WO2019223517A1 - 肿瘤标志物、甲基化检测试剂、试剂盒及其应用 - Google Patents

肿瘤标志物、甲基化检测试剂、试剂盒及其应用 Download PDF

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WO2019223517A1
WO2019223517A1 PCT/CN2019/085584 CN2019085584W WO2019223517A1 WO 2019223517 A1 WO2019223517 A1 WO 2019223517A1 CN 2019085584 W CN2019085584 W CN 2019085584W WO 2019223517 A1 WO2019223517 A1 WO 2019223517A1
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tumor
methylation
nucleotide sequence
sequence shown
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PCT/CN2019/085584
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French (fr)
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刘相林
赵荣淞
邹鸿志
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广州市康立明生物科技有限责任公司
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Priority to JP2020565479A priority Critical patent/JP7123177B2/ja
Priority to AU2019274887A priority patent/AU2019274887B2/en
Priority to CA3100858A priority patent/CA3100858A1/en
Priority to SG11202011368RA priority patent/SG11202011368RA/en
Priority to EP19807357.9A priority patent/EP3798315A4/en
Priority to KR1020207036321A priority patent/KR20210010564A/ko
Priority to US17/057,246 priority patent/US20210189461A1/en
Publication of WO2019223517A1 publication Critical patent/WO2019223517A1/zh
Priority to PH12020551982A priority patent/PH12020551982A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

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  • the invention relates to the field of biotechnology, in particular to tumor markers, methylation detection reagents, kits and applications thereof.
  • Colorectal cancer also known as colorectal cancer
  • colorectal cancer is a common malignant tumor of the digestive tract. Its incidence has been increasing year by year in China. In some coastal areas of China, such as Shanghai and Guangzhou, the incidence of colorectal cancer has jumped to the second place, behind lung cancer.
  • the formation of bowel cancer is currently thought to be the result of the accumulation of genetic defects and epigenetic defects.
  • the early onset of colorectal cancer is insidious and often without obvious symptoms. In the later stage, symptoms such as blood in the stool, abdominal pain, and diarrhea may occur. And when symptoms appear, it is often late, which brings great pain and expensive treatment costs to patients. Therefore, early detection, early diagnosis, and early treatment are important measures to reduce the incidence and mortality of colorectal cancer.
  • Screening can detect bowel cancer and precancerous lesions early and remove the lesions, thereby preventing the occurrence of bowel cancer.
  • the current screening methods for colorectal cancer include occult blood test and colonoscopy.
  • the occult blood test has the problems of being easily affected by food or having a low detection rate of adenomas.
  • Colonoscopy is the gold standard for the diagnosis of bowel cancer, but it is not highly compliant when used as a screening tool. Therefore, a highly accurate and compliant screening method for bowel cancer is urgently needed.
  • the present invention provides a tumor marker, capture sequence, primer pair, probe, methylation detection reagent, kit and application thereof.
  • the sensitivity of this tumor marker to bowel cancer in feces is close to that in tissues, and even higher than that in tissues.
  • Another object of the present invention is to provide a marker, a capture sequence, a primer pair, a probe, a methylation detection reagent, a kit and a method for non-invasively detecting tumors.
  • the present invention provides the following technical solutions:
  • the invention provides the application of the COL4A2 gene in preparing tumor markers.
  • the sequence of the COL4A2 gene is at least 97.8% identical to the sequence shown in Genebank Accession No. NC_000013.11.
  • the sequence of the COL4A2 gene is at least 98.9% identical to the sequence shown in Genebank Accession No. NC_000013.11.
  • the sequence of the COL4A2 gene is at least 99.9% identical to the sequence shown in Genebank Accession No. NC_000013.11.
  • the sequence of the COL4A2 gene is 100% identical to the sequence shown in Genebank Accession No. NC_000013.11.
  • the tumor is a colorectal tumor.
  • the tumor is colorectal cancer or adenoma.
  • the sample to be tested is tissue, body fluid or excrement.
  • the tissue is intestinal tissue.
  • the body fluid includes, but is not limited to, blood, serum, plasma, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid, or aqueous humor.
  • the excreta is sputum, urine, saliva or feces.
  • the invention also provides the application of the methylation detection reagent of the COL4A2 gene in preparing a tumor detection reagent or a kit.
  • the methylation detection reagent of the COL4A2 gene may be a methylation detection reagent in the prior art.
  • a methylation detection reagent in the prior art, there are various methods for detecting methylation of a target gene, such as methylation-specific PCR. (MSP), methylation-specific quantitative PCR (qMSP), PCR of methylated DNA-specific binding proteins, quantitative PCR and DNA chips, methylation-sensitive restriction enzymes, bisulfite sequencing, or Pyrosequencing and more.
  • MSP methylation-specific PCR.
  • qMSP methylation-specific quantitative PCR
  • PCR of methylated DNA-specific binding proteins methylated DNA-specific binding proteins
  • quantitative PCR and DNA chips methylation-sensitive restriction enzymes
  • bisulfite sequencing or Pyrosequencing and more.
  • Pyrosequencing methylation-sensitive restriction enzymes
  • the invention also provides a capture sequence, which has any one of the nucleotide sequences shown below:
  • the present invention also provides a primer pair, and the upstream primer has any one of the nucleotide sequences shown below:
  • the downstream primer has any of the nucleotide sequences shown below:
  • X A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 3;
  • XI A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 3 or having the core shown in SEQ ID NO: 3 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;
  • the present invention also provides a probe having any one of the nucleotide sequences shown below:
  • XIV A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 4;
  • XV a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 4 or having the core shown in SEQ ID NO: 4 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;
  • the present invention also provides a methylation detection reagent for the COL4A2 gene, including capture sequences, primers and / or probes for the COL4A2 gene.
  • capture sequences, primers, and / or probes obtained for CpG islands of the COL4A2 gene are included.
  • the primers and / or probes detect methylation of the COL4A2 gene by quantitative methylation-specific PCR (qMSP).
  • the methylation detection reagent provided by the present invention detects the methylation level of the COL4A2 gene genome, intergenic region or promoter region and the region near the promoter region.
  • the presence of methylation in tumor tissue is considered to be an apparent modification of DNA with potential clinical value.
  • methylations in the genome, intergenic regions, promoters, or nearby regions and methylation in these regions may be related to tumors.
  • the methylation detection reagent provided by the present invention includes capture sequences, primers, and / or probes obtained for a COL4A2 gene promoter region or a CpG island in a region near the promoter region.
  • the capture sequence in the methylation detection reagent provided by the present invention has any one of the nucleotide sequences shown below:
  • the upstream primer in the primer has any one of the nucleotide sequences shown below:
  • the downstream primer in the primer has any one of the nucleotide sequences shown below:
  • X A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 3;
  • XI A sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to the nucleotide sequence shown in SEQ ID NO: 3 or having the core shown in SEQ ID NO: 3 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;
  • the probe in the methylation detection reagent provided by the present invention has any one of the nucleotide sequences shown below:
  • XIV A nucleotide sequence obtained by modifying, replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO: 4;
  • XV a sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the nucleotide sequence shown in SEQ ID NO: 4 or having the core shown in SEQ ID NO: 4 Functionally similar nucleotide sequences obtained from CpG islands of nucleotide sequences;
  • the invention also provides a kit for detecting tumors, which comprises the capture sequence, primer pairs, probes or methylation detection reagents.
  • the kit provided by the present invention comprises: one or more containers divided into receiving reagents therein.
  • the kit provided by the present invention includes: a first container containing a capture sequence; a second container containing a primer pair for amplification; and a third container containing a probe.
  • the kit provided by the present invention further includes a common reagent in the kit, such as a transforming agent commonly used in qMSP, for converting unmethylated cytosine bases to uracil, The methylated cytosine bases remain unchanged.
  • the conversion agent includes, but is not limited to, bisulfite, bisulfite or hydrazine, and the like.
  • Another example is DNA polymerase, dNTPs, Mg 2+ ions and buffers commonly used in the amplification of COL4A2 gene.
  • the invention also provides the application of the aforementioned capture sequences, primer pairs, probes, methylation detection reagents, and kits in detecting tumors.
  • the invention also provides a tumor detection method, which distinguishes normal samples from tumor samples by detecting the methylation level of the COL4A2 gene.
  • the present invention detects the methylation level of the COL4A2 gene gene body, intergenic region or promoter region, and a region near the promoter region.
  • the present invention distinguishes normal samples from tumor samples by detecting methylation levels in the promoter region of the COL4A2 gene and a region near the promoter region.
  • the methylation level is detected using the capture sequence, primer pair, probe, methylation detection reagent or the kit.
  • the methylation level is determined by methylation-specific PCR, or methylation-specific quantitative PCR, or methylated DNA-specific binding protein PCR, quantitative PCR, and DNA. Chips, or methylation-sensitive restriction enzymes, or bisulfite sequencing, or pyrosequencing.
  • the methylation level is detected by methylation-specific quantitative PCR.
  • step (1) detecting the methylation level of the subject's COL4A2 gene comprises the following steps:
  • the DNA of the test sample is transformed with bisulfite, bisulfite or hydrazine;
  • step a) extracting DNA from a sample to be tested by using a magnetic bead capture method includes the following steps;
  • the magnetic beads were washed and transferred to a clean centrifuge tube, and the washing solution was added, and incubated at room temperature at 100-2000 rpm for 0.5-5min. The supernatant was placed on a magnetic stand and the supernatant was repeated 3 times;
  • the target gene DNA was eluted with a buffer.
  • the detection standard is: judging tumor specimens and normal specimens according to cutoff values, the cutoff value of Ct values in fecal specimens is 32 to 42, preferably, cutoff values of Ct value in feces specimens The value is 34.9. If the Ct value of the fecal specimen is less than or equal to the cutoff value of the Ct value, it is determined to be a tumor specimen, and the Ct value of the fecal specimen is greater than the cutoff value of the Ct value to be determined as a normal specimen.
  • the threshold value of the basalization level value is 1 to 10.
  • the threshold value of the methylation level value in the tissue specimen is 4.9, and the methylation level value of the tissue specimen is greater than or equal to the threshold value of the methylation level value.
  • the value is judged as a tumor specimen, and the methylation level value of the tissue specimen is less than the threshold value of the methylation level value as a normal specimen. This threshold can be adjusted according to the actual situation.
  • the invention also provides a tumor detection system, the system includes the following components;
  • the methylation detection component contains one or more of a fluorescent quantitative PCR instrument, a PCR instrument, and a sequencer;
  • the methylation detection component further comprises the capture sequence, a primer pair, a probe, a methylation detection reagent or a kit.
  • the data processing component is configured to: a. Receive test data of a test sample and a normal control sample; b. Store test data of the test sample and a normal control sample; c. Test data on the same type of test sample and normal control sample; d. According to the comparison result, respond to the probability or possibility of the tumor of the test subject.
  • the result output component is used to output the probability or possibility that the test subject has a tumor.
  • the judgment criterion of the data processing component is:
  • the tumor specimen and the normal specimen are judged according to the cutoff value.
  • the cutoff value of the Ct value in the stool specimen is 32 to 42, preferably, the cutoff value of the Ct value in the stool specimen is 34.9.
  • the cutoff value of the Ct value is judged as a tumor specimen, and the cutoff value of the Ct value of the fecal specimen is greater than the cutoff value of the Ct value as a normal specimen;
  • the cutoff value of the methylation level in the tissue specimen is 1 to 10, preferably
  • the cutoff value of the methylation level in the tissue specimen is 4.9, and the cutoff value of the methylation level of the tissue specimen is greater than or equal to the cutoff value of the methylation level value, which is determined as a tumor specimen, and the methylation level of the tissue specimen is methylated. If the value of the methylation level is smaller than the threshold value of the methylation level, it is judged as a normal specimen. This threshold can be adjusted according to the actual situation.
  • the tumor of the present invention is a colorectal tumor.
  • the tumor of the present invention is colorectal cancer or adenoma.
  • the sample to be tested or the sample type provided by the present invention is tissue, body fluid or excrement.
  • the tissue is intestinal tissue.
  • the body fluid includes blood, serum, plasma, extracellular fluid, interstitial fluid, lymph fluid, cerebrospinal fluid, or aqueous humor.
  • the excreta is sputum, urine, saliva or feces.
  • the present invention finds through research that by detecting the methylation level of the promoter region of the COL4A2 gene, colorectal cancer specimens can be distinguished from normal human stool specimens.
  • the present invention detects colorectal cancer by using a methylation detection reagent containing the gene, and the detection sensitivity and specificity for colorectal cancer are very high.
  • the markers and technical solutions provided by the present invention can detect colorectal cancer with high sensitivity and specificity, and detect colorectal cancer in feces.
  • the rate is higher than that of tissue specimens.
  • the methylation detection reagent of the COL4A2 gene can detect 85% of colorectal cancer when the specificity is 95.2%, and the detection rate of colorectal cancer is higher than that of tissues. Specimens can be easily used as test samples for reliable diagnosis of colorectal cancer. Obtaining stool samples is very easy, sampling is non-invasive and simple, and it will not cause any pain and inconvenience to the patient.
  • the methylation detection reagent of the COL4A2 gene can detect 80% of colorectal cancer when the specificity is 95.2%.
  • the methylation detection reagent and extraction detection method of the COL4A2 gene can easily and accurately determine colorectal cancer and normal people.
  • the methylation detection reagent of this gene is expected to be used for stool gene detection Kit and serve for clinical detection of bowel cancer.
  • the reagent / kit in one of the above technical solutions is to detect and diagnose cancer by methylation level. More and more studies have confirmed that methylation change is an early event in the process of tumorigenesis and detects abnormal methylation. It is easier to detect early lesions.
  • FIG. 1 shows the ROC curve of colorectal cancer detected by the COL4A2 gene in the stool test of Example 1;
  • FIG. 2 shows the amplification curve of the COL4A2 gene standard curve in the stool experiment of Example 1;
  • Figure 3 shows the ROC curve of colorectal cancer detected by the COL4A2 gene in the tissue experiment of Example 2;
  • Figure 4 shows the ROC curve of colorectal cancer detected by the SFRP1 gene in a 19-pair tissue experiment of Comparative Example 2;
  • Figure 5 shows the ROC curve of colorectal cancer detected by the SFRP1 gene in the 36 stool tests of Comparative Example 2.
  • the invention discloses a tumor marker, a methylation detection reagent, a kit and an application thereof. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be noted that all similar replacements and modifications will be apparent to those skilled in the art, and they are all considered to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant persons can modify or appropriately modify and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention. Apply the technology of the present invention.
  • the tumor markers, methylation reagents, kits and the raw materials, auxiliary materials and reagents used in the application can be purchased or synthesized on the market.
  • CpG islands are CpG-rich regions in a nucleic acid sequence. CpG islands start upstream of the promoter and extend downstream to the transcribed region. Methylation of CpG islands on the promoter usually suppresses gene expression.
  • the CpG island in the promoter is part of the methylation, and there is a conserved DNA methylation target in the CpG open sea of the genome. Recent studies have revealed the synergistic effects of methylation of non-promoter regions (such as genomic and UTR) on gene expression. Genomic methylation may be a potential therapeutic target in cancer.
  • CpG islands refer to some regions rich in CpG dinucleotides, which are usually located in the promoter and its vicinity.
  • the CpG islands in the present invention not only refer to the promoter and its vicinity are rich in CpG dinucleus.
  • Nucleotides also include hybrid methylated CpG sites, or isolated CpG sites.
  • the CpG-containing nucleic acid is DNA.
  • the present invention is applicable, for example, a sample containing DNA, or DNA and RNA containing mRNA, wherein the DNA or RNA may be single-stranded or double-stranded, or a DNA-RNA hybrid strand may be included in the sample.
  • a “primer” or “probe” in the present invention refers to an oligonucleotide comprising a region complementary to the sequence of at least 6 consecutive nucleotides of a target nucleic acid molecule (eg, a target gene).
  • the primer or probe comprises at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 with the target molecule.
  • a region of complementary sequence of consecutive or discontinuous block nucleotides When a primer or probe comprises a region that is complementary to at least x consecutive nucleotides of the target molecule, the primer or probe is at least 95% complementary to at least x consecutive nucleotides of the target molecule. In some embodiments, the primer or probe is at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule.
  • the “detection” in the present invention is the same as diagnosis. In addition to the early diagnosis of colorectal tumors, it also includes the diagnosis of middle and advanced stages of colorectal tumors. Selection of therapeutic targets.
  • colorectal tumor marker COL4A2 makes early diagnosis of colorectal tumors possible.
  • a methylated gene is determined to be methylated in a clinically or morphologically normal cell in a cancer cell, this indicates that the normally expressed cell is developing towards cancer.
  • colorectal cancer can be diagnosed at an early stage by methylation of the colorectal tumor-specific gene COL4A2 in normal-representing cells.
  • early diagnosis refers to the possibility of discovering cancer before metastasis, preferably before the morphological changes of tissues or cells can be observed.
  • the reagents / kits of the present invention are also promising for colorectal tumor screening, risk assessment, prognostic diagnosis, disease identification, diagnosis of the disease stage, and selection of therapeutic targets.
  • diagnosis can be made by measuring the progress of colorectal tumors at different stages or stages by measuring the degree of methylation of COL4A2 obtained from the sample.
  • degree of methylation of the COL4A2 gene of a nucleic acid isolated from a sample of each stage of colorectal cancer By comparing the degree of methylation of the COL4A2 gene of a nucleic acid isolated from a sample of each stage of colorectal cancer with the COL4A2 gene of one or more nucleic acids isolated from a sample of intestinal tissue without abnormal cell proliferation
  • the degree of basicization can detect the specific stage of colorectal tumor in the sample.
  • EZ DNA DNA Kit (Zymo Research) was used to methylate the DNA fragment in the previous step, and the final eluent was 15ul for qMSP detection.
  • qMSP reaction system 25ul (8.2ul nuclease-free water, 5 ⁇ Colorless GoTaq FlexiBuffer 5ul, MgCl2 (25mM) 5ul, dNTPs (10mM) 1ul, GoTaq Hot Start Polymerase 0.5ul, Forward Primer (100uM) 0.125ul, Reverse Primer (100uM) 0.125ul, Probe (100uM) 0.05ul, DNA 5ul). Reaction procedure: 95 ° C for 4 min, (95 ° C for 20s, 56 ° C for 30s, 72 ° C for 30s) ⁇ 45 Cycles, 37 ° C for 30s.
  • the COL4A2 gene methylation site is relatively constant, and is mainly located in the promoter region or nearby CpG islands. A set of capture sequences, primers and probes was designed for these regions and used in the COL4A2 gene methylation detection reagent.
  • the capture sequences and primer probes contained in the reagent are as follows:
  • the detection sensitivity of the COL4A2 gene is 85% (68/80), the specificity is 95.2% (79/83), and the area under the ROC curve is 0.966 (95% CI: 0.941-0.991, p ⁇ 0.0001)
  • the standard amplification efficiency was 99%, and the linearity R 2 was 0.994.
  • No amplification means no amplification curve and no Ct data, which belongs to a range greater than the cutoff.
  • the detection sensitivity of the COL4A2 gene is 80% (84/105), the specificity is 95.2% (100/105), and the area under the ROC curve is 0.857 (95% CI: 0.795-0.919, p ⁇ 0.0001). .
  • QIAamp DNA Stool Mini Kit QIAGEN is used to extract the DNA from stool samples, and then use methylation-specific PCR (MSP) or quantitative methylation-specific PCR (qMSP) to qualitatively or quantitatively detect the markers in the samples. Level. Among them, the detection of colorectal cancer by MSP requires running electrophoresis, which is more inconvenient and has the risk of product contamination.
  • the QIAamp DNA Stool Mini Kit is used to extract fecal DNA from human and bacterial total DNA. There is very little human tumor DNA. , Is not conducive to subsequent PCR detection.
  • the methylation level of SFRP1 gene was also detected in 19 pairs of tissues and 36 stool samples.
  • the target gene extraction method was the same as in Examples 1 and 2.
  • the ROC curve of colorectal cancer detected by SFRP1 gene is shown in Figure 4. As shown:
  • the detection sensitivity of SFRP1 gene was 89%, the specificity was 95%, and the area under the ROC curve was 0.972 (95% CI: 0.929-1, p ⁇ 0.001).
  • the detection sensitivity of the SFRP1 gene was 67%, the specificity was 94%, and the area under the ROC curve was 0.892 (95% CI: 0.790-0.994, p ⁇ 0.0001).
  • the SFRP1 gene has a higher detection sensitivity and specificity for colorectal cancer tissues, and its sensitivity has been greatly reduced in stool samples.

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Abstract

本发明属于生物技术领域，公开了肿瘤标志物、甲基化检测试剂、试剂盒及其应用。本发明披露：通过检测COL4A2基因启动子区的甲基化水平，可以从正常人的粪便标本中区分出结直肠癌标本。本发明利用该基因的甲基化检测试剂来检测结直肠癌。

Description

肿瘤标志物、甲基化检测试剂、试剂盒及其应用 技术领域
本发明涉及生物技术领域，特别涉及肿瘤标志物、甲基化检测试剂、试剂盒及其应用。
背景技术
结直肠癌，又称为大肠癌，是一种常见的消化道恶性肿瘤。其发病率在我国逐年升高，在我国部分沿海地区比如上海和广州，大肠癌发病率已跃居第二位，仅次于肺癌。目前认为肠癌的形成是遗传缺陷和表观遗传缺陷累积的结果。结直肠癌早期发病隐匿，常无明显症状，晚期可出现便血、腹痛、腹泻等症状。而当出现症状就诊时常常是晚期，这给病人带来极大的痛苦和昂贵的治疗费用。因此早发现、早诊断、早治疗是降低结直肠癌发病率和死亡率的一项重要措施。
筛查可以早期发现肠癌和癌前病变，并去除病灶，从而阻止肠癌的发生。目前大肠癌的筛查方法主要有隐血试验和肠镜检查。隐血试验存在易受食物影响或对腺瘤检出率不高的问题。肠镜虽是肠癌诊断金标准，但作为筛查手段使用时人群依从性不高。因此急需一种准确性高、依从性高的肠癌筛查方法。
粪便基因检测作为一种新的肠癌筛查方法，现在越来越受到重视。该方法
Figure PCTCN2019085584-appb-000001
于2016年纳入美国的肠癌筛查指南。该方法具有方便、无创、对肠癌和癌前病变腺瘤的检出率高等特点。要做成对肠癌检测高性能的粪便基因检测试剂盒，主要需要克服两大障碍：粪便DNA的提取和标志物选择。一方面，粪便中成分复杂，对下游反应的抑制物多，还有许多细菌DNA，要从这样的混合物中提取出人的目标基因，需要一套高敏的基因提取和纯化方法；另一方面，目前和肠癌相关的标志物很多，尤其是DNA甲基化标志物，因为研究表明，DNA甲基化是肿瘤形成的早期事件。但很多甲基化标志物在细胞、组织层面表现很好，当用于粪便、血液等筛查媒介时，其对肠癌的敏感性和特异性就降下来了，比如vimentin基因，其在组织中的敏感性有83％，在粪便标本中就降到了46％，类似的还有SFRP1、 SFRP2等基因，这样的标志物无法满足真正用于肠癌临床检测的需求。因此，挑选在粪便中对肠癌有极高检测敏感性和特异性的标志物是肠癌粪便基因检测的关键，并且这样的标志物有望真正用于肠癌的临床检测。
发明内容
有鉴于此，本发明提供一种肿瘤标志物、捕获序列、引物对、探针、甲基化检测试剂、试剂盒及其应用。该肿瘤标志物在粪便中对肠癌的敏感性与组织中敏感性接近，甚至高于组织中的敏感性。
本发明的另一个目的在于提供一种无创检测肿瘤的标志物、捕获序列、引物对、探针、甲基化检测试剂、试剂盒及方法。为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了COL4A2基因在制备肿瘤标志物中的应用。
在本发明的一些具体实施方案中，所述COL4A2基因的序列与Genebank Accession No.NC_000013.11所示的序列具有至少97.8％的同一性。
在本发明的一些具体实施方案中，所述COL4A2基因的序列与Genebank Accession No.NC_000013.11所示的序列具有至少98.9％的同一性。
在本发明的一些具体实施方案中，所述的COL4A2基因的序列与Genebank Accession No.NC_000013.11所示的序列具有至少99.9％的同一性。
在本发明的一些具体实施方案中，所述的COL4A2基因的序列与Genebank Accession No.NC_000013.11所示的序列具有100％的同一性。
在本发明的一些具体实施方案中，所述肿瘤为结直肠肿瘤。
在本发明的一些具体实施方案中，所述肿瘤为结直肠癌或腺瘤。
在本发明的一些具体实施方案中，待测样本为组织、体液或排泄物。
在本发明的一些具体实施方案中，所述组织为肠组织。
在本发明的一些具体实施方案中，所述体液包括但不限于血液、血清、血浆、细胞外液、组织液、淋巴液、脑脊液或房水。在本发明的一些具体实施方案中，所述排泄物为痰液、尿液、唾液或粪便。
本发明还提供了COL4A2基因的甲基化检测试剂在制备肿瘤检测试剂或者试剂盒中的应用。
所述的COL4A2基因的甲基化检测试剂可以是现有技术中的甲基化检测试剂，现有技术中，已经有多种方法可以检测目的基因的甲基化，如甲基化特异性PCR(MSP)、甲基化特异性定量PCR(qMSP)、甲基化DNA特异性结合蛋白的PCR，定量PCR以及DNA芯片、甲基化敏感的限制性内切酶、重亚硫酸盐测序法或者焦磷酸测序等等。每种检测方法均有其相对应的试剂，这些试剂均可以用本发明检测COL4A2基因的甲基化。
本发明还提供了一种捕获序列，所述捕获序列具有如下所示的核苷酸序列中的任意一项：
Ⅰ、具有SEQ ID NO:1所示的核苷酸序列；
Ⅱ、具有SEQ ID NO:1所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列或具有SEQ ID NO:1所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
III、与SEQ ID NO:1所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:1所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
IV、如Ⅰ、Ⅱ或III所示序列的互补序列。
本发明还提供了一种引物对，上游引物具有如下所示的核苷酸序列中的任意一项：
Ⅴ、具有SEQ ID NO:2所示的核苷酸序列；
Ⅵ、具有SEQ ID NO:2所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
Ⅶ、与SEQ ID NO:2所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:2所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
Ⅷ、如Ⅴ、Ⅵ或Ⅶ所示序列的互补序列；
下游引物具有如下所示的核苷酸序列中的任意一项：
Ⅸ、具有SEQ ID NO:3所示的核苷酸序列；
Ⅹ、具有SEQ ID NO:3所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
Ⅺ、与SEQ ID NO:3所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:3所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
Ⅻ、如Ⅸ、Ⅹ或Ⅺ所示序列的互补序列。
本发明还提供了一种探针，所述探针具有如下所示的核苷酸序列中的任意一项：
XIII、具有SEQ ID NO:4所示的核苷酸序列；
XIV、具有SEQ ID NO:4所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
XV、与SEQ ID NO:4所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:4所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
XVI、如XIII、XIV或XV所示序列的互补序列。
本发明还提供了COL4A2基因的甲基化检测试剂，包括针对COL4A2基因的捕获序列、引物和/或探针。
在本发明的一些具体的实施方案中，包括针对COL4A2基因的CpG岛获得的捕获序列、引物和/或探针。
在本发明的一些具体的实施方案中，引物和/或探针通过甲基化特异性定量PCR(quantitative Methylation-Specific PCR,qMSP)检测COL4A2基因的甲基化。
在本发明的一些具体的实施方案中，本发明提供的甲基化检测试剂通过检测COL4A2基因的基因体、基因间区或启动子区及启动子区附近区域的甲基化水平。
肿瘤组织中存在的甲基化被认为是具有潜在临床价值的DNA表观修饰。基因体以、基因间区、启动子或其附近区域均存在甲基化，且这些区域的甲基化均可能与肿瘤相关。目前，在多种肿瘤中已经证实，抑癌基因启动子或其附近区域的CpG岛异常甲基化导致了转录失活。在本发明的一些具体实施方案中，本发明提供的甲基化检测试剂包括针对COL4A2基因的启动子区或所述启动子区附近区域的CpG岛获得的捕获序列、引物和/或探针。
在本发明的一些具体实施方案中，本发明提供的甲基化检测试剂中所述捕获序列具有如下所示的核苷酸序列中的任意一项：
Ⅰ、具有SEQ ID NO:1所示的核苷酸序列；
Ⅱ、具有SEQ ID NO:1所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列或具有SEQ ID NO:1所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
III、与SEQ ID NO:1所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:1所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
IV、如Ⅰ、Ⅱ或III所示序列的互补序列。
在本发明的一些具体实施方案中，本发明提供的甲基化检测试剂中，所述引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
Ⅴ、具有SEQ ID NO:2所示的核苷酸序列；
Ⅵ、具有SEQ ID NO:2所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
Ⅶ、与SEQ ID NO:2所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:2所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
Ⅷ、如Ⅴ、Ⅵ或Ⅶ所示序列的互补序列。
在本发明的一些具体实施方案中，本发明提供的甲基化检测试剂中，所述引物中的下游引物具有如下所示的核苷酸序列中的任意一项：
Ⅸ、具有SEQ ID NO:3所示的核苷酸序列；
Ⅹ、具有SEQ ID NO:3所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
Ⅺ、与SEQ ID NO:3所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:3所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
Ⅻ、如Ⅸ、Ⅹ或Ⅺ所示序列的互补序列。
在本发明的一些具体实施方案中，本发明提供的甲基化检测试剂中所述探针具有如下所示的核苷酸序列中的任意一项：
XIII、具有SEQ ID NO:4所示的核苷酸序列；
XIV、具有SEQ ID NO:4所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
XV、与SEQ ID NO:4所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:4所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
XVI、如XIII、XIV或XV所示序列的互补序列。
本发明还提供了一种检测肿瘤的试剂盒，包括所述的捕获序列、引物对、探针或甲基化检测试剂。
在本发明的一些具体实施方案中，本发明提供的试剂盒包括：划分成其内接收试剂的一个或多个容器。
在本发明的一些具体实施方案中，本发明提供的试剂盒包括：第一容器，其包含捕获序列；第二容器，其包含用于扩增的引物对；第三容器，其包含探针。
在本发明的一些具体实施方案中，本发明提供的试剂盒还包括试剂盒中的常用试剂，如qMSP中常用转化剂，用于将非甲基化的胞嘧啶碱基转化为尿嘧啶，而甲基化的胞嘧啶碱基保持不变。所述的转化剂包括但不限于亚硫酸氢盐、重亚硫酸氢盐或肼盐等等。又如扩增COL4A2基因中常用的DNA聚合酶、dNTPs、Mg 2+离子和缓冲液等等。
本发明还提供了上述的捕获序列、引物对、探针、甲基化检测试剂、试剂盒在检测肿瘤中的应用。
本发明还提供了一种肿瘤的检测方法，通过检测COL4A2基因的甲基化水平，区分正常样本和肿瘤样本。
在本发明的一些具体实施方案中，其包含以下步骤：
(1)检测受试者COL4A2基因的甲基化水平；
(2)将受试者COL4A2基因的甲基化水平与正常对照样本的甲基化水平相比较；
(3)根据所述受试者COL4A2基因的水平与所述正常对照样本的甲基化水平相比的升高，指示所述受试者患有或者有风险患上肿瘤，以区分正常样本和肿瘤样本；.
在本发明的一些具体实施方案中，本发明通过检测COL4A2基因的基因体、基因间区或启动子区及启动子区附近区域的甲基化水平。
在本发明的一些具体实施方案中，本发明通过检测COL4A2基因启动子区及启动子区附近区域的甲基化水平，区分正常样本和肿瘤样本。
在本发明的一些具体实施方案中，甲基化水平采用所述的捕获序列、引物对、探针、甲基化检测试剂或所述的试剂盒检测。
在本发明的一些具体实施方案中，所述甲基化水平通过甲基化特异性PCR，或者甲基化特异性定量PCR，或者甲基化DNA特异性结合蛋白的PCR、定量PCR、以及DNA芯片，或者甲基化敏感的限制性内切酶，或者重亚硫酸盐测序法，或者焦磷酸测序检测。
在本发明的一些具体实施方案中，所述的甲基化水平通过甲基化特异性定量PCR检测。
在本发明的一些具体实施方案中，步骤(1)中，检测受试者COL4A2基因的甲基化水平包含以下步骤：
a)采用磁珠捕获法提取待测样品的DNA；
b)待测样品的DNA采用亚硫酸氢盐、重亚硫酸氢盐或肼盐进行转化；
c)甲基化特异性定量PCR(qMSP)检测。
在本发明的一些具体实施方案中，步骤a)中，采用磁珠捕获法提取待测样品的DNA包括以下步骤；
取待测样本在保护液中混合研磨、离心、取上清；
上清再离心，取上清，加入裂解液和带有特定互补寡核苷酸捕获序列的磁珠至上清液中孵育；
弃部分上清后洗下磁珠转移至干净离心管，加入洗液，室温100-2000rpm孵育0.5-5min，置于磁力架上吸去上清，重复3次；
用缓冲液将目标基因DNA洗脱。
在本发明的一些具体实施方案中，检测标准为：根据界值判断肿瘤标本和正常标本，粪便标本中的Ct值的界值为32～42，优选地，粪便标本中的Ct值的界值为34.9，所述粪便标本的Ct值小于等于所述Ct值的界值则判断为肿瘤标本，所述粪便标本的Ct值大于所述Ct值的界值则判断为正常标本；组织标本中甲基化水平值的界值为1～10，优选地，组织标本中甲基化水平值的界 值为4.9，所述组织标本的甲基化水平值大于等于所述甲基化水平值的界值则判断为肿瘤标本，所述组织标本的甲基化水平值小于所述甲基化水平值的界值则判断为正常标本。该界值可根据实际情况进行调整。
本发明还提供了一种肿瘤的检测系统，所述的系统包含有以下构件；
(1)COL4A2基因的甲基化检测构件；
(2)数据处理构件；
(3)结果输出构件；
在本发明的一些具体实施方案中，所述的甲基化检测构件含有荧光定量PCR仪、PCR仪、测序仪中的一种或多种；
在本发明的一些具体实施方案中，所述的甲基化检测构件还含有所述的捕获序列、引物对、探针、甲基化检测试剂或试剂盒。
在本发明的一些具体实施方案中，所述的数据处理构件被配置于a.接收待测样本以及正常对照样本的测试数据；b.储存待测样本以及正常对照样本的测试数据；c.比对同种类型的待测样本以及正常对照样本的测试数据；d.根据比对结果，响应于测试者罹患肿瘤的概率或者可能性。
在本发明的一些具体实施方案中，所述的结果输出构件用于输出测试者罹患肿瘤的概率或者可能性。
在本发明的一些具体实施方案中，数据处理构件的判断标准为：
根据界值判断肿瘤标本和正常标本，粪便标本中的Ct值的界值为32～42，优选地，粪便标本中的Ct值的界值为34.9，所述粪便标本的Ct值小于等于所述Ct值的界值则判断为肿瘤标本，所述粪便标本的Ct值大于所述Ct值的界值则判断为正常标本；组织标本中甲基化水平值的界值为1～10，优选地，组织标本中甲基化水平值的界值为4.9，所述组织标本的甲基化水平值大于等于所述甲基化水平值的界值则判断为肿瘤标本，所述组织标本的甲基化水平值小于所述甲基化水平值的界值则判断为正常标本。该界值可根据实际情况进行调整。
在本发明的一些具体实施方案中，本发明所述肿瘤为结直肠肿瘤。
在本发明的一些具体实施方案中，本发明所述肿瘤为结直肠癌或腺瘤。
在本发明的一些具体实施方案中，本发明提供的待测样本或者样本类型为组织、体液或排泄物。
在本发明的一些具体实施方案中，所述组织为肠组织。
在本发明的一些具体实施方案中，所述体液包括血液、血清、血浆、细胞外液、组织液、淋巴液、脑脊液或房水。
在本发明的一些具体实施方案中，所述排泄物为痰液、尿液、唾液或粪便。
本发明通过研究发现：通过检测COL4A2基因启动子区的甲基化水平，可以很好的从正常人的粪便标本中区分出结直肠癌标本。本发明就是利用含有该基因的甲基化检测试剂来检测结直肠癌的，并且对肠癌的检测敏感性和特异性非常高。
与现有的检测肠癌的标志物相比，本发明提供的标志物和技术方案能够以很高的敏感性和特异度来检测出结直肠癌，并且在粪便中对结直肠癌的检出率高于组织标本，具体有以下几点：
1、上述的一个技术方案中，COL4A2基因的甲基化检测试剂在粪便标本中能够在特异性为95.2％时，检测出85％的结直肠癌，对结直肠癌的检出率高于组织标本，可以简便地粪便做为检测样本，对结直肠癌进行可靠的诊断。粪便样品获得非常容易，取样无创简单，而且不会对病人造成任何的痛苦和不便。
2、上述的一个技术方案中，COL4A2基因的甲基化检测试剂在组织标本中能够在特异性为95.2％时，检测出80％的结直肠癌。
3、上述的一个技术方案中，COL4A2基因的甲基化检测试剂和提取检测方法能非常方便、准确地判断出结直肠癌和正常人，该基因的甲基化检测试剂有望用于粪便基因检测试剂盒，并服务于肠癌的临床检测。
4、上述的一个技术方案中的试剂/试剂盒是通过甲基化水平来检测和诊断癌症，越来越多的研究证实甲基化改变是肿瘤发生过程中的早期事件，检测甲基化异常更易发现早期病变。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示实施例1粪便实验中，COL4A2基因检测结直肠癌的ROC曲线；
图2示实施例1粪便实验中，COL4A2基因标准曲线扩增图谱；
图3示实施例2组织实验中，COL4A2基因检测结直肠癌的ROC曲线；
图4示对比例2的19对组织实验中，SFRP1基因检测结直肠癌的ROC曲线；
图5示对比例2的36例粪便实验中，SFRP1基因检测结直肠癌的ROC曲线。
具体实施方式
本发明公开了一种肿瘤标志物、甲基化检测试剂、试剂盒及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的肿瘤标志物、甲基化试剂、试剂盒及其应用中所用原料、辅料及试剂均可由市场购得或者合成。
只要有可检测差异性甲基化的CpG位点，COL4A2基因的任何核酸片段可以用于本发明。CpG岛是核酸序列中CpG富集区域。CpG岛开始于启动子的上游，向下游延伸至转录区域。在启动子上的CpG岛的甲基化通常抑制基因的表达。启动子内的CpG岛为甲基化的一部分，基因体中的CpG open sea存在保守的DNA甲基化靶标。最近的研究揭示了非启动子区域(如基因体和UTR)甲基化对基因表达的协同效应，基因体甲基化可能是癌症中潜在的治疗靶点。
通常来说，CpG岛是指富含CpG二核苷酸的一些区域，通常位于启动子及其附近的区域，本发明中的CpG岛，不仅指启动子及其附近的区域富含CpG二核苷酸，也包括杂合甲基化的CpG位点，或者是孤立的CpG位点。
通常，含CpG的核酸是DNA。然而，本发明可适用，例如，包含DNA、或者DNA和含有mRNA的RNA的样品，其中DNA或者RNA可以是单链的或者双链的，或者DNA-RNA杂交链也可能包括在样品中。
本发明中的“引物”或“探针”是指一种寡核苷酸，其包含与靶核酸分子(例如靶基因)的至少6个连续核苷酸的序列互补的区域。在一些实施方案 中，引物或探针包含与靶分子的至少9、至少10、至少11、至少12、至少13、至少14、至少15、至少16、至少17、至少18、至少19或至少20个连续或不连续的分块核苷酸的序列互补的区域。当引物或探针包含与靶分子的至少x个连续核苷酸互补的区域时，所述引物或探针与靶分子的至少x个连续核苷酸至少95％互补。在一些实施方案中，引物或探针与靶分子至少96％、至少97％、至少98％、至少99％或100％互补。
本发明中的“检测”同诊断，除了结直肠肿瘤的早期诊断，还包括结直肠肿瘤中期和晚期的诊断，且也包括结直肠肿瘤筛选、风险评估、预后、疾病识别、病症阶段的诊断和治疗性靶标的选择。
结直肠肿瘤标志物COL4A2的应用使得结直肠肿瘤的早期诊断成为可能。当确定在癌症细胞中甲基化的基因在临床上或形态学上正常表象的细胞中甲基化时，这就表明该正常表象的细胞向癌症发展。这样，结直肠癌可在早期通过在正常表象的细胞中的结直肠肿瘤特异性基因COL4A2的甲基化而诊断。
其中，早期诊断指的是在转移之前发现癌症的可能性，优选在可观察到组织或者细胞的形态学变化之前。
除了结直肠肿瘤的早期诊断，本发明的试剂/试剂盒还有希望用于结直肠肿瘤筛选、风险评估、预后诊断、疾病识别、病症阶段的诊断和治疗性靶标的选择。
作为病症阶段可选的实施方式，可通过在结直肠肿瘤在不同阶段或时期的进展可通过从样品中获取的COL4A2的甲基化程度的测量进行诊断。通过比较从结直肠癌的每个阶段的样品中分离出的核酸的COL4A2基因甲基化程度与从没有细胞增殖性异常的肠组织中的样品中分离出的一个或多个核酸的COL4A2基因甲基化程度，可检测样品中结直肠肿瘤的具体阶段。
下面结合实施例，进一步阐述本发明：
实施例1
选取163例粪便标本(80例结直肠癌，83例正常，均经肠镜或病理确诊)，进行研磨离心，加入100ul捕获磁珠(含有COL4A2基因的捕获序列)，并按 下属技术方案操作，最后得到Bisulfite转化后的DNA 15ul。然后进行qMSP检测COL4A2的甲基化水平。
技术方案如下：
1)收集有肠镜结果的正常人和结直肠肿瘤病人的粪便标本，按1g粪便：4mL保护液混合研磨后5000rpm离心10min，取上清弃沉淀；
2)取出10mL上清再次离心，取上清3.2mL加入2mL裂解液和100ul捕获磁珠M1，92℃孵育10min，然后室温放置1h；
3)置于磁力架上弃部分上清后洗下磁珠转移至2mL离心管，加入800ul洗液W1，室温1300rpm孵育1min，置于磁力架上吸去上清，重复3次；
4)加入55ul洗脱液，92℃1300rpm孵育10min，置于磁力架上，3min内转移50ul洗脱液至新的EP管中；
5)用EZ DNA Methylation Kit(Zymo Research)对上一步骤中的DNA片段进行甲基化处理，最后的洗脱液15ul用于qMSP检测。
qMSP反应体系：25ul(无核酸酶水8.2ul，5×Colorless GoTaq Flexi Buffer 5ul，MgCl2(25mM)5ul，dNTPs(10mM)1ul，GoTaq Hot Start polymerase 0.5ul，Forward primer(100uM)0.125ul，Reverse primer(100uM)0.125ul，Probe(100uM)0.05ul，DNA 5ul)。反应程序：95℃4min，(95℃20s，56℃30s，72℃30s)×45Cycles，37℃30s。
最后根据标准曲线计算基因在标本中的拷贝数。
COL4A2基因发生甲基化的位点相对恒定，主要位于启动子区或附近的CpG岛上。针对这些区域设计了其中一组捕获序列、引物和探针，并用于COL4A2基因甲基化检测试剂中。
试剂中含有的捕获序列、引物探针如下：
COL4A2的捕获序列(SEQ ID NO:1)：
5’-GCTGCTGCCCGAACGCATTGGCCCTTCCAGAAGCA-3’
COL4A2的qMSP引物探针：
Forward Primer(SEQ ID NO:2)：5’-AGAGAGTTTAGTAAGGTCGGTC-3’
Reverse Primer(SEQ ID NO:3)：5’-GACTTCAAAAACTACTACCCG-3’
Probe(SEQ ID NO:4)：5’-TGTCGGTGTGTCGTCGGC-3’
粪便实验中，COL4A2基因检测结直肠癌的ROC曲线如图1所示：
对于结直肠癌，COL4A2基因的检测敏感性是85％(68/80)，特异性为95.2％(79/83)，ROC曲线下面积是0.966(95％CI：0.941-0.991，p<0.0001)
粪便实验中，COL4A2基因标准曲线扩增图谱如图2所示：
标曲扩增效率为99％，线性度R 2＝0.994。
表1
Figure PCTCN2019085584-appb-000002
Figure PCTCN2019085584-appb-000003
Figure PCTCN2019085584-appb-000004
Figure PCTCN2019085584-appb-000005
表1注：“无扩增”表示无扩增曲线，无Ct数据，属于大于界值的范围。
实施例2
选取105对结直肠癌与癌旁正常组织标本(均经肠镜或病理确诊)，根据Protocol，分别用QIAamp DNA Kit(QIAGEN)提取组织DNA，然后用EZ DNA Methylation Kit(Zymo Research)转化DNA。
然后进行qMSP检测COL4A2的甲基化水平。
qMSP反应体系和反应步骤同实施例1的粪便实验。最后根据标准曲线计算基因在标本中的甲基化值：(Target/ACTB)*100。所用的qMSP引物探针同
实施例1。
组织实验中，COL4A2基因检测结直肠癌的ROC曲线如图3所示：
对于结直肠癌，COL4A2基因的检测敏感性是80％(84/105)，特异性为95.2％(100/105)，ROC曲线下面积是0.857(95％CI：0.795-0.919，p<0.0001)。
表2
Figure PCTCN2019085584-appb-000006
Figure PCTCN2019085584-appb-000007
Figure PCTCN2019085584-appb-000008
Figure PCTCN2019085584-appb-000009
Figure PCTCN2019085584-appb-000010
对比例1
目前有研究用QIAamp DNA Stool Mini Kit(QIAGEN)对粪便标本的DNA进行提取，然后用甲基化特异性PCR(MSP)或定量甲基化特异性PCR(qMSP)来定性或定量检测标本中标志物的水平。其中通过MSP来检测结直肠癌因需要跑电泳，操作更不方便，且有产物污染风险；通过QIAamp DNA Stool Mini Kit来提取粪便中的DNA为人和细菌的总DNA，真正人的肿瘤DNA极少，不利于后续PCR检测。
对比例2
有研究表明SFRP1基因甲基化与肠癌有关，在粪便中检测该基因的甲基化程度，可以检出结直肠癌。在53例粪便标本(29例肠癌、7例腺瘤、17例正常)实验中，在特异性为86％时，可检出89％的结直肠肿瘤。(Zhang W,Bauer M,Croner RS,Pelz JO,Lodygin D,Hermeking H,Sturzl M,Hohenberger W,Matzel KE.DNA stool test for colorectal cancer:Hypermethylation of the secreted frizzled-related protein-1 gene.DISEASES OF THE COLON&RECTUM 2007；50(10):1618-26；discussion 1626-7.)
在19对组织和36例粪便标本中同样检测了SFRP1基因的甲基化水平，靶基因提取方法同实施例1和2。19对组织实验中，SFRP1基因检测结直肠癌的ROC曲线如图4所示：
对于结直肠癌组织，SFRP1基因的检测敏感性是89％，特异性为95％，ROC曲线下面积是0.972(95％CI：0.929-1，p<0.001)。
36例粪便实验中，SFRP1基因检测结直肠癌的ROC曲线如图5所示：
对于结直肠癌，SFRP1基因的检测敏感性是67％，特异性为94％，ROC曲线下面积是0.892(95％CI：0.790-0.994，p<0.0001)。
由此可见，SFRP1基因对结直肠癌组织具有较高的检测敏感性和特异性，而在粪便标本中其敏感性就大幅降低了。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Figure PCTCN2019085584-appb-000011
Figure PCTCN2019085584-appb-000012

Claims (14)

  1. COL4A2基因在制备肿瘤标志物中的应用；
    优选地，所述COL4A2基因的序列与Genebank Accession No.NC_000013.11所示的序列具有至少97.8％，至少98.9％，至少99.9％或100％的同一性；
    优选地，所述肿瘤为结直肠肿瘤；
    优选地，所述肿瘤为结直肠癌或腺瘤；
    优选地，所述标志物所针对的待测样本为组织、体液或排泄物；
    优选地，所述组织为肠组织；
    优选地，所述体液为血液、血清、血浆、细胞外液、组织液、淋巴液、脑脊液或房水；
    优选地，所述排泄物为痰液、唾液、尿液或粪便。
  2. COL4A2基因的甲基化检测试剂在制备肿瘤检测试剂或者试剂盒中的应用；
    优选地，所述COL4A2基因的序列与Genebank Accession No.NC_000013.11所示的序列具有至少97.8％，至少98.9％，至少99.9％或100％的同一性；
    优选地，所述肿瘤为结直肠肿瘤；
    优选地，所述肿瘤为结直肠癌或腺瘤；
    优选地，检测试剂所针对的待测样本为组织、体液或排泄物；
    优选地，所述组织为肠组织；
    优选地，所述体液为血液、血清、血浆、细胞外液、组织液、淋巴液、脑脊液或房水；
    优选地，所述排泄物为痰液、尿液、唾液或粪便。
  3. 一种捕获序列，其特征在于，所述捕获序列具有如下所示的核苷酸序列中的任意一项：
    Ⅰ、具有SEQ ID NO:1所示的核苷酸序列；
    Ⅱ、具有SEQ ID NO:1所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列或具有SEQ ID NO:1所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    III、与SEQ ID NO:1所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:1所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    IV、如Ⅰ、Ⅱ或III所示序列的互补序列。
  4. 一种引物对，其特征在于，上游引物具有如下所示的核苷酸序列中的任意一项：
    Ⅴ、具有SEQ ID NO:2所示的核苷酸序列；
    Ⅵ、具有SEQ ID NO:2所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
    Ⅶ、与SEQ ID NO:2所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:2所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    Ⅷ、如Ⅴ、Ⅵ或Ⅶ所示序列的互补序列；
    下游引物具有如下所示的核苷酸序列中的任意一项：
    Ⅸ、具有SEQ ID NO:3所示的核苷酸序列；
    Ⅹ、具有SEQ ID NO:3所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
    Ⅺ、与SEQ ID NO:3所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:3所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    Ⅻ、如Ⅸ、Ⅹ或Ⅺ所示序列的互补序列。
  5. 一种探针，其特征在于，所述探针具有如下所示的核苷酸序列中的任意一项：
    XIII、具有SEQ ID NO:4所示的核苷酸序列；
    XIV、具有SEQ ID NO:4所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
    XV、与SEQ ID NO:4所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:4所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    XVI、如XIII、XIV或XV所示序列的互补序列。
  6. COL4A2基因的甲基化检测试剂，其特征在于，包括COL4A2基因的甲基化检测的捕获序列、引物和/或探针；
    优选地，包括针对COL4A2基因的CpG岛获得的捕获序列、引物和/或探针；
    优选地，包括针对COL4A2基因的基因体、基因间区、启动子区或所述启动子区附近区域的CpG岛获得的捕获序列、引物和/或探针；
    优选地，所述捕获序列具有如下所示的核苷酸序列中的任意一项：
    Ⅰ、具有SEQ ID NO:1所示的核苷酸序列；
    Ⅱ、具有SEQ ID NO:1所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列或具有SEQ ID NO:1所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    III、与SEQ ID NO:1所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:1所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    IV、如Ⅰ、Ⅱ或III所示序列的互补序列；
    所述引物中的上游引物具有如下所示的核苷酸序列中的任意一项：
    Ⅴ、具有SEQ ID NO:2所示的核苷酸序列；
    Ⅵ、具有SEQ ID NO:2所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
    Ⅶ、与SEQ ID NO:2所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:2所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    Ⅷ、如Ⅴ、Ⅵ或Ⅶ所示序列的互补序列；
    所述引物中的下游引物具有如下所示的核苷酸序列中的任意一项：
    Ⅸ、具有SEQ ID NO:3所示的核苷酸序列；
    Ⅹ、具有SEQ ID NO:3所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
    Ⅺ、与SEQ ID NO:3所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:3所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    Ⅻ、如Ⅸ、Ⅹ或Ⅺ所示序列的互补序列；
    优选地，所述探针具有如下所示的核苷酸序列中的任意一项：
    XIII、具有SEQ ID NO:4所示的核苷酸序列；
    XIV、具有SEQ ID NO:4所示的核苷酸序列经修饰、取代、缺失或添加一个或多个碱基获得的核苷酸序列；
    XV、与SEQ ID NO:4所示的核苷酸序列具有至少80％、至少85％、至少90％、至少95％或至少99％同一性的序列或具有SEQ ID NO:4所示的核苷酸序列的CpG岛获得的功能相近的核苷酸序列；
    XVI、如XIII、XIV或XV所示序列的互补序列。
  7. 一种试剂盒，其特征在于，包括如权利要求3所述的捕获序列，或者权利要求4所述的引物对，或者权利要求5所述的探针，或者如权利要求6所述的甲基化检测试剂；
    优选地，所述的试剂盒包括：第一容器，其包含捕获序列；第二容器，其包含用于扩增的引物对；第三容器，其包含探针。
  8. 如权利要求3所述的捕获序列，或者权利要求4所述的引物对，或者权利要求5所述的探针，或者权利要求6所述的甲基化检测试剂，在制备检测肿瘤的试剂盒中的应用；
    优选地，所述肿瘤为结直肠肿瘤；
    优选地，所述肿瘤为结直肠癌或腺瘤；
    优选地，检测所针对的待测样本为组织、体液或排泄物；
    优选地，所述组织为肠组织；
    优选地，所述体液为血液、血清、血浆、细胞外液、组织液、淋巴液、脑脊液或房水；
    优选地，所述排泄物为痰液、唾液、尿液或粪便。
  9. 权利要求3所述的捕获序列，或者权利要求4所述的引物对，或者权利要求5所述的探针，或者权利要求6所述的甲基化检测试剂，或者权利要求7所述的试剂盒在检测肿瘤中的应用；
    优选地，所述肿瘤为结直肠肿瘤；
    优选地，所述肿瘤为结直肠癌或腺瘤；
    优选地，检测所针对的样本为组织、体液或排泄物；
    优选地，所述组织为肠组织；
    优选地，所述体液为血液、血清、血浆、细胞外液、组织液、淋巴液、脑脊液或房水；
    优选地，所述排泄物为痰液、尿液、唾液或粪便。
  10. 一种肿瘤的检测方法，其特征在于，所述的方法包括，
    (1)检测受试者COL4A2基因的甲基化水平；
    (2)将受试者COL4A2基因的甲基化水平与正常对照样本的甲基化水平相比较；
    (3)根据所述受试者COL4A2基因的水平与所述正常对照样本的甲基化水平相比的升高，指示所述受试者患有或者有风险患上肿瘤，以区分正常样本和肿瘤样本；
    优选地，通过检测COL4A2基因的基因体、基因间区或启动子区及启动子区附近区域的甲基化水平；
    优选地，通过检测COL4A2基因启动子区及启动子区附近区域的甲基化水平，区分正常样本和肿瘤样本；
    优选地，所述甲基化水平通过甲基化特异性PCR，或者甲基化特异性定量PCR，或者甲基化DNA特异性结合蛋白的PCR、定量PCR、以及DNA芯片，或者甲基化敏感的限制性内切酶，或者重亚硫酸盐测序法，或者焦磷酸测序检测；
    优选地，所述的甲基化水平通过甲基化特异性定量PCR检测；
    优选地，甲基化水平采用如权利要求3所述的捕获序列，或者权利要求4所述的引物对，或者权利要求5所述的探针，或者如权利要求6所述的甲基化检测试剂，或者如权利要求7所述的试剂盒检测；
    优选地，步骤(1)中，检测受试者COL4A2基因的甲基化水平包含以下步骤：
    a)采用磁珠捕获法提取待测样品的DNA；
    b)待测样品的DNA采用亚硫酸氢盐、重亚硫酸氢盐或肼盐进行转化；
    c)甲基化特异性定量PCR检测；
    优选地，步骤a)中，采用磁珠捕获法提取待测样品的DNA包括以下步骤；
    取待测样本在保护液中混合研磨、离心、取上清；
    上清再离心，取上清，加入裂解液和带有特定互补寡核苷酸捕获序列的磁珠至上清液中孵育；
    弃部分上清后洗下磁珠转移至干净离心管，加入洗液，室温100-2000rpm孵育0.5-5min，置于磁力架上吸去上清，重复3次；
    用缓冲液将目标基因DNA洗脱。
  11. 如权利要求10所述的方法，其特征在于，检测标准为：根据界值判断肿瘤标本和正常标本，粪便标本中的Ct值的界值为32～42，所述粪便标本的Ct值小于等于所述Ct值的界值则判断为肿瘤标本，所述粪便标本的Ct值大于所述Ct值的界值则判断为正常标本；
    组织标本中甲基化水平值的界值为1～10，所述组织标本的甲基化水平值大于等于所述甲基化水平值的界值则判断为肿瘤标本，所述组织标本的甲基化水平值小于所述甲基化水平值的界值则判断为正常标本。
  12. 一种肿瘤的检测系统，其特征在于，所述的系统包含有以下构件；
    (1)COL4A2基因的甲基化检测构件；
    (2)数据处理构件；
    (3)结果输出构件；
    优选地，所述的甲基化检测构件含有荧光定量PCR仪、PCR仪、测序仪中的一种或多种；
    优选地，所述的甲基化检测构件还含有如权利要求3所述的捕获序列，或者权利要求4所述的引物对，或者权利要求5所述的探针，或者如权利要求6所述的甲基化检测试剂，或者如权利要求7所述的试剂盒；
    优选地，所述的数据处理构件被配置于a.接收待测样本以及正常对照样本的测试数据；b.储存待测样本以及正常对照样本的测试数据；c.比对同种类型的待测样本以及正常对照样本的测试数据；d.根据比对结果，响应于测试者罹患肿瘤的概率或者可能性；
    优选地，所述的结果输出构件用于输出测试者罹患肿瘤的概率或者可能性；
    优选地，数据处理构件的判断标准为：根据界值判断肿瘤标本和正常标本；
    粪便标本中的Ct值的界值为32～42，所述粪便标本的Ct值小于所述Ct值的界值则判断为肿瘤标本，所述粪便标本的Ct值大于等于所述Ct值的界值则判断为正常标本；
    组织标本中甲基化水平值的界值为1～10，所述组织标本的甲基化水平值大于所述甲基化水平值的界值则判断为肿瘤标本，所述组织标本的甲基化水平值小于等于所述甲基化水平值的界值则判断为正常标本。
  13. 如权利要求10-11任一所述的方法，或者权利要求12所述的系统，其特征在于，所述肿瘤为结直肠肿瘤；
    优选地，所述肿瘤为结直肠癌或腺瘤。
  14. 如权利要求10-11任一所述的方法，或者权利要求12所述的系统，其特征在于，所述的系统或者方法检测样本类型为组织、体液或排泄物；
    优选地，所述组织为肠组织；
    优选地，所述体液为血液、血清、血浆、细胞外液、组织液、淋巴液、脑脊液或房水；
    优选地，所述排泄物为痰液、尿液、唾液或粪便。
PCT/CN2019/085584 2018-05-22 2019-05-05 肿瘤标志物、甲基化检测试剂、试剂盒及其应用 WO2019223517A1 (zh)

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