WO2021013140A1 - 一种双基因监控反应体系、试剂盒及其应用 - Google Patents

一种双基因监控反应体系、试剂盒及其应用 Download PDF

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WO2021013140A1
WO2021013140A1 PCT/CN2020/103154 CN2020103154W WO2021013140A1 WO 2021013140 A1 WO2021013140 A1 WO 2021013140A1 CN 2020103154 W CN2020103154 W CN 2020103154W WO 2021013140 A1 WO2021013140 A1 WO 2021013140A1
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gene
methylation
probe
monitoring
actb
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French (fr)
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焦晶晶
秦闯华
唐四元
徐根明
潘艺
赵谦
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中南大学
湖南大地同年生物科技有限公司
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

  • the invention relates to a method for improving the detection accuracy of a methylated gene enzymatic digestion method, in particular to a dual-gene monitoring reaction system, a kit using the dual-gene monitoring reaction system and its application in detecting human colorectal cancer.
  • DNA methylation refers to the process in which organisms use s-adenosylmethionine as a methyl donor to transfer methyl groups to specific bases under the catalysis of DNA methyltransferase.
  • DNA methylation in mammals mainly occurs at the C of 5'-CpG-3' to generate 5-methylcytosine.
  • CpG exists in two forms: one is dispersed in the DNA sequence, and the other is highly aggregated, called CpG islands. In normal tissues, 70%-90% of scattered CpG is modified by methyl groups, while CpG islands are often unmethylated.
  • the local hypermethylation of CpG islands is an important factor in tumorigenesis. Because the local hypermethylation of CpG islands is earlier than cell malignant proliferation, the methylation detection can be used for tumors. Prediction.
  • the methods for methylation detection on the market all use heavy salt conversion method to process the samples before detection. This method causes great damage to the sample nucleic acid, resulting in low sensitivity of detection, and false results caused by incomplete conversion. Positive result.
  • the DNA methylation detection method-methylation-sensitive restriction endonuclease method can avoid damage to the target nucleic acid, thereby greatly improving the accuracy of detection.
  • the restriction enzyme method is incomplete. The resulting false positive results are also a major problem that limits the accuracy of this method.
  • the purpose of the present invention is to provide a method for improving the accuracy of the detection of methylation by the enzyme digestion method, and achieve the purpose of improving the detection accuracy through a creative dual-gene monitoring reaction system and its application.
  • a dual-gene monitoring reaction system which includes a target gene primer pair and probe, an internal reference gene GAPDH primer pair and probe, an enzyme digestion monitoring gene primer pair and probe, the target gene and an enzyme digestion monitoring gene ACTB primer
  • the amplified region contains multiple methylation-sensitive restriction endonuclease cleavage sites.
  • the probe of the target gene, the probe of the internal reference gene GAPDH, and the probe of the enzyme digestion monitoring gene ACTB are individually labeled with one of FAM, VIC, Cy3, Cy5, ROX, and HEX.
  • the target gene, the internal reference gene GAPDH, and the enzyme digestion monitoring gene ACTB use different fluorescent labels. More preferably, the target gene SFRP1 probe uses FAM fluorescent label, the internal reference gene GAPDH probe uses VIC fluorescent label, and the enzyme digestion monitoring gene ACTB The probe is fluorescently labeled with Cy5.
  • the target gene primer probe, the internal reference gene GAPDH primer probe and the enzyme digestion monitoring gene ACTB primer probe are reacted in one tube.
  • a method for improving the accuracy of methylation detection by the enzyme digestion method by adopting the above-mentioned dual-gene monitoring reaction system including the following steps:
  • Step 1 Use a nucleic acid extraction kit to extract the sample nucleic acid DNA
  • Step 2 Take 10-100ng of the extracted nucleic acid and perform restriction endonuclease digestion treatment with methylation-sensitive restriction enzymes;
  • Step three the enzyme digestion product obtained in step two is detected by a dual-gene monitoring reaction system.
  • the amount of the methylation-sensitive restriction endonuclease described in step 2 is 10-100 U.
  • the methylation-sensitive restriction endonuclease described in step 2 is selected from one or more of the following endonucleases: AatII, AciI, AclI, AfeI, AgeI, AscI, AsiSI, AvaI, BceAI , BmgBI, BsaAI, BsaHI, BsiEI, BsiWI, BsmBI, BspDI, BspEI, BsrFI, BssHII, BstBI, BstUI, BtgZI, ClaI, EagI, FauI, FseI, FspI, HaeII, HgaI, HhaI, I, HinP1I , KasI, MluI, NaeI, NarI, NgoMIV, NotI, NruI, Nt.BsmAI, Nt.CviPII, PaeR7I, PluTI, PmlI, PvuI, Rs
  • the present invention also provides a DNA methylation detection kit used in the above method for improving the accuracy of detection of methylation by the enzyme digestion method, which includes the following components:
  • the sequence of the primer pair for SFRP1 gene is:
  • SFRP1-F GTTGGATCCCAGGAAGAGCG (SEQ ID NO. 2);
  • SFRP1-R CATGCTGTGCACGTGATTCC (SEQ ID NO.3);
  • the probe sequence for SFRP1 is:
  • SFRP1-P CGCGAGGGGCGCTGAGCGATACC (SEQ ID NO.4);
  • the primer pair sequence for VIM is:
  • VIM-F GCTCCTCTGCCGTGCG (SEQ ID NO.13);
  • VIM-R TAGTTGGCGAAGCGGTCATT (SEQ ID NO.14);
  • the probe sequence for VIM is:
  • VIM-P CAGGACTCGGTGGACTTCTCGCTGGCCG (SEQ ID NO.15);
  • the primer pair sequence for TWIST1 is:
  • TWIST1-F GCACATTCGTGGGCTCTCAA (SEQ ID NO.16);
  • TWIST1-R ATGTCGTAAAGAGTGCGCCG (SEQ ID NO.17);
  • the probe sequence for TWIST1 is:
  • TWIST1-P CCGATTTCTCCTTCCACTTTGCCAGTGCAC (SEQ ID NO.18);
  • the concentration of each primer in the aforementioned primer pair is 0.1-10 uM.
  • the concentration of the above probe is 0.1-10 uM.
  • Step one sample DNA extraction
  • Step 2 Purify the DNA extracted in Step 1, and remove inhibitory components
  • Step 3 Take 1-20ug of purified DNA and digest it with methylation-sensitive restriction enzymes
  • Step 4 Use a dual-gene monitoring reaction system to detect the methylation of SFRP1 gene, VIM gene, and TWIST1 gene respectively.
  • the invention also provides an application based on the above kit in detecting human colorectal cancer.
  • the amplified region of the designed primer of the present invention contains multiple methylation-sensitive restriction endonuclease cutting sites, and the wild-type sequence is cut into fragments under the action of the methylation-sensitive restriction endonuclease.
  • the subsequent PCR reaction cannot be amplified, and the methylated sequence can remain intact under the action of the methylation-sensitive restriction endonuclease, and can be amplified in the subsequent PCR reaction.
  • the specific process is shown in Figure 1.
  • the amplified region of the target gene contains n selected methylation-sensitive restriction endonuclease sites, and the internal reference gene GAPDH amplification region does not contain the selected methylation-sensitive restriction endonuclease sites Point, the amplified region of the restriction enzyme monitoring gene ACTB contains m selected methylation sensitive restriction endonuclease cut sites, and m ⁇ n, because the restriction enzyme cutting monitoring gene ACTB must be digested to ensure the target gene It can also be digested by restriction enzymes, so m ⁇ 1.
  • the target gene , The internal reference gene GAPDH, restriction enzyme digestion monitoring gene ACTB are all reacted in a reaction tube, and the target gene, internal reference gene GAPDH, restriction restriction enzyme monitoring gene ACTB are all from the human genome.
  • the restriction enzyme digestion monitoring gene ACTB is all digested, It means that the amplified region of the target gene can also be digested.
  • the digestion monitoring gene ACTB is not digested, it means that the reaction system is affected by digestion, and the target gene may not be digested.
  • the amplified region of the internal reference gene GAPDH does not contain a methylation-sensitive restriction endonuclease cleavage site, which is not affected by restriction enzyme cleavage, and can monitor the human genome content in the reaction system.
  • the nucleic acid loss is less and the detection sensitivity is higher;
  • the dual-gene monitoring reaction system is adopted, which can monitor the sample quality during the detection reaction and the digestion efficiency of the enzyme digestion reaction, which greatly improves the accuracy of the detection;
  • Figure 1 is the cleavage process of the methylated sequence and wild-type sequence of the present invention under the action of methylation-sensitive restriction endonucleases;
  • Figure 2 shows the results of 50ng, 40ng, 30ng, 20ng, 10ng Human Non-Methylated DNA digestion of SFRP1 gene according to the detection system in Table 2;
  • Figure 3 shows the results of 50ng, 40ng, 30ng, 20ng, 10ng Human Non-Methylated DNA digestion of SFRP1 gene according to the detection system in Table 3;
  • Figure 4 shows the results of 50ng, 40ng, 30ng, 20ng, 10ng Human Non-Methylated DNA digestion of ACTB gene according to the detection system in Table 3;
  • Figure 5 is a graph showing the amplification curve of the target gene SFRP1 of restriction product A in Example 2;
  • Fig. 6 is a graph showing the amplification curve of the digestion monitoring gene ACTB of the digestion product A in Example 2;
  • Fig. 7 is a graph showing the amplification curve of the target gene SFRP1 of restriction product B in Example 2;
  • Figure 8 is a graph showing the amplification curve of the digestion monitoring gene ACTB of the digestion product B in Example 2;
  • FIG. 10 is a graph showing the amplification curve of the enzyme digestion monitoring gene ACTB of 34 effective samples in Example 3;
  • FIG. 11 is a graph showing the amplification curve of the internal reference gene GAPDH in 3 cases of invalid samples without GAPDH amplification in the detection system 3 in Example 3;
  • FIG. 12 is a graph showing the amplification curve of the enzyme digestion monitoring gene ACTB of 3 cases of GAPDH non-amplified invalid samples in the detection system 3 in Example 3;
  • FIG. 13 is a graph showing the amplification curve of the internal reference gene GAPDH in 3 cases of invalid samples with amplification of ACTB in the detection system 3 in Example 3;
  • FIG. 14 is a graph showing the amplification curve of the enzyme digestion monitoring gene ACTB of 3 invalid samples with amplification of ACTB in the detection system 3 in Example 3;
  • Figure 15 is S1, S2, S3, S4, S5, S6 enzyme digestion method FAM channel detection results;
  • Figure 16 shows the FAM channel detection results of S1, S2, S3, S4, S5, S6 heavy salt treatment method
  • Figure 17 is a sequence diagram of the wild-type SFRP1
  • Figure 18 is a sequence diagram of SFRP1 methylation
  • Figure 19 is a VIM wild-type sequence diagram
  • Figure 20 is a sequence diagram of VIM methylation
  • Figure 21 is the TWIST1 wild-type sequence diagram
  • FIG. 22 is a methylation sequence diagram of TWIST1
  • Figure 23 is a graph showing the amplification curve of SFRP1 gene, VIM gene and TWIST1 gene in negative samples of colorectal cancer
  • Figure 24 is a graph showing the amplification curve of the internal reference gene GAPDH in the detection of SFRP1, VIM, and TWIST1 genes in negative samples of colorectal cancer;
  • Figure 25 is a graph showing the amplification curve of SFRP1 gene, VIM gene and TWIST1 gene in positive samples of colorectal cancer
  • Figure 26 is a graph showing the amplification curve of the internal reference gene GAPDH in the detection of SFRP1, VIM, and TWIST1 genes in positive samples of colorectal cancer;
  • Fig. 27 is a graph showing that 28 cases of colorectal cancer positive samples detected by restriction enzyme digestion monitoring gene ACTB were all digested by restriction enzymes and could not be amplified.
  • the CpG island sequence in the promoter region of SFRP1 gene is:
  • the primer pair and probe sequence designed for the promoter region of SFRP1 gene are:
  • SFRP1-F GTTGGATCCCAGGAAGAGCG (SEQ ID NO. 2);
  • SFRP1-R CATGCTGTGCACGTGATTCC (SEQ ID NO.3);
  • SFRP1-P FAM-CGCGAGGGGCGCTGAGCGATACC (SEQ ID NO.4);
  • the sequence of the amplified region of the primer pair is:
  • This sequence contains two restriction sites for methylation-sensitive restriction endonuclease HpaII.
  • the wild-type sample template sequence will be Enzyme digestion is divided into 3 segments, and the amplification cannot be completed when performing fluorescent quantitative PCR detection.
  • GAPDH-F GCAACTAGGATGGTGTGGCT (SEQ ID NO.6);
  • GAPDH-R TCGCCCCACTTGATTTTGGA (SEQ ID NO.7);
  • GAPDH-P VIC-ACCTTGTGTCCCTCAATATGGTCCTGT (SEQ ID NO. 8);
  • the sequence of the amplified region of the primer pair is:
  • This sequence contains 0 restriction sites for methylation-sensitive restriction endonuclease HpaII.
  • the sample template sequence will not be affected by the enzyme. It is possible to complete the amplification by fluorescence quantitative PCR detection. Since it is not affected by restriction enzyme digestion, and GAPDH is used as a housekeeping gene, the human nucleic acid content in the sample can be monitored.
  • the present invention also designs the primer pair and probe sequence of the enzyme cutting monitoring gene ACTB:
  • ACTB-F AATGAGGCAGGACTTAGCTT (SEQ ID NO.10);
  • ACTB-R TTCCTTCCTGGGTGAGTGGAG (SEQ ID NO.11);
  • ACTB-P Cy5-CAGCCCCGAGGGGTAACCCTCATGTCA (SEQ ID NO.12);
  • the sequence of the amplified region of the primer pair is:
  • This sequence contains a restriction site for methylation-sensitive restriction endonuclease HpaII.
  • the sample template sequence will be digested It is two stages, and the amplification cannot be completed when the fluorescent quantitative PCR detection is performed.
  • the target gene SFRP1 probe is FAM fluorescently labeled
  • the internal reference gene GAPDH probe is VIC fluorescently labeled
  • the digestion monitoring gene ACTB probe is Cy5 fluorescently labeled, so it can be detected in one tube.
  • ACTB Since the number of restriction sites (1) in the amplification region of the restriction enzyme control gene ACTB is less than the number of restriction sites in the amplification region of the target gene SFRP1 (2), and the number of ACTB genes is the same as the number of SFRP1 genes, when ACTB When the gene is digested, it means that SFRP1 can also be digested, which can achieve the purpose of monitoring the digestion system and prevent false positive results due to some target fragments not being digested.
  • the implementation of this method includes the following steps:
  • the detection system of Table 2 only adds the primer pair and probe of the target gene SFRP1, and the primer pair and probe of the internal reference gene GAPDH; the detection system of Table 3 The primer pair and probe of the target gene SFRP1, the primer pair and probe of the internal reference gene GAPDH, and the primer pair and probe of the restriction enzyme control gene ACTB are added inside;
  • Serial number SFRP1 GAPDH ACTB determination 1 No amplification No amplification No amplification Invalid sample 2 No amplification No amplification With amplification Invalid sample 3 No amplification With amplification No amplification SFRP1 unmethylated 4 With amplification No amplification No amplification Invalid sample 5 With amplification No amplification With amplification Invalid sample 6 With amplification With amplification No amplification SFRP1 methylation 7 With amplification With amplification With amplification Incomplete digestion, invalid detection
  • Figure 2 shows the results of 50ng, 40ng, 30ng, 20ng, 10ng Human Non-Methylated DNA digestion of the SFRP1 gene according to the detection system in Table 2.
  • Figure 3 shows the detection system of 50ng, 40ng, 30ng, 20ng, 10ng Human according to Table 3 The result of SFRP1 gene after non-Methylated DNA digestion. From Figure 2 and Figure 3, it can be seen that 50ng and 40ng Human Non-Methylated DNA were amplified after detection of SFRP1 methylation, indicating that 50ng and 40ng Human Non-Methylated DNA was not digested cleanly.
  • the method of the present invention can improve the accuracy of methylation detection and prevent the occurrence of false positive results.
  • the restriction enzyme cut monitor gene can be used as the target gene monitor gene, and the following test:
  • the target gene SFRP1 can be digested in the following 4 situations: Case 1: Both restriction sites are cut; Case 2: One of the 2 restriction sites is cut; Case 3: Two restriction sites The other was digested by restriction enzymes; Case 4: Both restriction sites were not cut by restriction enzymes. In the above 4 cases, only case 4 can be amplified, so the probability of the target gene being digested and unable to complete the amplification is 3/4.
  • the restriction enzyme cutting monitoring gene ACTB has only one restriction site.
  • the restriction enzyme cutting monitoring gene ACTB has the following two conditions: Case 1: 1 restriction site is cut; Case 2: 1 restriction site is not cut Enzyme digestion. In the above two cases, only case 2 can be amplified. Therefore, the probability that the restriction enzyme digestion monitoring gene ACTB is digested and cannot be amplified is 1/2, which is less than the probability of the target gene being digested.
  • the target gene SFRP1 and the digestion monitoring gene ACTB are reacted in one tube.
  • the digestion conditions are the same. Therefore, whether the digestion monitoring gene ACTB will be digested and cannot be amplified can be used as whether the target gene will be digested.
  • the monitoring genes that cannot be amplified are the following two conditions: Case 1 restriction site is cut; Case 2: 1 restriction site is not cut Enzyme digestion. In the above two cases, only case 2 can be amplified. Therefore, the probability that the restriction enzyme digestion monitoring gene ACTB is digested and cannot be amplified is 1/2, which is less than the probability of the target gene
  • the amplified region of the selected monitoring gene ACTB contains the restriction enzyme cut sites of the selected restriction enzyme, and the number of restriction enzyme cut sites needs to be less than or equal to the number of restriction enzyme cut sites of the target gene to ensure that The two monitoring genes reacting in one tube can play the role of monitoring the target gene.
  • the detection method of the present invention can ensure the accuracy of detection and solve the technical problem of improving the accuracy of detection to be solved by the present invention.
  • Embodiment 3 Increasing the sample size further proves the reliability of the solution of the present invention; 40 samples are taken for processing according to the following steps:
  • Step one sample DNA extraction
  • Step 2 Purify the DNA extracted in Step 1, and remove inhibitory components
  • Step 3 Take 40ng of purified DNA and digest it with methylation-sensitive restriction enzymes.
  • the digestion system is as follows:
  • Step 4 Use the following detection system 1 (target gene + internal reference gene), detection system 2 (target gene + restriction enzyme cutting monitoring gene), and detection system 3 (target gene + restriction enzyme cutting monitoring gene + internal reference gene) to detect the alpha of SFRP1 gene. Base.
  • test results and test results are interpreted as follows:
  • methylated and non-methylated standard DNA (Human Methylated&Non-Methylated DNA Set, Catalog#D5014, ZYMORESEARCH) were used for blending, and 10ng of sample DNA with different mutation frequencies was blended, as shown in Table 13. :
  • the detection steps of the method of the present invention are as follows:
  • the present invention (enzyme digestion method) can detect at least 0.5% methylation in 10ng nucleic acid, while the heavy salt method can only detect 1% methylation in 10ng nucleic acid.
  • the sensitivity of the) method to detect methylation is much higher than that of the heavy salt method.
  • the methylation detection kit includes: primer pairs and probes designed for the promoter region of SFRP1 gene, VIM gene, and TWIST1 gene.
  • the sequence of the primer pair for SFRP1 gene is:
  • SFRP1-F GTTGGATCCCAGGAAGAGCG (SEQ ID NO. 2);
  • SFRP1-R CATGCTGTGCACGTGATTCC (SEQ ID NO.3);
  • the probe sequence for SFRP1 is:
  • SFRP1-P CGCGAGGGGCGCTGAGCGATACC (SEQ ID NO.4);
  • the primer pair sequence for VIM is:
  • VIM-F GCTCCTCTGCCGTGCG (SEQ ID NO.14);
  • VIM-R TAGTTGGCGAAGCGGTCATT (SEQ ID NO.15);
  • the probe sequence for VIM is:
  • VIM-P CAGGACTCGGTGGACTTCTCGCTGGCCG (SEQ ID NO.16);
  • the primer pair sequence for TWIST1 is:
  • TWIST1-F GCACATTCGTGGGCTCTCAA (SEQ ID NO.17);
  • TWIST1-R ATGTCGTAAAGAGTGCGCCG (SEQ ID NO.18);
  • the probe sequence for TWIST1 is:
  • TWIST1-P CCGATTTCTCCTTCCACTTTGCCAGTGCAC (SEQ ID NO.19);
  • the concentration of each primer in the above primer pair is 0.1-10uM
  • the concentration of the above-mentioned probe is 0.1-10uM.
  • QiaGen Fecal DNA Extraction Kit to extract human genomic DNA from stool samples of normal people or patients with bowel cancer (for specific extraction methods, refer to QiaGen Fecal DNA Extraction Kit Instructions).
  • digestion time is 37°C16h; digestion system is shown in Table 15 below (the selected endonuclease was purchased from NEB company, and the digestion system refers to the purchased endonuclease Manual)
  • composition volume 10x Methylation Sensitive Restriction Enzyme Buffer 5ul Human genomic DNA/negative control/positive quality control 1-10ug Methylation sensitive restriction endonuclease HpaII 5-20U Nuclease-free water Make up to 50ul
  • the amplified region sequence and restriction principle of the selected target genes SFRP1, VIM, TWIST1 are as follows:
  • the SFRP1 wild-type sequence is shown in Figure 17.
  • the SFRP1 wild-type sequence has 2 restriction sites.
  • the methylation-sensitive restriction endonuclease HpaII specifically cuts it into 3 segments.
  • the sequence after restriction is:
  • the SFRP1 methylation sequence is shown in Figure 18. Because in the sequence middle HpaII is a methylation-sensitive restriction endonuclease. After digestion, the SFRP1 methylation sequence will not be cut into fragments. The sequence after digestion is:
  • VIM wild type sequence is shown in Figure 19.
  • VIM wild-type sequence has 1 restriction site, and methylation sensitive restriction endonuclease HpaII specifically cuts it into 2 segments.
  • VIM methylation sequence is shown in Figure 20.
  • the wild-type sequence of TWIST1 has one restriction site.
  • the methylation-sensitive restriction endonuclease HpaII specifically cuts it into two segments.
  • the sequence after restriction is:
  • TWIST1 The methylation sequence of TWIST1 is shown in Figure 22.
  • step 3 Take 10ul of the digested product in step 3 for real-time quantitative qPCR detection to detect the methylation of SFRP1 gene, VIM gene, and TWIST1 gene respectively.
  • the qPCR detection system is shown in Table 16 below.
  • S is for the detection of SFRP1 gene
  • D is for detection of VIM gene
  • N is for detection of TWIST1 gene
  • PC is a positive quality control
  • NC is a negative control
  • NTC is a blank control.
  • a single sample can be interpreted. If the sample detects SFRP1 gene, VIM gene, and TWIST1 gene, the Cp value of the internal reference gene GAPDH (VIC channel) is less than or equal to 40, and the Cp value of the enzyme digestion monitoring gene ACTB (Cy5 channel) is all If it is equal to 50, it means that the amount of DNA input in the sample is sufficient and all of it is digested by restriction enzymes.
  • a single sample can be interpreted as shown in Table 20 below:
  • the stool samples collected above were tested and interpreted. In 30 normal people, no methylation of three genes was detected. 20 patients with precancerous adenoma followed this reagent The box interpretation method detected positive 18 cases (accounting for 90%), and 30 patients with bowel cancer were detected positive according to this kit interpretation method (accounting for 93.3%).
  • Figure 23 is the amplification curve of SFRP1 gene, VIM gene, and TWIST1 gene in negative samples of colorectal cancer
  • Figure 24 negative samples of colorectal cancer are detecting SFRP1 gene, Amplification curve of internal reference gene GAPDH when VIM gene and TWIST1 gene
  • Figure 25 shows the amplification curve of SFRP1 gene, VIM gene, and TWIST1 gene in positive colorectal cancer samples
  • Figure 26 shows the detection of SFRP1 gene and VIM gene in positive colorectal cancer samples , TWIST1 gene time reference gene GAPDH amplification curve. It can be seen from Figure 27 that the 28 cases of colorectal cancer positive samples that were detected by restriction enzyme digestion monitoring gene ACTB were all digested by restriction enzymes and could not be amplified.

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Abstract

一种提高甲基化基因酶切法检测准确性的方法，具体涉及一种双基因监控反应体系、应用双基因监控反应体系的试剂盒及其在检测人结直肠癌中的应用。采用酶切法处理所需检测样本核酸，与重盐法相比，核酸损失更少，检测灵敏度更高；采用双基因监控反应体系，既能监控检测反应时的样本质量，又能监控酶切反应的酶切效率，使检测的准确性大大提高；该方法的应用操作简单，成本低。

Description

一种双基因监控反应体系、试剂盒及其应用 技术领域
本发明涉及一种提高甲基化基因酶切法检测准确性的方法，具体涉及一种双基因监控反应体系、应用双基因监控反应体系的试剂盒及其在检测人结直肠癌中的应用。
背景技术
DNA甲基化是指生物体在DNA甲基转移酶的催化下，以s-腺苷甲硫氨酸为甲基供体，将甲基转移到特定的碱基上的过程。哺乳动物中DNA甲基化主要发生在5’-CpG-3’的C上生成5-甲基胞嘧啶。在哺乳动物中CpG以两种形式存在：一种是分散在DNA序列中，另一种呈现高度聚集状态，称之为CpG岛。在正常组织里，70％-90％散在的CpG是被甲基修饰的，而CpG岛往往是非甲基化的。目前，越来越多的研究表明，CpG岛局部的高甲基化是肿瘤发生的一个重要因素，由于CpG岛的局部高度甲基化要早于细胞恶性增生，故其甲基化的检测可用于肿瘤的预测。
目前市面上针对甲基化检测的方法均采用重盐转化法对检测前的样本进行处理，该方法对样本核酸的损伤很大，导致检测的灵敏度不高，而且存在转化不完全而导致的假阳性结果。
与重盐转化法相比，DNA甲基化检测方法--甲基化敏感性限制性内切酶法可以避免对目的核酸的损伤，从而使检测的准确度大大提高，然而，酶切不完全而导致的假阳性结果也是限制该方法准确性的一个主要问题。
发明内容
针对现有技术的不足，本发明目的在于提供一种提高酶切法检测甲基化准确性的方法，通过创造性的双基因监控反应体系及其应用，达到提高检测准确度的目的。
为了解决上述技术问题，本发明的技术方案如下：
一种双基因监控反应体系，体系中包括目的基因引物对和探针，内参基因 GAPDH引物对和探针，酶切监控基因引物对和探针，所述目的基因和酶切监控基因ACTB的引物扩增区域包含多个甲基化敏感性限制性内切酶酶切位点。
优选的，目的基因的探针、内参基因GAPDH的探针和酶切监控基因ACTB的探针分别单独采用FAM、VIC、Cy3、Cy5、ROX、HEX中的一种标记。
优选的，目的基因、内参基因GAPDH、酶切监控基因ACTB采用不同的荧光标记，更优选的，目的基因SFRP1探针采用FAM荧光标记，内参基因GAPDH探针采用VIC荧光标记，酶切监控基因ACTB探针采用Cy5荧光标记。
优选的，目的基因引物探针、内参基因GAPDH引物探针和酶切监控基因ACTB引物探针在1管中进行反应。
一种采用上述的双基因监控反应体系提高酶切法检测甲基化准确性的方法，包括以下步骤：
步骤一、采用核酸提取试剂盒提取样本核酸DNA；
步骤二、取10-100ng所提取的核酸用甲基化敏感性限制性内切酶进行酶切处理；
步骤三、对步骤二中得到的酶切产物采用双基因监控反应体系进行检测。
优选的，步骤二中所述的甲基化敏感性限制性内切酶的量为10-100U。
优选的，步骤二中所述的甲基化敏感性限制性内切酶选自以下内切酶中的一种或多种：AatII、AciI、AclI、AfeI、AgeI、AscI、AsiSI、AvaI、BceAI、BmgBI、BsaAI、BsaHI、BsiEI、BsiWI、BsmBI、BspDI、BspEI、BsrFI、BssHII、BstBI、BstUI、BtgZI、ClaI、EagI、FauI、FseI、FspI、HaeII、HgaI、HhaI、HinP1I、HpaII、Hpy99I、HpyCH4IV、KasI、MluI、NaeI、NarI、NgoMIV、NotI、NruI、Nt.BsmAI、Nt.CviPII、PaeR7I、PluTI、PmlI、PvuI、RsrII、SacII、SalI、SfoI、SgrAI、SmaI、SnaBI、TliI、TspMI、XhoI、XmaI、ZraI。
本发明还提供了一种用于上述提高酶切法检测甲基化准确性方法的DNA甲基化检测试剂盒，包括以下成分：
1)SFRP1基因的引物对和探针；
2)VIM基因的引物对和探针；
3)TWIST1基因的引物对和探针；
4)GAPDH的引物对和探针；
5)ACTB的引物对和探针；
6)阴性和阳性质控品；
7)PCR反应液。
每次检测时，阴性质控品，阳性质控品都需要和样本一起同步参与检测，以保证实验的准确性和有效性；
所述针对SFRP1基因的引物对的序列为：
SFRP1-F：GTTGGATCCCAGGAAGAGCG(SEQ ID NO.2)；
SFRP1-R：CATGCTGTGCACGTGATTCC(SEQ ID NO.3)；
所述针对SFRP1的探针序列为：
SFRP1-P：CGCGAGGGGCGCTGAGCGATACC(SEQ ID NO.4)；
所述针对VIM的引物对序列为：
VIM-F：GCTCCTCTGCCGTGCG(SEQ ID NO.13)；
VIM-R：TAGTTGGCGAAGCGGTCATT(SEQ ID NO.14)；
所述针对VIM的探针序列为：
VIM-P：CAGGACTCGGTGGACTTCTCGCTGGCCG(SEQ ID NO.15)；
所述针对TWIST1的引物对序列为：
TWIST1-F：GCACATTCGTGGGCTCTCAA(SEQ ID NO.16)；
TWIST1-R：ATGTCGTAAAGAGTGCGCCG(SEQ ID NO.17)；
所述针对TWIST1的探针序列为：
TWIST1-P：CCGATTTCTCCTTCCACTTTGCCAGTGCAC(SEQ ID NO.18)；
优选的，上述引物对中各引物的浓度为0.1-10uM。
优选的，上述探针的浓度为0.1-10uM。
上述的试剂盒的具体使用步骤包括如下步骤：
步骤一、样本DNA提取；
步骤二、纯化步骤一中提取的DNA，去除抑制成分；
步骤三、取1-20ug纯化后DNA用甲基化敏感性限制性内切酶进行酶切处理；
步骤四、采用双基因监控反应体系分别检测SFRP1基因，VIM基因，TWIST1基因的甲基化。
本发明还提供了一种基于上述试剂盒在检测人结直肠癌中的应用。
本发明设计引物的扩增区域包含多个甲基化敏感性限制性内切酶酶切位点， 野生型的序列在甲基化敏感性限制性内切酶的作用下被酶切为片段，后续PCR反应时无法扩增，而甲基化序列在甲基化敏感性限制性内切酶的作用下能保持完整，后续PCR反应时可以扩增。具体过程如图1所示。
目的基因扩增区域含有n个所选取的甲基化敏感的限制性内切酶酶切位点，内参基因GAPDH扩增区域不含有所选取的甲基化敏感性限制性内切酶酶切位点，酶切监控基因ACTB扩增区域含有m个所选取的甲基化敏感性限制性内切酶酶切位点，且m≤n，由于酶切监控基因ACTB必须被酶切才能保证目的基因也能被酶切，因此，m≥1。因为酶切检测基因ACTB的甲基化敏感性限制性内切酶酶切位点个数m小于或等于目的基因的甲基化敏感性限制性内切酶酶切位点个数n，目的基因，内参基因GAPDH，酶切监控基因ACTB均在一个反应管中进行反应，且目的基因，内参基因GAPDH，酶切监控基因ACTB均来自于人基因组，当酶切监控基因ACTB均被酶切时，则说明目的基因扩增区域也能被酶切，当酶切监控基因ACTB没有被酶切，则说明反应体系酶切受到影响，目的基因或者没有被酶切。内参基因GAPDH扩增区域不含甲基化敏感性限制性内切酶酶切位点，不受酶切的影响，可以监控反应体系中的人基因组含量。
与现有技术相比，本发明的有益效果如下：
1、采用酶切法处理所需检测样本核酸，与重盐法相比，核酸损失更少，检测灵敏度更高；
2、采用双基因监控反应体系，既能监控检测反应时的样本质量，又能监控酶切反应的酶切效率，使检测的准确性大大提高；
3、该发明方法的应用操作简单，成本低。
附图说明
图1是本发明的甲基化序列和野生型序列在甲基化敏感性限制性内切酶作用下的切割过程；
图2是按照表2检测体系检测50ng，40ng，30ng，20ng，10ng Human Non-Methylated DNA酶切后SFRP1基因的结果；
图3是按照表3检测体系检测50ng，40ng，30ng，20ng，10ng Human Non-Methylated DNA酶切后SFRP1基因的结果；
图4是按照表3检测体系检测50ng，40ng，30ng，20ng，10ng Human  Non-Methylated DNA酶切后ACTB基因的结果；
图5是实施例2中酶切产物A目的基因SFRP1扩增曲线图；
图6是实施例2中酶切产物A酶切监控基因ACTB扩增曲线图；
图7是实施例2中酶切产物B目的基因SFRP1扩增曲线图；
图8是实施例2中酶切产物B酶切监控基因ACTB扩增曲线图；
图9是实施例3中34例有效样本内参基因GAPDH扩增曲线图；
图10是实施例3中34例有效样本酶切监控基因ACTB扩增曲线图；
图11是实施例3中检测体系3的3例GAPDH无扩增的无效样本内参基因GAPDH扩增曲线图；
图12是实施例3中检测体系3的3例GAPDH无扩增的无效样本酶切监控基因ACTB扩增曲线图；
图13是实施例3中检测体系3的3例ACTB有扩增的无效样本内参基因GAPDH扩增曲线图；
图14是实施例3中检测体系3的3例ACTB有扩增的无效样本酶切监控基因ACTB扩增曲线图；
图15是S1,S2,S3,S4,S5,S6酶切法FAM通道检测结果；
图16是S1,S2,S3,S4,S5,S6重盐处理法FAM通道检测结果；
图17是SFRP1野生型序列图；
图18是SFRP1甲基化序列图；
图19是VIM野生型序列图；
图20是VIM甲基化序列图；
图21是TWIST1野生型序列图；
图22是TWIST1甲基化序列图；
图23是结直肠癌阴性样本SFRP1基因，VIM基因，TWIST1基因扩增曲线图；
图24是结直肠癌阴性样本在检测SFRP1基因，VIM基因，TWIST1基因时内参基因GAPDH扩增曲线图；
图25是结直肠癌阳性样本SFRP1基因，VIM基因，TWIST1基因扩增曲线图；
图26是结直肠癌阳性样本在检测SFRP1基因，VIM基因，TWIST1基因时内参基因GAPDH扩增曲线图；
图27是实施例6检出的28例结直肠癌阳性样本酶切监控基因ACTB均被酶切而无法完成扩增的曲线图。
具体实施方式
以下将结合实施例来详细说明本发明。需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。
实施例1
检测SFRP1基因甲基化
SFRP1基因启动子区域CpG岛序列为：
Figure PCTCN2020103154-appb-000001
Figure PCTCN2020103154-appb-000002
针对SFRP1基因启动子区域设计的引物对和探针序列为：
SFRP1-F：GTTGGATCCCAGGAAGAGCG(SEQ ID NO.2)；
SFRP1-R：CATGCTGTGCACGTGATTCC(SEQ ID NO.3)；
SFRP1-P：FAM-CGCGAGGGGCGCTGAGCGATACC(SEQ ID NO.4)；
该引物对扩增区域序列为：
Figure PCTCN2020103154-appb-000003
Figure PCTCN2020103154-appb-000004
该序列包含2个甲基化敏感性限制性内切酶HpaII的酶切位点，当用甲基化敏感性限制性内切酶对样本核酸进行酶切处理时，野生型样本模板序列会被酶切为3段，做荧光定量PCR检测时无法完成扩增。
同时还设计了内参基因GAPDH的引物对和探针序列：
GAPDH-F：GCAACTAGGATGGTGTGGCT(SEQ ID NO.6)；
GAPDH-R：TCGCCCCACTTGATTTTGGA(SEQ ID NO.7)；
GAPDH-P：VIC-ACCTTGTGTCCCTCAATATGGTCCTGT(SEQ ID NO.8)；
该引物对扩增区域序列为：
Figure PCTCN2020103154-appb-000005
该序列包含0个甲基化敏感性限制性内切酶HpaII的酶切位点，当用甲基化敏感性限制性内切酶对样本核酸进行酶切处理时，样本模板序列不会被酶切，做荧光定量PCR检测是可以完成扩增，由于不受酶切的的影响，且GAPDH作为管家基因，可以监控样本里面的人源核酸含量。
本发明还设计了酶切监控基因ACTB的引物对和探针序列：
ACTB-F：AATGAGGGCAGGACTTAGCTT(SEQ ID NO.10)；
ACTB-R：TTCCTTCCTGGGTGAGTGGAG(SEQ ID NO.11)；
ACTB-P：Cy5-CAGCCCCGAGGGGTAACCCTCATGTCA(SEQ ID NO.12)；
该引物对扩增区域序列为：
Figure PCTCN2020103154-appb-000006
该序列包含1个甲基化敏感性限制性内切酶HpaII的酶切位点，当用甲基化敏感性限制性内切酶对样本核酸进行酶切处理时，样本模板序列会被酶切为2段，做荧光定量PCR检测时无法完成扩增。
目的基因SFRP1探针采用FAM荧光标记，内参基因GAPDH探针采用VIC 荧光标记，酶切监控基因ACTB探针采用Cy5荧光标记，因此可以在1管中完成检测。
由于酶切监控基因ACTB扩增区域酶切位点个数(1个)小于目的基因SFRP1扩增区域酶切位点个数(2个)，而ACTB基因数目与SFRP1基因数目相同，因此当ACTB基因被酶切时，则说明SFRP1也能被酶切，可以达到监控酶切体系的目的，防止由于有的目的片段没有被酶切而导致的假阳性结果。
本方法的实施包括以下几个步骤：
1、取10ng,20ng,30ng,40ng,50ng的Human Non-Methylated DNA用10U的HpaII进行酶切处理，按照下表1配置酶切体系，在PCR仪上37℃酶切16h；
表1
Figure PCTCN2020103154-appb-000007
2、分别按照下表2和下表3配置检测体系，其中表2的检测体系里面只加了目的基因SFRP1的引物对和探针，内参基因GAPDH的引物对和探针；表3的检测体系里面加了目的基因SFRP1的引物对和探针，内参基因GAPDH的引物对和探针，酶切监控基因ACTB引物对和探针；
表2
Figure PCTCN2020103154-appb-000008
Figure PCTCN2020103154-appb-000009
表3
组分 体积(ul)
上一步酶切产物 10
2x TaqMan TM Universal Master Mix II,with UNG 25
SFRP1-F(10uM) 1
SFRP1-R(10uM) 1
SFRP1-P(10uM) 1
GAPDH-F(10uM) 1
GAPDH-R(10uM) 1
GAPDH-P(10uM) 1
ACTB-F(10uM) 1
ACTB-R(10uM) 1
ACTB-P(10uM) 1
ddH 2O 6
3、分别按照上述表2和表3中的体系将样本加入
Figure PCTCN2020103154-appb-000010
Multiwell Plate 96,white中，封上膜后置于
Figure PCTCN2020103154-appb-000011
仪器上进行荧光定量qPCR检测。
qPCR反应程序为表4：
表4
Figure PCTCN2020103154-appb-000012
4、检测结果分析
按照下表5进行判定，检测出SFRP1基因该启动子CpG岛扩增区域是否甲基化。
表5
序号 SFRP1 GAPDH ACTB 判定
1 无扩增 无扩增 无扩增 无效样本
2 无扩增 无扩增 有扩增 无效样本
3 无扩增 有扩增 无扩增 SFRP1非甲基化
4 有扩增 无扩增 无扩增 无效样本
5 有扩增 无扩增 有扩增 无效样本
6 有扩增 有扩增 无扩增 SFRP1甲基化
7 有扩增 有扩增 有扩增 酶切不完全，无效检测
图2为按照表2检测体系检测50ng，40ng，30ng，20ng，10ng Human Non-Methylated DNA酶切后SFRP1基因的结果，图3为按照表3检测体系检测50ng，40ng，30ng，20ng，10ng Human Non-Methylated DNA酶切后SFRP1基因的结果。由图2，图3可以看出，50ng，40ng的Human Non-Methylated DNA酶切后检测SFRP1甲基化时，均有扩增，说明50ng，40ng的Human Non-Methylated DNA没有被酶切干净。当我们按照表2中的检测体系进行检测时，由于没有酶切监控基因ACTB监测酶切体系，我们只能判定这2个样本(50ng Human Non-Methylated DNA，40ng Human Non-Methylated DNA)为阳性样本，导致假阳性的发生；然而，当我们按照表3中的检测体系进行检测时，由于加入了酶切监控基因ACTB监测酶切体系，如图4所示，50ng，40ng的Human Non-Methylated DNA酶切后检测时ACTB基因均有扩增，说明50ng，40ng的Human Non-Methylated DNA没有被酶切干净，因此可以判定这2个样本(50ng Human Non-Methylated DNA，40ng Human Non-Methylated DNA)是由于有片段没有被酶切而导致的假阳性样本。
通过以上结果分析可以看出，本发明方法可以提高甲基化检测的准确性，防止假阳性结果的发生。
实施例2：
为了进一步证明：酶切位点越多的目的基因碰撞到内切酶的概率要大于酶切位点更少的酶切监控基因，酶切监控基因可以作为目的基因的监控基因，还进行了以下试验：
1)取50ng的Human Non-Methylated DNA分别按照下表进行酶切处理，
表6
Figure PCTCN2020103154-appb-000013
表7
Figure PCTCN2020103154-appb-000014
表6和表7中的反应体系配好后，在PCR仪上37℃酶切16h，按照表6的体系酶切的产物命名为酶切产物A,按照表7的体系酶切的产物命名为酶切产物B；
2)按照实施例1中的表3的检测体系分别对酶切产物A和酶切产物B进行检测,检测结果如图5-图8所示。由图可以看出，在相同条件下进行酶切后，目的基因SFRP1被酶切干净而无法扩增，而酶切监控基因ACTB没有被酶切干净，可以完成扩增。目的基因酶切位点为2个，酶切监控基因酶切位点个数为1个，因此，相比于酶切监控基因ACTB，酶切位点更多的目的基因SFRP1碰撞到内切酶而被酶切的概率更大，而非甲基化的目的基因SFRP1的2个酶切位点中只要有不少于1个位点被酶切，就无法完成扩增。目的基因SFRP1的酶切存在下面4种情况：情况1：2个酶切位点均被酶切；情况2：2个酶切位点其中一个被酶切；情况3：2个酶切位点其中另一个被酶切；情况4：2个酶切位点均没被酶切。上面4种情况，只有情况4能发生扩增，因此目的基因被酶切而无法完成扩增的概率是3/4。酶切监控基因ACTB只有一个酶切位点，酶切监控基因ACTB的酶切存在下面2种情况：情况1:1个酶切位点被酶切；情况2:1个酶切位点没有被酶切。上面2种情况，只有情况2能发 生扩增，因此酶切监控基因ACTB被酶切而无法完成扩增的概率是1/2，概率小于目的基因被酶切的概率。而目的基因SFRP1和酶切监控基因ACTB在一管中进行反应，酶切条件是相同的，因此，酶切监控基因ACTB的是否会被酶切而无法扩增可以作为目的基因是否会被酶切而无法扩增的监控基因。因此，保证所选择的监控基因ACTB的扩增区域包含所选择限制性内切酶的酶切位点，且酶切位点个数需要小于或等于目的基因酶切位点个数，从而保证在一个管中反应的两个监控基因能起到监控目的基因的作用，采用本发明的检测方法，才能保证检测的准确性，才能解决本发明要解决的提高检测的准确性的技术问题。
实施例3：增大样本量进一步证明本发明方案的可靠性；各取40例样本按照以下步骤进行处理：
步骤一、样本DNA提取；
步骤二、纯化步骤一中提取的DNA，去除抑制成分；
步骤三、取40ng纯化后DNA用甲基化敏感性限制性内切酶进行酶切处理，酶切体系如下：
表8
Figure PCTCN2020103154-appb-000015
步骤四、分别用以下检测体系1(目的基因+内参基因)，检测体系2(目的基因+酶切监控基因)，检测体系3(目的基因+酶切监控基因+内参基因)检测SFRP1基因的甲基化。
检测体系1：
表9
组分 体积(μL)
酶切产物 10
2x TaqMan TM Universal Master Mix II,with UNG 25
SFRP1-F(10uM) 1
SFRP1-R(10uM) 1
SFRP1-P(10uM) 1
GAPDH-F(10uM) 1
GAPDH-R(10uM) 1
GAPDH-P(10uM) 1
ddH2O 9
检测体系2：
表10
组分 体积(μL)
酶切产物 10
2x TaqMan TM Universal Master Mix II,with UNG 25
SFRP1-F(10uM) 1
SFRP1-R(10uM) 1
SFRP1-P(10uM) 1
ACTB-F(10uM) 1
ACTB-R(10uM) 1
ACTB-P(10uM) 1
ddH2O 9
检测体系3：
表11
组分 体积(μL)
酶切产物 10
2x TaqMan TM Universal Master Mix II,with UNG 25
SFRP1-F(10uM) 1
SFRP1-R(10uM) 1
SFRP1-P(10uM) 1
GAPDH-F(10uM) 1
GAPDH-R(10uM) 1
GAPDH-P(10uM) 1
ACTB-F(10uM) 1
ACTB-R(10uM) 1
ACTB-P(10uM) 1
ddH2O 6
40例样本分别用检测体系1，检测体系2，检测体系3检测，检测结果和检测结果判读如下:
表12
Figure PCTCN2020103154-appb-000016
检测结果见附图9-14。
实施例4
本发明方法与重盐法在检测基因甲基化灵敏性方面的对比
本实施例使用甲基化和非甲基化标准品DNA(Human Methylated&Non-Methylated DNA Set，Catalog#D5014，ZYMO RESEARCH)进行掺和，掺和10ng的不同突变频率的样本DNA，如表13所示：
表13
Figure PCTCN2020103154-appb-000017
本发明方法酶切法检测步骤为：
1)取10U的HpaI按照实施例1的步骤对S1,S2,S3,S4,S5,S6进行酶切处理，
37℃酶切16h；
2)按照实施例1的步骤对酶切后的产物进行检测，检测结果如图15所示。
重盐法检测步骤：
1)取S1,S2,S3,S4,S5,S6用EZ DNA Methylation-Gold Kits进行硫化；
2)用设计的SFRP1基因重盐法检测的引物检测，检测结果如图16所示。
通过对比可以看出，本发明(酶切法)最低能检出10ng核酸中0.5％甲基化，而重盐法最低只能检出10ng核酸中1％甲基化，本发明(酶切法)方法检测甲基化灵敏度要远高于重盐法。
实施例5
基于本发明方法开发的粪便样本结直肠癌早筛甲基化检测试剂盒
本实施例基于粪便样本结直肠癌早筛甲基化检测试剂盒主要成分如表14所 示：
表14
Figure PCTCN2020103154-appb-000018
每次检测时，阴性质控品，阳性质控品都需要和样本一起同步参与检测，以保证实验的准确性和有效性；
该甲基化检测试剂盒包括：针对SFRP1基因，VIM基因，TWIST1基因启动子区域设计的引物对和探针。
所述针对SFRP1基因的引物对的序列为：
SFRP1-F：GTTGGATCCCAGGAAGAGCG(SEQ ID NO.2)；
SFRP1-R：CATGCTGTGCACGTGATTCC(SEQ ID NO.3)；
所述针对SFRP1的探针序列为：
SFRP1-P：CGCGAGGGGCGCTGAGCGATACC(SEQ ID NO.4)；
所述针对VIM的引物对序列为：
VIM-F：GCTCCTCTGCCGTGCG(SEQ ID NO.14)；
VIM-R：TAGTTGGCGAAGCGGTCATT(SEQ ID NO.15)；
所述针对VIM的探针序列为：
VIM-P：CAGGACTCGGTGGACTTCTCGCTGGCCG(SEQ ID NO.16)；
所述针对TWIST1的引物对序列为：
TWIST1-F：GCACATTCGTGGGCTCTCAA(SEQ ID NO.17)；
TWIST1-R：ATGTCGTAAAGAGTGCGCCG(SEQ ID NO.18)；
所述针对TWIST1的探针序列为：
TWIST1-P：CCGATTTCTCCTTCCACTTTGCCAGTGCAC(SEQ ID NO.19)；
上面所述引物对中各引物的浓度为0.1-10uM；
上面所述探针的浓度为0.1-10uM。
本发明试剂盒具体操作步骤如下：
1、粪便样本DNA提取
采用QiaGen粪便DNA提取试剂盒提取正常人或肠癌患者粪便样本里面的人基因组DNA(具体提取方法参照QiaGen粪便DNA提取试剂盒说明书)。
2、纯化1中提取的人基因组DNA
采用ZYMO RESEARCH公司的DNA Clean&Concentrator纯化试剂盒纯化1中提取的人基因组DNA，以进一步去除抑制成分。
3、酶切处理
取1-20ug纯化后的人基因组DNA进行酶切，酶切时间为37℃16h；酶切体系如下表15所示(所选内切酶购买于NEB公司，酶切体系参照所购内切酶说明书)
表15
组成 体积
10x甲基化敏感性限制性内切酶Buffer 5ul
人基因组DNA/阴性质控品/阳性质控品 1-10ug
甲基化敏感性限制性内切酶HpaII 5-20U
无核酶水 补足至50ul
所选靶基因SFRP1，VIM，TWIST1的扩增区域序列和酶切原理如下：
SFRP1野生型序列为图17所示。
SFRP1野生型序列有2个酶切位点，甲基化敏感限制性内切酶HpaII特异性的将其酶切为3段，酶切后序列为：
Figure PCTCN2020103154-appb-000019
Figure PCTCN2020103154-appb-000020
SFRP1甲基化序列为图18所示。由于序列中
Figure PCTCN2020103154-appb-000021
中的
Figure PCTCN2020103154-appb-000022
为甲基化，而HpaII为甲基化敏感的限制性内切酶，经过酶切后，SFRP1甲基化序列不会被酶切为片段，酶切后序列为：
Figure PCTCN2020103154-appb-000023
VIM野生型序列为图19所示。
VIM野生型序列有1个酶切位点，甲基化敏感限制性内切酶HpaII特异性的将其酶切为2段，
酶切后序列为：
Figure PCTCN2020103154-appb-000024
VIM甲基化序列为图20所示。
由于序列中
Figure PCTCN2020103154-appb-000025
中的
Figure PCTCN2020103154-appb-000026
为甲基化，而HpaII为甲基化敏感的限制性内切酶，经过酶切后，SFRP1甲基化序列不会被酶切为片段，酶切后序列为：
Figure PCTCN2020103154-appb-000027
TWIST1野生型序列为图21所示。
TWIST1野生型序列有1个酶切位点，甲基化敏感限制性内切酶HpaII特异性的将其酶切为2段，酶切后序列为：
Figure PCTCN2020103154-appb-000028
TWIST1甲基化序列为图22所示。
由于序列中
Figure PCTCN2020103154-appb-000029
中的
Figure PCTCN2020103154-appb-000030
为甲基化，而HpaII为甲基化敏感的限制性内切酶，经过酶切后，TWIST1甲基化序列不会被酶切为片段，酶切后序列为：
Figure PCTCN2020103154-appb-000031
由上可知，靶基因SFRP1，VIM，TWIST1野生型序列会被甲基化敏感性限制性内切酶酶切为片段，在qPCR检测时无法完成扩增，而甲基化序列不会被甲基化敏感性限制性内切酶酶切，在qPCR检测时可以完成扩增，从而达到检测甲基化的目的。
4、实时定量qPCR检测
取10ul步骤3中酶切后的产物进行实时定量qPCR检测，分别检测SFRP1基因，VIM基因，TWIST1基因的甲基化，qPCR检测的体系如下表16所示。
表16
Figure PCTCN2020103154-appb-000032
Figure PCTCN2020103154-appb-000033
每个样本做三个孔，分别检测SFRP1基因，VIM基因，TWIST1基因的甲基化情况，检测时的推荐布局如下表17所示：
表17
  1 2 3 4 5 6 7 8 9 10 11 12
A PC1 PC2 PC3 S7-S S7-D S7-N            
B NC1 NC2 NC3 S… S… S…            
C S1-S S1-D S1-N NTC NTC NTC            
D S2-S S2-D S2-N                  
E S3-S S3-D S3-N                  
F S4-S S4-D S4-N                  
G S5-S S5-D S5-N                  
H S6-S S6-D S6-N                  
注：S为检测SFRP1基因，D为检测VIM基因，N为检测TWIST1基因，PC为阳性质控品，NC为阴性质控品，NTC为空白对照。
将点好样后覆好膜的96孔板瞬时离心，置于Lightcycler480Ⅱ上进行qPCR检测，反应程序如表18所示：
表18
Figure PCTCN2020103154-appb-000034
5、检测结果的判读(以Lightcycler 480II为例)
判读前，在Noise Band界面将Noiseband(Auto)改为Noiseband(Fluoresc)， 将Noiseband后面方框中的数字改成2.0000，阴性质控品，阳性质控品的Cp值按下表19进行判读时，满足表19中所示，则说明qPCR反应有效。
表19
Figure PCTCN2020103154-appb-000035
接下来可以对单个样本进行判读，如果样本检测SFRP1基因，VIM基因，TWIST1基因时内参基因GAPDH(VIC通道)的Cp值均小于或等于40，酶切监控基因ACTB(Cy5通道)的Cp值均等于50，则说明检测时样本DNA的投入量足够且均被酶切，可以对单个样本按下表20所示进行判读：
表20
Figure PCTCN2020103154-appb-000036
实施例6
基于本发明方法所开发的试剂盒在检测人结直肠癌中的应用
收集下面三种粪便样本：
(1)30例肠镜确诊为正常人的粪便样本；
(2)20例肠镜及病理检查确诊为癌前腺瘤患者的粪便样本；
(3)30例肠镜及病理检查确诊为肠癌患者的粪便样本。
按照实施例3中试剂盒的使用方法，对上面收集的粪便样本进行检测及判读，在30例正常人中均未检出三个基因的甲基化，20例癌前腺瘤患者按照本试剂盒判读方法检出阳性18例(占90％)，30例肠癌患者按照本试剂盒判读方法检出阳性28例(占93.3％)。
本实施例的具体实验数据如图23-图27所示，其中图23为结直肠癌阴性样本SFRP1基因，VIM基因，TWIST1基因扩增曲线图；图24结直肠癌阴性样本在检测SFRP1基因，VIM基因，TWIST1基因时内参基因GAPDH扩增曲线图；图25为结直肠癌阳性样本SFRP1基因，VIM基因，TWIST1基因扩增曲线图；图26为结直肠癌阳性样本在检测SFRP1基因，VIM基因，TWIST1基因时内参基因GAPDH扩增曲线图。由图27可以看出，检出的28例结直肠癌阳性样本酶切监控基因ACTB均被酶切而无法完成扩增。
上述实施例阐明的内容应当理解为这些实施例仅用于更清楚地说明本发明，而不用于限制本发明的范围，在阅读了本发明之后，本领域技术人员对本发明的各种等价形式的修改均落入本申请所附权利要求所限定的范围。
Figure PCTCN2020103154-appb-000037
Figure PCTCN2020103154-appb-000038
Figure PCTCN2020103154-appb-000039
Figure PCTCN2020103154-appb-000040
Figure PCTCN2020103154-appb-000041
Figure PCTCN2020103154-appb-000042
Figure PCTCN2020103154-appb-000043
Figure PCTCN2020103154-appb-000044
Figure PCTCN2020103154-appb-000045
Figure PCTCN2020103154-appb-000046

Claims (11)

  1. 一种双基因监控反应体系，其特征在于，体系中包括目的基因引物对和探针，内参基因GAPDH引物对和探针，酶切监控基因ACTB引物对和探针，所述目的基因和酶切监控基因ACTB的引物扩增区域包含多个甲基化敏感性限制性内切酶酶切位点。
  2. 根据权利要求1所述的双基因监控反应体系，其特征在于，目的基因扩增区域含有n个所选取的甲基化敏感的限制性内切酶酶切位点，内参基因GAPDH扩增区域不含有所选取的甲基化敏感的限制性内切酶酶切位点，酶切监控基因ACTB扩增区域含有m个所选取的甲基化敏感的限制性内切酶酶切位点，且m≦n。
  3. 根据权利要求1所述的双基因监控反应体系，其特征在于，目的基因的探针、内参基因GAPDH的探针和酶切监控基因ACTB的探针分别单独采用FAM、VIC、Cy3、Cy5、ROX、HEX中的一种标记。
  4. 根据权利要求1所述的双基因监控反应体系，其特征在于，目的基因、内参基因GAPDH、酶切监控基因ACTB采用不同的荧光标记，优选的，目的基因SFRP1探针采用FAM荧光标记，内参基因GAPDH探针采用VIC荧光标记，酶切监控基因ACTB探针采用Cy5荧光标记。
  5. 根据权利要求1所述的双基因监控反应体系，其特征在于，目的基因引物探针、内参基因GAPDH引物探针和酶切监控基因ACTB引物探针在1管中进行反应。
  6. 一种采用权利要求1-5任一项所述的双基因监控反应体系提高酶切法检测甲基化准确性的方法，其特征在于，包括以下步骤：
    步骤一、采用核酸提取试剂盒提取样本核酸DNA；
    步骤二、取10-100ng所提取的核酸用甲基化敏感性限制性内切酶进行酶切处理；
    步骤三、对步骤二中得到的酶切产物采用双基因监控反应体系进行检测。
  7. 根据权利要求6所述的方法，其特征在于，步骤二中所述的甲基化敏感性限制性内切酶的量为10-100U。
  8. 根据权利要求6所述的方法，其特征在于，步骤二中所述的甲基化敏感 性限制性内切酶选自以下内切酶中的一种或多种：AatII、AciI、AclI、AfeI、AgeI、AscI、AsiSI、AvaI、BceAI、BmgBI、BsaAI、BsaHI、BsiEI、BsiWI、BsmBI、BspDI、BspEI、BsrFI、BssHII、BstBI、BstUI、BtgZI、ClaI、EagI、FauI、FseI、FspI、HaeII、HgaI、HhaI、HinP1I、HpaII、Hpy99I、HpyCH4IV、KasI、MluI、NaeI、NarI、NgoMIV、NotI、NruI、Nt.BsmAI、Nt.CviPII、PaeR7I、PluTI、PmlI、PvuI、RsrII、SacII、SalI、SfoI、SgrAI、SmaI、SnaBI、TliI、TspMI、XhoI、XmaI、ZraI。
  9. 一种DNA甲基化检测试剂盒，其特征在于，包括以下成分：
    1)SFRP1基因的引物和探针；
    2)VIM基因的引物和探针；
    3)TWIST1基因的引物和探针；
    4)GAPDH的引物和探针；
    5)ACTB的引物和探针；
    6)阴性和阳性质控品；
    7)PCR反应液。
  10. 根据权利要求9所述的试剂盒的具体使用步骤包括如下步骤：
    步骤一、样本DNA提取；
    步骤二、纯化步骤一中提取的DNA，去除抑制成分；
    步骤三、取1-20μg纯化后DNA用甲基化敏感性限制性内切酶进行酶切处理；
    步骤四、采用双基因监控反应体系分别检测SFRP1基因，VIM基因，TWIST1基因的甲基化。
  11. 一种基于权利要求9或10所述试剂盒在检测人结直肠癌中的应用。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116970705A (zh) * 2023-09-13 2023-10-31 上海奕谱生物科技有限公司 用于尿路上皮癌基因甲基化检测的核酸产品、试剂盒及应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110283889B (zh) * 2019-07-22 2020-10-13 中南大学 一种双基因监控反应体系、试剂盒及其应用
CN110669831B (zh) * 2019-11-11 2023-09-19 益善生物技术股份有限公司 人sgip1、scand3和myo1g基因甲基化检测试剂盒
CN110923300A (zh) * 2019-11-18 2020-03-27 人和未来生物科技（长沙）有限公司 一种基因甲基化的检测方法和应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011015519A1 (en) * 2009-08-05 2011-02-10 Diasorin S.P.A. Highly sensitive rapid isothermal method for the detection of methylation patterns
CN104131070A (zh) * 2014-05-22 2014-11-05 苏州工业园区为真生物医药科技有限公司 一种对基因甲基化检测的方法
CN105441556A (zh) * 2015-12-29 2016-03-30 上海赛安生物医药科技有限公司 Dna甲基化转化效率的判断方法及其试剂盒和应用
CN107164524A (zh) * 2017-06-27 2017-09-15 深圳市优圣康生物科技有限公司 用于基因甲基化检测的引物和探针、采样方法、试剂盒
CN108103163A (zh) * 2018-01-17 2018-06-01 湖南大地同年生物科技有限公司 一种基因甲基化的检测方法
CN108139385A (zh) * 2015-07-27 2018-06-08 豪夫迈·罗氏有限公司 用于诊断和治疗炎症性肠病的方法
CN109097471A (zh) * 2018-08-21 2018-12-28 杭州和壹基因科技有限公司 一种用于结直肠癌及癌前病变检测的试剂盒及其使用方法
CN110283889A (zh) * 2019-07-22 2019-09-27 中南大学 一种双基因监控反应体系、试剂盒及其应用

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080261217A1 (en) * 2006-10-17 2008-10-23 Melnikov Anatoliy A Methylation Profile of Cancer
US9896730B2 (en) * 2011-04-25 2018-02-20 OSI Pharmaceuticals, LLC Use of EMT gene signatures in cancer drug discovery, diagnostics, and treatment
US20140227694A1 (en) * 2013-02-08 2014-08-14 Zymo Research Corporation Epigenetic markers of pluripotency
WO2016138105A2 (en) * 2015-02-24 2016-09-01 Zymo Research Corporation Assays to determine dna methylation and dna methylation markers of cancer
CN109929919B (zh) * 2018-09-14 2020-02-14 深圳市晋百慧生物有限公司 Dna甲基化检测方法及相关应用
CN109694905A (zh) * 2019-01-29 2019-04-30 深圳市亚辉龙生物科技股份有限公司 检测待测基因甲基化的核酸组合物及其试剂盒和待测基因甲基化的检测方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011015519A1 (en) * 2009-08-05 2011-02-10 Diasorin S.P.A. Highly sensitive rapid isothermal method for the detection of methylation patterns
CN104131070A (zh) * 2014-05-22 2014-11-05 苏州工业园区为真生物医药科技有限公司 一种对基因甲基化检测的方法
CN108139385A (zh) * 2015-07-27 2018-06-08 豪夫迈·罗氏有限公司 用于诊断和治疗炎症性肠病的方法
CN105441556A (zh) * 2015-12-29 2016-03-30 上海赛安生物医药科技有限公司 Dna甲基化转化效率的判断方法及其试剂盒和应用
CN107164524A (zh) * 2017-06-27 2017-09-15 深圳市优圣康生物科技有限公司 用于基因甲基化检测的引物和探针、采样方法、试剂盒
CN108103163A (zh) * 2018-01-17 2018-06-01 湖南大地同年生物科技有限公司 一种基因甲基化的检测方法
CN109097471A (zh) * 2018-08-21 2018-12-28 杭州和壹基因科技有限公司 一种用于结直肠癌及癌前病变检测的试剂盒及其使用方法
CN110283889A (zh) * 2019-07-22 2019-09-27 中南大学 一种双基因监控反应体系、试剂盒及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MELNIKOV, A.A. ET AL.: "Array-Based Multiplex Analysis of DNA Methylation in Breast Cancer Tissues", JOURNAL OF MOLECULAR DIAGNOSTICS, vol. 10, no. 1,, 31 January 2008 (2008-01-31), XP002573261 *
WANG, JIAN: "The Quantitative Analysis of Fetal RASSF1A Sequences in Maternal Plasma between Normal Pregnancy and Pre-eclampsia", CHINA MASTER’S THESES FULL-TEXT DATABASE, no. 2,, 15 February 2011 (2011-02-15) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116970705A (zh) * 2023-09-13 2023-10-31 上海奕谱生物科技有限公司 用于尿路上皮癌基因甲基化检测的核酸产品、试剂盒及应用
CN116970705B (zh) * 2023-09-13 2024-05-28 上海奕谱生物科技有限公司 用于尿路上皮癌基因甲基化检测的核酸产品、试剂盒及应用

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