM) 0.5 μL per probe NucleaseFreeWater To 50μL

[0169] 3. Mix thoroughly and centrifuge briefly to place all reaction solution at the bottom of the qPCR reaction tube.

[0170] 4. Start the following reaction program on the Stepone Plus qPCR instrument: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, 45 cycles.

[0171] Fluorescence signal acquisition was set at 60℃ with a 30s extension step.

[0172] Example 6: Optimization of Detection Reagent Components

[0173] The primers and probes listed in Example 4 were tested using the detection reaction system and process of Example 5. Primers and probes for the target gene and the internal reference gene ACTB were added to each tube of qPCR reaction. The internal reference gene ACTB was labeled with TAMRA fluorescent label; BMP3, IKZF1, and SDC2 were labeled with FAM fluorescent label; and NDRG4, Septin 9, FBN1, and SPG20 were labeled with VIC fluorescent label.

[0174] The qPCR results are shown in Table 12.

[0175] Table 12 qPCR reaction results for different target genes

[0176]

[0177] Note: "Undetermined" indicates that no amplification signal was detected and no Ct value was found.

[0178] To ensure detection sensitivity, the initial input of 10 ng Promega male gDNA standard was used. Primers and probes with a target gene Ct ≤ 31 under the Non-MSRE group condition of Ct(ACTB) < 28, and a target gene Ct ≥ 37.5 under the MSRE group condition of Ct(ACTB) < 28, were defined as qualified. Results showed that qualified primers and probes targeted the following genes: BMP3, SDC2, Septin 9, and SPG20.

[0179] Example 7 MSRE-qPCR Sensitivity Test

[0180] Based on the genes and corresponding primers and probes selected in Example 6, the FAM channel used a combination of BMP3 and SDC2, and the VIC channel used a combination of Septin9 and SPG20. The plan was to determine the lower limit of detection (LOD) of the constructed MSRE-qPCR detection system. Promega's male gDNA standard, with an initial input of 10 pg-100 ng, was used for the experiment, following the same detection procedure as in Example 5. The results are shown in Table 13.

[0181] Table 13 qPCR results (Ct values) with different amounts of standard input.

[0182]

[0183] The criteria for effective amplification were ACTB Ct≤35 and target gene Ct<38.

[0184] The constructed MSRE-qPCR can effectively distinguish between the Non-MSRE group and the MSRE group with a template input as low as 0.1 ng.

[0185] Example 8: MSRE-qPCR applied to the detection of methylation standards

[0186] Human HCT116 DKO non-Methylated DNA and Human HCT116 DK OMethylated DNA from ZYMO were mixed at weight percentages to prepare templates with methylation levels of 25%, 12.5%, 6.25%, 3.125%, 1.5625%, 0.78125%, and 0.390625%. The initial input was 10 ng, and the detection procedure was the same as in Example 5. TAMRA was used to detect ACTB, FAM was used to detect SDC2 and BMP3, and VIC was used to detect Septin 9 and SPG20. Results are shown in [Figure number missing]. Figure 1 And Table 14.

[0187] Table 14 qPCR results of standards with different methylation levels

[0188]

[0189]

[0190] Note: In Table 14, ΔCt1 = Ct(FAM) - Ct(TAMRA); ΔCt2 = Ct(VIC) - Ct(TAMRA).

[0191] The results show that the method constructed in this invention can effectively distinguish samples with different methylation levels. Based on the Ct values ​​corresponding to the detection results of the three fluorescence channels, the Ct difference between the target gene and the internal reference gene was calculated. ΔCt1 and ΔCt2 both showed a negative correlation with methylation level; that is, the higher the methylation level, the smaller the ΔCt.

[0192] Based on the relationship between ΔCt and methylation level, a logarithmic model was constructed with ΔCt defined as the independent variable and methylation level as the dependent variable. The results are shown in […]. Figure 2 and Figure 3 The results showed that the goodness of fit for ΔCt1 and ΔCt2 were 0.9879 and 0.9852, respectively.

[0193] Example 9: MSRE-qPCR applied to clinical sample detection performance

[0194] Twenty clinical samples were obtained, including 11 colorectal cancer tumor tissue samples and 9 adjacent normal tissue samples.

[0195] DNA extraction was performed according to the Qiagen DNA extraction kit procedure, with 10 ng and 1 ng of DNA used as the initial sample for each sample. The detection reaction procedure was the same as in Example 5.

[0196] ΔCt=0.5×ΔCt1(Ct FAM -Ct TAMRA )+0.5×ΔCt2(Ct VIC -Ct TAMRA The cut-off value is defined as ΔCt = 10.00. ΔCt ≥ 10 indicates a negative result; ΔCt < 10 indicates a positive result.

[0197] See results Figure 4 and Figure 5 The results showed a highly significant difference in ΔCt between tumor tissue samples and adjacent normal tissue samples, indicating that this method has the potential to detect clinical samples.

[0198] Example 10 MSRE-qPCR and NGS concordance analysis

[0199] Using the same 20 clinical samples from Example 9, with an initial dosage of 10 ng, the ΔCt value (Ct(Septin 9)-Ct(ACTB)) was obtained by MSRE-qPCR targeting Septin 9, and the detection method was the same as in Example 5. The obtained ΔCt was substituted into the fitting equation between ΔCt and methylation level, and the methylation level was calculated as in Example 8.

[0200] The same 20 clinical samples, using Nano-Tech's... Methylation library construction kit and A methylated library was constructed using a DNA methylation-bisulfite conversion module, with an input of 50 ng. The constructed library was used by Nano-Tech. Hybridization capture sequencing was performed using EMS Panel v1.0 to analyze the methylation level of Septin 9 (hg19:chr17:75368884-75369052) (denoted as μCaler-EMS Panel). Additionally, Nanotech's [Company Name] ... The DNA Full Screen System allows for library construction using a 50 ng input of the same sample. REMS Panel hybridization capture was used to analyze the methylation level of Septin 9 (hg19:chr17:75368884-75369052) (denoted as μCalerFull Screen).

[0201] The methylation levels of Septin 9 target obtained by detecting 20 clinical samples using three methods were compared. The results are shown in [Figure Number]. Figure 6 The results showed that, for the same sample, MSRE-qPCR, μCaler-EMS Panel, and μCalerFull Screen exhibited consistent discrimination and trends in methylation signals at the same target site, indicating reliable data.

[0202] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A kit for detecting methylation of colorectal cancer-related genes, characterized in that, include: Methylation-sensitive restriction endonucleases and primers and probes for detecting genes; the genes include target genes and internal reference genes; Both the probes for detecting the target gene and the internal reference gene are modified with fluorescent groups and quenching groups; the fluorescent groups modified on the probes for detecting the target gene are different from those modified on the probes for detecting the internal reference gene. The target genes include one or more of the following: BMP3 gene, SDC2 gene, Septin 9 gene, and SPG20 gene. The nucleotide sequences of the primers and probes for detecting the BMP3 gene are shown in SEQ ID NO. 23-25; The nucleotide sequences of the primers and probes for detecting the SDC2 gene are shown in SEQ ID NO.32-34; The nucleotide sequences of the primers and probes for detecting the Septin 9 gene are shown in SEQ ID NO. 35–37; The nucleotide sequences of the primers and probes for detecting the SPG20 gene are shown in SEQ ID NO.41-43.

2. The reagent kit according to claim 1, characterized in that, The internal reference gene includes the ACTB gene.

3. The reagent kit according to claim 2, characterized in that, The nucleotide sequences of the primers and probes corresponding to the ACTB gene are shown in SEQ ID NO.11-13.

4. The reagent kit according to claim 1, characterized in that, The methylation-sensitive restriction endonucleases include one or more of AciI, HpaⅡ, and HhaI.

5. The reagent kit according to claim 1, characterized in that, The fluorescent group includes a TAMRA group, a VIC group, or a FAM group; the quenching group includes a BHQ1 quenching group or a BHQ2 quenching group.

6. The reagent kit according to claim 1, characterized in that, The kit also includes one or more of Probe qPCRMixMultiPlus, dimethyl sulfoxide, and betaine.

7. The use of the kit according to any one of claims 1 to 6 in detecting the methylation level of colorectal cancer-related genes, wherein the direct purpose of the use is non-diagnostic and non-therapeutic.

8. A method for detecting the methylation level of rectal cancer-related genes using the kit according to any one of claims 1 to 6, wherein the direct purpose of the method is non-diagnostic and non-therapeutic, characterized in that, Includes the following steps: The DNA of the sample to be tested was digested using a methylation-sensitive restriction endonuclease to obtain the reaction product; The reaction product was subjected to qPCR with primers and probes to obtain the Ct values ​​of the target gene and internal reference gene. Calculate the value of ΔCt according to Equation I: ΔCt = Target gene Ct value - Internal reference gene Ct value (Equation I); The smaller the ΔCt value, the higher the methylation level of the sample being tested.

9. The method according to claim 8, characterized in that, When performing enzyme digestion, the amount of DNA used in the sample to be tested is ≥1 ng.

10. The method according to claim 8, characterized in that, In addition to the reaction products, primers, and probes, the qPCR reaction system also includes: 25 μL Probe qPCRMix MultiPlus, 1 μL ROXReference Dye, 5 μL GCEnhancer, and nuclease-free water to a final volume of 50 μL; the concentration of the primers in the reaction system is 20 μM; and the concentration of the probes in the reaction system is 10 μM. The qPCR reaction program was as follows: 95℃ for 30s; 95℃ for 10s, 60℃ for 30s, for 45 cycles.

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