Colorectal cancer markers and detection kits

Abstract

The present invention relates to the field of molecular diagnosis, and in particular to colorectal cancer markers and detection kits. The present invention provides markers, primer probe sets, and products and detection methods for colorectal cancer, realizes multi-marker joint detection, improves detection performance, and helps to use blood for early screening and early diagnosis of colorectal cancer. The detection method is affordable and has good compliance, and is expected to increase the screening detection rate of colorectal cancer, improve the survival rate of colorectal cancer, and improve prognosis.

Description

Technical Field

[0001] The present invention relates to the field of molecular diagnosis, and in particular to a colorectal cancer marker and a detection kit. Background Art

[0002] Early screening and diagnosis of colorectal cancer are crucial to improving the survival rate and prognosis of patients. There are currently a variety of testing options, including tumor markers in the blood, fecal occult blood testing, endoscopic testing, etc. However, each of the traditional options has its own problems, so people have conducted research on testing methods based on molecular biology methods, among which molecular testing based on fecal DNA plus fecal occult blood testing is the most well-known.

[0003] Most current colorectal cancer detection programs are based on the morphology of stool samples, using the exfoliated cells of colorectal cancer in them for DNA extraction and molecular biological testing, such as methylation PCR programs. However, domestic and foreign literature shows that this method has the problem of insufficient compliance, which affects the improvement of screening rates; and stool samples are collected from the users themselves rather than professionals, which may lead to collection failure due to sample contamination. Many documents show that, in contrast, the use of blood tests can help improve the overall compliance of the subjects, thereby increasing the proportion of the tested population.

[0004] The overall sensitivity and specificity of blood methylation PCR testing are not ideal, especially the sensitivity is slightly improved compared to traditional tumor markers such as CEA. The FDA has excluded the only approved blood methylation PCR testing product from its recommendation. Another technical form of blood colorectal cancer detection is high-throughput NGS testing, which can detect more markers and has the potential to improve detection sensitivity. However, the NGS solution is expensive, the operation steps are complicated, the bioinformatics department is required to analyze and process the data, and the overall turnover period is long. These factors have affected its promotion and use in clinical practice. Summary of the invention

[0005] In view of this, the present invention provides a detection marker and a detection kit for colorectal cancer, the purpose of which is to efficiently detect colorectal cancer in blood specimens with the help of a methylation PCR scheme, the key of which lies in the selection of corresponding methylation markers, and the selection of primers and probe sequences for detecting markers; in addition, a corresponding detection kit is provided according to the number of markers. For example, when multiple markers are detected together, pre-amplification of multiple markers is first carried out, and then the pre-amplification products are tested for multiple markers. In the case where multiple markers need to be detected, especially when the number of fluorescent channels of a fluorescent PCR instrument is exceeded, there is no need to divide a sample into several parts for detection to avoid signal loss, break through the channel limitation of the fluorescent PCR instrument, achieve detection of more markers, and then superimpose and enhance the sensitivity of the overall detection.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides markers, including one or more of the following:

[0008] i), chr12:104851089-104851189;

[0009] ii), chr1:108507613-108507712;

[0010] iii), chr2:145274418-145274511;

[0011] iv), chr7:50343911-50343986;

[0012] v), chr2:182321972-182322057;

[0013] vi), chr9:132382692-132382799;

[0014] vii), chr17:75369757-75369864;

[0015] viii), chr10:7451050-7451154.

[0016] In some specific embodiments of the present invention, the above-mentioned markers are chr7:50343911-50343986 and chr2:182321972-182322057.

[0017] In some specific embodiments of the present invention, the above-mentioned markers are chr7:50343911-50343986 and chr9:132382692-132382799.

[0018] In some specific embodiments of the present invention, the above-mentioned markers are chr2:182321972-182322057 and chr9:132382692-132382799.

[0019] In some specific embodiments of the present invention, the above-mentioned markers are chr7:50343911-50343986, chr2:182321972-182322057 and chr9:132382692-132382799.

[0020] In some specific embodiments of the present invention, the above-mentioned markers are chr12:104851089-104851189, chr1:108507613-108507712, chr2:145274418-145274511, chr7:50343911-50343986, chr2:182321972-182322057, chr9:132382692-132382799, chr17:75369757-75369864 and chr10:7451050-7451154.

[0021] The present invention also provides the use of the marker in any of the following:

[0022] a), preparing primers and / or probes for detecting colorectal cancer;

[0023] b) preparing colorectal cancer detection products;

[0024] The product includes a reagent, a kit or a device.

[0025] In some specific embodiments of the present invention, the detection in the above application uses a blood sample as the sample to be tested.

[0026] The present invention also provides a primer probe set, which is designed based on the above markers.

[0027] In some specific embodiments of the present invention, the primer-probe set comprises a forward primer, a reverse primer and a probe;

[0028] The sequence of the forward primer is:

[0029] (1), the nucleotide sequence shown in SEQ ID NO: (3X-2); or

[0030] (2) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (1), and having the same or similar function as (1); or

[0031] (3) a nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (1) or (2);

[0032] The sequence of the reverse primer is:

[0033] (4) the nucleotide sequence shown in SEQ ID NO: (3X-1); or

[0034] (5) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (4), and having the same or similar function as (4); or

[0035] (6) a nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (4) or (5);

[0036] The sequence of the probe is:

[0037] (7) the nucleotide sequence shown in SEQ ID NO: 3X; or

[0038] (8) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (7), and having the same or similar function as (7); or

[0039] (9) a nucleotide sequence having at least 90% homology to the nucleotide sequence shown in (7) or (8);

[0040] Wherein, X is selected from any integer from 1 to 8;

[0041] The plurality is 2 to 5.

[0042] In some specific embodiments of the present invention, the primer probe set is a CHST11 primer probe set, a VAV3-AS1 primer probe set, a ZEB2 primer probe set, an IKZF1 primer probe set, an ITGA4 primer probe set, a C9orf50 primer probe set, a SEPT9-2 primer probe set or a SFMBT2 primer probe set;

[0043] The primer sequences of the CHST11 primer-probe set are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, and the probe sequence is shown in SEQ ID NO: 3;

[0044] The primer sequences of the VAV3-AS1 primer-probe set are shown in SEQ ID NO:4 and SEQ ID NO:5, respectively, and the probe sequence is shown in SEQ ID NO:6;

[0045] The primer sequences of the ZEB2 primer-probe set are shown in SEQ ID NO:7 and SEQ ID NO:8, respectively, and the probe sequence is shown in SEQ ID NO:9;

[0046] The primer sequences of the IKZF1 primer-probe set are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the probe sequence is shown in SEQ ID NO: 12;

[0047] The primer sequences of the ITGA4 primer-probe set are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and the probe sequence is shown in SEQ ID NO: 15;

[0048] The primer sequences of the C9orf50 primer probe set are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the probe sequence is shown in SEQ ID NO: 18;

[0049] The primer sequences of the SEPT9-2 primer-probe set are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively, and the probe sequence is shown in SEQ ID NO: 21;

[0050] The primer sequences of the SFMBT2 primer-probe set are shown in SEQ ID NO:22 and SEQ ID NO:23, respectively, and the probe sequence is shown in SEQ ID NO:24.

[0051] In some specific embodiments of the present invention, in the above primer probe set:

[0052] The CHST11 primer probe set also includes pre-amplification primers having sequences of SEQ ID NO: 28 and SEQ ID NO: 29;

[0053] The VAV3-AS1 primer probe set also includes pre-amplification primers having sequences of SEQ ID NO: 30 and SEQ ID NO: 31;

[0054] The ZEB2 primer probe set also includes pre-amplification primers having sequences of SEQ ID NO:32 and SEQ ID NO:33;

[0055] The IKZF1 primer probe set also includes pre-amplification primers having sequences of SEQ ID NO:34 and SEQ ID NO:35;

[0056] The ITGA4 primer probe set also includes pre-amplification primers having sequences of SEQ ID NO: 36 and SEQ ID NO: 37;

[0057] The C9orf50 primer probe set also includes pre-amplification primers having sequences of SEQ ID NO:38 and SEQ ID NO:39;

[0058] The SEPT9-2 primer probe set also includes pre-amplification primers having sequences of SEQ ID NO:40 and SEQ ID NO:41;

[0059] The SFMBT2 primer probe set also includes pre-amplification primers with sequences of SEQ ID NO:42 and SEQ ID NO:43 respectively.

[0060] The present invention also provides a primer-probe set combination, which is a combination of two or more of the following:

[0061] A), the CHST11 primer probe set in the above primer probe set;

[0062] B), the VAV3-AS1 primer probe set in the above primer probe set;

[0063] C), the ZEB2 primer probe set in the above primer probe set;

[0064] D), the IKZF1 primer probe set in the above primer probe set;

[0065] E), the ITGA4 primer probe set in the above primer probe set;

[0066] F), the C9orf50 primer probe set in the above primer probe set;

[0067] G), the SEPT9-2 primer probe set in the above primer probe set;

[0068] H), the SFMBT2 primer probe set in the above primer probe set.

[0069] The present invention also provides a product for detecting colorectal cancer, comprising any one of the above primer probe sets or the above primer probe set combination;

[0070] The product includes a reagent, a kit or a device.

[0071] In some specific embodiments of the present invention, the primer probe group combination is the IKZF1 primer probe group in the primer probe group and the ITGA4 primer probe group in the primer probe group.

[0072] In some specific embodiments of the present invention, the primer probe group combination is the IKZF1 primer probe group in the primer probe group and the C9orf50 primer probe group in the primer probe group.

[0073] In some specific embodiments of the present invention, the primer probe group combination is the ITGA4 primer probe group in the primer probe group and the C9orf50 primer probe group in the primer probe group.

[0074] In some specific embodiments of the present invention, the primer probe group combination is the IKZF1 primer probe group in the primer probe group and the C9orf50 primer probe group in the primer probe group.

[0075] In some specific embodiments of the present invention, the above-mentioned primer probe group combination is the CHST11 primer probe group in the above-mentioned primer probe group, the VAV3-AS1 primer probe group in the above-mentioned primer probe group, the ZEB2 primer probe group in the above-mentioned primer probe group, the IKZF1 primer probe group in the above-mentioned primer probe group, the ITGA4 primer probe group in the above-mentioned primer probe group, the C9orf50 primer probe group in the above-mentioned primer probe group, the SEPT9-2 primer probe group in the above-mentioned primer probe group and the SFMBT2 primer probe group in the above-mentioned primer probe group.

[0076] In some specific embodiments of the present invention, the above-mentioned product uses a blood sample as the sample to be tested.

[0077] The present invention also provides a detection method, which detects a blood sample based on the primer probe set, the primer probe set combination or the product to obtain a detection result.

[0078] The present invention also provides a method for diagnosing colorectal cancer, which detects and analyzes a blood sample based on the above primer probe set, the above primer probe set combination or the above product to obtain a diagnosis result.

[0079] The markers and detection products of the present invention have the following effects:

[0080] 1. The present invention adopts a blood PCR solution, which can overcome the problem of poor compliance of stool samples and avoid the high cost and complicated process of NGS;

[0081] 2. In order to ensure the performance of the scheme, non-obvious selections were made for the marker regions, and the corresponding primer-probe combinations were also combined to achieve the purpose of multiplex testing to improve detection sensitivity;

[0082] 3. In the case of multiplex detection, in order to avoid the limitation of fluorescence channels of current fluorescence PCR instruments, for example, when more than 5 markers need to be detected, the probes in the marker region are combined according to the fluorescence channel combination of the instrument and equipment. After multi-marker amplification is achieved by pre-amplification, the probes can be divided into small portions for corresponding probe combination detection;

[0083] 4. Based on the selection of marker regions and the corresponding probe primer design, combined with multi-marker joint testing, the detection performance can be improved, which is helpful for the early screening and diagnosis of colorectal cancer using blood, thereby achieving affordable and high-compliance testing, and achieving an increase in the screening detection rate of colorectal cancer, and achieving an increase in the survival rate and prognosis of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0085] Figure 1 The tissue methylation differences of each marker are shown. DETAILED DESCRIPTION

[0086] The present invention discloses a detection marker and a detection kit for colorectal cancer. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0087] In order to improve the compliance of colorectal cancer detection, the present invention is committed to providing a colorectal cancer detection method using blood as a sample. However, unlike stool, tissue or cell samples, the signal-to-noise ratio of blood samples is generally not high enough. In order to solve this problem, the present invention uses the data of the blood buffy coat layer for screening, selects the marker with the strongest distinguishing ability from the massive markers that produce high background, and performs a good specificity test in blood samples with benign diseases. Among the markers involved in the present invention, the detection performance shown by various probes designed based on different methylation sites is not consistent, so some creative work has been devoted to obtain the best primer probe set. Taking the SEPT gene as an example: primers and probes were designed for SEPT-1, and the specific region was: chr17:75369608-75369687. Some of the test results were very different from the test results of other regions. For example, it was only 70bp different from the currently selected SEPT9-2: chr17:75369757-75369864, but in the white film layer of 51 positive samples based on SEPT-1: chr17:75369608-75369687 detection, background signals were detected in 41 samples, as high as 80.4%; and in the white film layer of the same 51 positive samples based on SEPT9-2: chr17:75369757-75369864 detection, only 2 samples had background signals. For example, SFMBT2 also has another region: chr10:7450992-7451109, where basically no signal can be detected in all tumor tissues. There is no reliable rule to follow for the selection of marker regions.

[0088] Specific embodiments of the present invention include:

[0089] 1. Through the analysis of existing databases and literature review, as well as the analysis of internal databases, certain methylation markers (specific regions) are screened out;

[0090] 2. According to the corresponding methylation regions, design corresponding primer and probe combinations, verify and optimize them on colorectal cancer tissue samples, and further narrow down the corresponding methylation regions;

[0091] 3. According to the screened markers and corresponding detection schemes, tests are performed on blood samples to obtain the final performance parameters.

[0092] Specific inspection methods can be:

[0093] i. Centrifuge the blood sample to obtain a plasma sample;

[0094] ii. Extract cfDNA from plasma samples using a cfDNA extraction kit;

[0095] iii. Perform direct detection or pre-amplification detection based on the selected marker or marker combination:

[0096] a. For direct detection, add the corresponding forward and reverse primers, the corresponding probe, the corresponding cfDNA sample and the PCR reaction buffer into a PCR reaction system to form a reaction system, perform qPCR / digital PCR reaction, collect the corresponding signals, and finally obtain the positive and negative judgment results of the detection in a certain calculation;

[0097] b. If multiple marker regions are to be detected together, then design peripheral primer pairs for each region, perform PCR amplification in a multiplex reaction, obtain corresponding PCR amplification products, design internal primer pairs for each region, and corresponding probe sequences, add corresponding internal primers and probe sequences, and a small portion of the amplification product, such as 1 / 50 or 1 / 25, to a qPCR system to obtain specific Ct values ​​for several marker regions of the system. If the number of marker regions detected is more than the number of fluorescent channels of the fluorescent PCR instrument, the corresponding regional detection probes can be distinguished and detected in different combinations.

[0098] The specific regions of the markers of the present invention are as follows (reference genome: hg19):

[0099] CHST11:chr12:104851089-104851189;

[0100] VAV3-AS1:chr1:108507613-108507712;

[0101] ZEB2:chr2:145274418-145274511;

[0102] IKZF1:chr7:50343911-50343986;

[0103] ITGA4:chr2:182321972-182322057;

[0104] C9orf50:chr9:132382692-132382799;

[0105] SEPT9-2:chr17:75369757-75369864;

[0106] SFMBT2:chr10:7451050-7451154.

[0107] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0108] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0109] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0110] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0111] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0112] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0113] The present invention will be further described below in conjunction with embodiments:

[0114] Example 1

[0115] For the methylation level detection of the specific regions of the target genes ZEB2, CHST11, VAV3-AS1, SFMBT2, IKZF1, ITGA4, C9orf50, and SEPT9-2 in the genomic DNA of the buffy coat or the genomic DNA of the tissue, methylation-specific primers (MSP) and Taqman probes were designed, and ACTB was selected as the internal reference gene. Primers and Taqman probes were also designed for ACTB. The fluorescent groups of the following probes are only used as examples, and other fluorescent groups can be replaced according to specific needs. The fluorescent groups are not bound to specific bases and can be adjusted according to actual needs. The sequences of the primers and probes for the 9 genes are shown in Table 1.

[0116] Table 1: Methylation marker primers and probe sequences

[0117]

[0118]

[0119] Example 2

[0120] For the detection of methylation levels in plasma cfDNA of specific regions of target genes ZEB2, CHST11, VAV3-AS1, SFMBT2, KZF1, ITGA4, C9orf50, and SEPT9-2, pre-amplification external primers and qPCR internal primers were designed, and ACTB was selected as the internal reference gene. Primers and Taqman probes were also designed for ACTB. The fluorescent groups of the following probes are only used as examples, and other fluorescent groups can be replaced according to specific needs. The fluorescent groups are also not bound to specific bases and can be adjusted according to actual needs. The external and internal primer probe sequences of the 9 genes are shown in Table 2.

[0121] Table 2: Primer and probe sequences for methylation marker detection in cfDNA

[0122]

[0123]

[0124] Example 3: Detection of target gene methylation levels in 51 normal human buffy coat cells

[0125] 1. Nucleic Acid Extraction

[0126] The buffy coat cells were isolated from the whole blood of 51 normal subjects, and the genomic DNA of the buffy coat cells was isolated according to the operating instructions of the Meiki kit (Meiki, Cat#D311102). The specific steps can be referred to the operating instructions of the Meiki kit. TM dsDNA HS Assay Kit was used for quantification of genomic DNA.

[0127] 2. Bisulfite treatment

[0128] According to the Zymo kit EZ-96 DNA Methylation-Lightning TM The operating requirements of MagPrep (Zymo Research, Cat#D5046) are to treat the genomic DNA of each normal human buffy coat cell with bisulfite, so that the C in the unmethylated CpG in the cell genomic DNA is converted to T, while the C in the methylated CpG remains C after conversion, thereby making the methylation or not become the difference in specific nucleic acid bases.

[0129] 3.qPCR detection

[0130] The methylation level of the transformed genomic DNA was detected. The system is shown in Table 3, and the reaction procedure is shown in Table 4:

[0131] Table 3

[0132] Components volume EpiTect MethyLight Master Mix (Qiagen, Cat#59496) 10μl 9 gene primers, probe oligo combinations 3μl Transforming DNA 7μl Total 20μl

[0133] Table 4: Reaction Procedure

[0134]

[0135] 4. Results

[0136] The methylation levels of the corresponding regions of the above 8 genes in 51 cases of buffy coat DNA are shown in Table 5.

[0137] Table 5

[0138]

[0139]

[0140] Example 4: Detection of target gene methylation levels in 20 paired colorectal cancer and adjacent tissues

[0141] 1. Nucleic Acid Extraction

[0142] Corresponding tissue DNA was extracted from 20 groups of fresh frozen colorectal cancer tissues and adjacent tissues, and DNA from colorectal cancer and adjacent tissues was obtained according to the operating requirements of the Meiji kit (Meiji, Cat#IVD3018). For specific steps, please refer to the operating instructions of the Meiji kit. TM dsDNA HS Assay Kit was used for quantification of genomic DNA.

[0143] 2. Bisulfite treatment

[0144] According to the Zymo kit EZ-96 DNA Methylation-Lightning TM The operating requirements of MagPrep (Zymo Research, Cat#D5046) are to treat each tissue DNA with bisulfite so that the C in the unmethylated CpG in the genomic DNA is converted to T, while the C in the methylated CpG remains C after conversion, thus making the methylation or not become the difference in specific nucleic acid bases.

[0145] 3.qPCR detection

[0146] The methylation level of the transformed genomic DNA was detected, the system is shown in Table 6, and the reaction procedure is shown in Table 7:

[0147] Table 6

[0148] Components volume EpiTect MethyLight Master Mix (Qiagen, Cat#59496) 10μL 9 gene primers, probe oligo combinations 3μL Transforming DNA 7μL Total 20μL

[0149] Table 7: Reaction Procedure

[0150]

[0151] 4. Results

[0152] The methylation level of each gene in each sample was calibrated using the Ct of the internal reference gene to obtain the corresponding △Ct = Ct marker –Ct ACTB. For genes with no specific signal detected in the DNA of the tissue in this case, △Ct was set to 25.0. Chi-square test was performed for the difference in methylation levels. For each gene, the △△Ct (△Ct adjacent to cancer - △Ct cancer) in the paired colorectal cancer and paracancerous tissues was greater than 4 and was considered to have specific distinguishing ability. The specific results are shown in Table 8:

[0153] Table 8

[0154] Gene Discrimination(%) P-value CHST11 55％ 0.049 C9orf50 90％ <0.0001 SFMBT2 70％ <0.0001 ZEB2 70％ <0.0001 IKZF1 75％ 0.0064 VAV3-AS1 50％ 0.0589 ITGA4 75％ <0.0001 SEPT9-2 30％ <0.0001

[0155] Specific paired tissue methylation differences such as Figure 1 shown.

[0156] Example 5: Performance Verification in Plasma Samples

[0157] To prove that the methylation levels of these gene regions can be used to detect colorectal cancer in plasma samples, we tested 250 plasma samples, including 117 blood samples from colorectal cancer patients, 5 blood samples from high-grade adenomas, 60 blood samples from patients with benign diseases, and 68 blood samples from normal people.

[0158] 1. cfDNA extraction

[0159] 10 ml of whole blood was drawn from the subject, and the corresponding plasma was separated. The patient's cfDNA was obtained according to the operating requirements of the Meiji kit (Meiji, Cat# D318203D). For specific steps, please refer to the operating instructions of the Meiji kit. The Qubit TM The dsDNA HS Assay Kit was used to quantify genomic DNA, and Qsep100 was used to perform a quality check on the length distribution of cfDNA to ensure that there was no genomic DNA contamination.

[0160] 2. Bisulfite treatment

[0161] According to the Zymo kit EZ-96 DNA Methylation-Lightning TM The operating requirements of MagPrep (Zymo Research, Cat#D5046) are to treat each tissue DNA with bisulfite so that the C in the unmethylated CpG in the genomic DNA is converted to T, while the C in the methylated CpG remains C after conversion, thus making the methylation or not become the difference in specific nucleic acid bases.

[0162] 3. Pre-amplification

[0163] The converted cfDNA was pre-amplified, the system is shown in Table 9, and the reaction procedure is shown in Table 10:

[0164] Table 9

[0165]

[0166]

[0167] Table 10: Reaction schedule

[0168]

[0169] After the pre-amplification, the amplified product was diluted 4 times, and 8 μL of each diluted product was taken for qPCR detection. The system is shown in Table 11, and the reaction procedure is shown in Table 12:

[0170] Table 11

[0171] Components volume Luna Universal qPCR Master Mix (NEB, Cat#M3003) 10μL 9 Gene primers, probe oligo internal combination 2μL Pre-amplification dilution product 8μL Total 20μL

[0172] Table 12: Reaction Procedure

[0173]

[0174] 4. Results

[0175] For each sample, the internal reference Ct is first determined to be less than 26, then the test result of this sample is valid, and the other genes are judged as follows: CHST11+ZEB2≤40, IKZF1≤40, ITGA4≤30, C9orf50≤40, VAV3-AS1+SFMBT2≤40, SEPT9-2≤40, then the channel (the result of the gene or gene combination is positive) is judged respectively. If any of the genes is positive, the sample is judged to be colorectal cancer. The specific results are shown in Table 13:

[0176] Table 13

[0177]

[0178]

[0179] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer-probe set combination, characterized in that: are IKZF1 primer probe set, ITGA4 primer probe set, and C9orf50 primer probe set; The primer sequences of the IKZF1 primer-probe set are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the probe sequence is shown in SEQ ID NO: 12; The primer sequences of the ITGA4 primer-probe set are shown in SEQ ID NO: 13 and SEQ ID NO: 14, respectively, and the probe sequence is shown in SEQ ID NO: 15; The primer sequences of the C9orf50 primer probe set are shown in SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the probe sequence is shown in SEQ ID NO:

18.

2. A product for detecting colorectal cancer, including a reagent, a kit or a device, characterized in that: Comprising the primer-probe set combination according to claim 1.

3. The product according to claim 2, characterized in that A blood sample is used as a sample to be tested.

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