A combined marker for colorectal cancer detection and use thereof

Abstract

The application discloses a combined marker for colorectal cancer detection and application thereof, and the combined marker is composed of ALX4 genes, SFRP2 genes, SEPT9 genes, SDC2 genes and TFPI2 genes. Experiments prove that the five genes of the ALX4 genes, the SFRP2 genes, the SEPT9 genes, the SDC2 genes and the TFPI2 genes are used in a combined manner to diagnose early colorectal cancer, and the operation is simple, time-consuming is short, the sensitivity and the specificity are high, meanwhile, the detection rate can be effectively improved, and the false positive of the result is reduced. The application has important application value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedicine, and particularly relates to a combined marker for colorectal cancer detection and application thereof. BACKGROUND

[0002] Colorectal cancer is a common malignant tumor in the gastrointestinal tract, and early symptoms are not obvious. With the enlargement of the cancer, symptoms such as change in bowel habits, hematochezia, diarrhea, alternating diarrhea and constipation, and local abdominal pain appear, and in the advanced stage, anemia, weight loss and other systemic symptoms appear. Colorectal cancer grows very slowly, and the incubation period is long, 93% of colorectal cancer is derived from adenoma, and it takes 5-7 years from adenoma to cancer. Studies have shown that annual fecal occult blood testing can reduce the mortality rate of colorectal cancer by 33%. Although colorectal cancer can be prevented and treated, the early diagnosis rate is only 10-15%. At present, the diagnosis and treatment of colorectal cancer still faces major problems, such as difficulty in early diagnosis, high recurrence and metastasis rate, radiotherapy and chemotherapy resistance, and lack of effective treatment targets. Early detection, early diagnosis and early treatment of colorectal cancer can effectively improve the treatment effect of colorectal cancer.

[0003] Most colorectal cancers start as growths on the inner wall of the colon or rectum, which are called polyps. The common types of polyps are hyperplastic polyps and colorectal adenomatous polyps, which can develop into cancer as they progress. Therefore, adenoma is called a cancerous disease. Intestinal adenomas can be screened by endoscopy, but the technical requirements for the examiner are high, and the clinical missed detection rate of endoscopic screening is high. Therefore, genetic screening of adenoma has important significance for the early diagnosis and screening of colorectal cancer.

[0004] It takes 5-10 years for colorectal cancer to develop from a polyp to colorectal cancer. This stage is low in cost and good in effect; but once this stage is broken through, as the disease progresses, the lesion speed increases, the treatment cost is high, and the effect is poor. There is no specific clinical manifestation in the early stage of colorectal cancer, and there is a lack of high sensitivity and specificity in early diagnosis technology. Most patients are diagnosed with advanced cancer, thus missing the best treatment opportunity. Therefore, early diagnosis and treatment is the best way to deal with colorectal cancer. Currently, the diagnosis of cancer mainly relies on imaging CT and MRI detection, but it cannot determine its benign or malignant nature and needs long-term image follow-up. Pathological examination requires tumor tissue, which is often in the middle and late stages. Early diagnosis of colorectal cancer has always been a clinical difficulty. Electronic colonoscopy is the gold standard for diagnosing colorectal cancer, which can remove and biopsy the lesion, but it is not suitable for early screening; CA199 can be used for colorectal cancer screening, but it has poor specificity and sensitivity. Tumor markers can be detected in body fluids or tissues, which can reflect the presence of tumors, differentiation degree, prognosis estimation and judgment of treatment effect, etc. Compared with tissue biopsy, colon bubble lavage fluid, plasma, sputum and other specimens are easy to obtain and do not cause trauma to the examinee, and can be continuously sampled for comparison. However, the current serum markers such as CEA, CA125, CA199, etc. not only have low sensitivity, but also lack specificity. Therefore, it is urgent to develop new early screening biomarkers for colorectal cancer.

[0005] It is found that DNA methylation occurs in almost all tumors, which is an early event in tumorigenesis, can be detected before clinical diagnosis, and is a potential reliable early screening indicator for early diagnosis, risk prediction, clinical course monitoring and efficacy evaluation of tumors. It is related to the early activation of oncogene demethylation in carcinogenesis, and also related to the inactivation of tumor suppressor genes caused by gene methylation in normal cells; its sensitivity is better than that of existing protein serum markers, and it is more specific than serum markers. As a new molecular marker, DNA methylation has received more and more attention in tumor diagnosis, and its advantages include: (1) promoter hypermethylation frequently occurs during tumor formation, even more than gene mutation, and many of them are important genes related to tumor formation; (2) methylation is an important event in the early stage of tumorigenesis; (3) DNA methylation is stable and can be detected by PCR amplification effect. Therefore, methylation detection has potential application value in early diagnosis of tumors.

[0006] Common methods for detecting DNA methylation include methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), high-resolution melting (HRM), direct sequencing of the genome, etc. SUMMARY

[0007] The present application aims to early diagnose colorectal cancer.

[0008] The present application first protects the use of the combination marker in the preparation of a colorectal cancer detection kit.

[0009] The combination marker can consist of ALX4 gene, SFRP2 gene, SEPT9 gene, SDC2 gene and TFPI2 gene.

[0010] The GeneBank of ALX4 gene is 60529.

[0011] The GeneBank of SFRP2 gene is 6423.

[0012] The GeneBank of SEPT9 gene is 10801.

[0013] The GeneBank of SDC2 gene is 6383.

[0014] The GeneBank of TFPI2 gene is 7980.

[0015] The present application also protects a colorectal cancer detection kit, which comprises detection reagents of the combination marker.

[0016] The kit specifically can consist of detection reagents of the combination marker.

[0017] In any of the above kits, the detection reagents of the combination marker can comprise primers and / or probes for detecting the methylation level of each gene in the combination marker.

[0018] In any of the above kits, the detection reagents of the combination marker specifically can consist of primers and / or probes for detecting the methylation level of each gene in the combination marker.

[0019] The primer for detecting the methylation level of each gene in the combination marker can be ALX4-F shown in SEQ ID NO: 1, ALX4-R shown in SEQ ID NO: 2, SERP2-F shown in SEQ ID NO: 4, SERP2-R shown in SEQ ID NO: 5, SEPTIN9-F shown in SEQ ID NO: 7, SEPTIN9-R shown in SEQ ID NO: 8, SDC2-F shown in SEQ ID NO: 10, SDC2-R shown in SEQ ID NO: 11, TFPI2-F shown in SEQ ID NO: 13, and TFPI2-R shown in SEQ ID NO: 14.

[0020] The probe for detecting the methylation level of each gene in the combination marker can be ALX4-P shown in SEQ ID NO: 3, SERP2-P shown in SEQ ID NO: 6, SEPTIN9-P shown in SEQ ID NO: 9, SDC2-P shown in SEQ ID NO: 12, and TFPI2-P shown in SEQ ID NO: 15.

[0021] The kit can further comprise a reagent for detecting a reference gene.

[0022] The kit can specifically consist of the reagent for detecting the combination marker and the reagent for detecting the reference gene.

[0023] The reagent for detecting the reference gene comprises a primer and / or a probe for detecting the reference gene.

[0024] The reagent for detecting the reference gene can specifically consist of a primer and / or a probe for detecting the reference gene.

[0025] The primer for detecting the reference gene is ACTB-F shown in SEQ ID NO: 16 and ACTB-R shown in SEQ ID NO: 17.

[0026] The probe for detecting the reference gene is ACTB-P shown in SEQ ID NO: 18.

[0027] One end of each of the probes (such as the probe for detecting the reference gene and the probe for detecting the methylation level of each gene in the combination marker) has a fluorescent label, and the other end has a fluorescent quenching label.

[0028] The specific target of detection for any of the above-mentioned kits can be plasma cfDNA, fecal cfDNA, or genomic DNA from colon tissue.

[0029] The kit described above may further include a data processing system; the data processing system converts the methylation levels of each gene in the combined biomarkers into the dCT of the subject. X It is used to determine whether a person being tested has colorectal cancer.

[0030] The subject's dCT X The calculation method is as follows: The genomic DNA from the plasma, fecal cfDNA, or colon tissue of the test subject is chemically modified (e.g., modified with bisulfite). Then, using this as a template, fluorescent PCR amplification is performed using any of the primers and probes described above. Fluorescence signals are collected, and the CT values ​​of ALX4, SFRP2, SEPT9, SDC2, TFPI2, and ACTB are obtained, and denoted as CT values ​​respectively. ALX4 CT scan SFRP2 CT scan SEPT9 CT scan SDC2 CT scan TFPI2 and CT ACTB If the amplification curve is not S-shaped or the CT value is blank, the CT value is recorded as 45; further calculate the dCT values ​​of each gene ALX4, SFRP2, SEPT9, SDC2, or TFPI2. X =CT x -CT ACTB ;

[0031] The method for determination is as follows: if at least two of the ALX4, SFRP2, SEPT9, SDC2, and TFPI2 genes of the test subject are methylated, then the test subject is a colorectal cancer patient; otherwise, the test subject is not a colorectal cancer patient; whether a gene is methylated is determined by comparing the dCT and dCT threshold values ​​of the genes in the test sample.

[0032] If the dCT of the ALX4, SFRP2, SEPT9, SDC2, or TFPI2 genes in the subject is less than or equal to the dCT threshold, then the subject has methylated based on that gene.

[0033] The dCT threshold value for each gene is the average statistical value obtained by comparing the dCT values ​​of colorectal cancer tissue and adjacent normal tissue. It is a threshold value that can best distinguish between tumors and non-tumors (for example, the median value of the dCt values ​​of each target gene in all validated colorectal cancer positive and negative samples is the dCt threshold value; a Ct value of methylation region detection of the gene ≤ the threshold value is positive; a Ct value of methylation region detection of the gene ≥ the threshold value is negative).

[0034] Experiments prove that the five genes of ALX4 gene, SFRP2 gene, SEPT9 gene, SDC2 gene and TFPI2 gene in the colon cancer tissue can be used for diagnosing early colorectal cancer, and the operation is simple, the time consumption is short, the sensitivity and the specificity are high, meanwhile, the detection rate can be effectively improved, and the false positive of the result can be reduced. The present application has important application value. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 The detection results of the ROC method in Example 2. DETAILED DESCRIPTION

[0036] The present application is further described in detail below with specific embodiments, and the examples given are only for illustrating the present application, but not for limiting the scope of the present application. The examples provided below can be used as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application.

[0037] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0038] In the following examples, the “to-be-tested sample” refers to a nucleic acid sample to be detected. Specifically, the to-be-tested sample can be isolated blood cells, one or more of the cells isolated from blood, a cell line, a tissue section, a biopsy tissue, a paraffin-embedded tissue, a body fluid, feces, urine, plasma, serum, whole blood, etc. gDNA, cfDNA and ctDNA.

[0039] In the following examples, the “target nucleic acid” refers to the nucleic acid fragments of the five colorectal cancer-related genes, i.e. the methylation DNA-specific fragments of human ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes.

[0040] In the following examples, the “probe” refers to a single-stranded nucleic acid with a known nucleotide sequence, which is basically complementary to the target nucleic acid in terms of nucleotide sequence structure, and can form a double strand with the “target nucleic acid”. The 5' end of the probe can carry a fluorescent group and / or a 3' end carrying a quenching group marker. The binding of the primer and the probe to the methylation-specific sequence in the sample DNA enables the molecular marker to detect the disease-colorectal cancer.

[0041] In the method of the present application, chemical modification needs to be performed on the extracted DNA, and bisulfite, bisulfite or hydrazine salt modification can be applied to the chemical modification.

[0042] In view of the need for clinical detection, the method of liquid biopsy can effectively reduce the harm to patients. When the real-time fluorescent PCR is applied for detection, the probe is connected with a fluorescent group suitable for judging different gene methylation DNA fragments. One end of the probe is labeled with a fluorescent group, and the other end is labeled with a quenching group; wherein the quenching group can quench the fluorescence emitted by the fluorescent group. When the PCR amplification reaction is carried out, the forward exonuclease activity of the polymerase is used to cut off the base with the fluorescent group, and the free fluorescent group is no longer affected by the quenching group, and can emit a certain wavelength of fluorescent signal under the action of excitation light. With the continuous accumulation of PCR products, the fluorescent signal is continuously enhanced, so that the presence of specific methylation DNA can be detected. As a preferred mode of the present application, when detecting, 5 specific probes labeled with 5 different fluorescent groups are added in the same reaction tube, which correspond to ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes and the internal reference gene ACTB respectively, and the presence of 5 target methylation DNA fragments is indicated in the same reaction tube. As a preferred mode of the present application, the fluorescent group labeled by the detection probe can be VIC, ROX, FAM, Cy5, Cy5.5, VIC, HEX, TET, JOE, NED or TAMRA, etc.; and the quenching group can be BHQ, MGB or Dabcy1. The present application is suitable for the multi-channel PCR detection technology commonly used in current clinical detection, and realizes multi-channel fluorescent detection in one reaction tube.

[0043] In the following examples, all patients gave informed consent and the experiments were approved by the ethics committee.

[0044] In the following examples, the human whole gene methylation DNA standard (EpiTect PCR Control DNA Set, Qiagen CatNo. / ID:59695, wherein the methylation DNA is indicated by +ve ctr) is the positive control DNA. The human whole gene non-methylation DNA standard (EpiTect PCR Control DNA Set, Qiagen CatNo. / ID:59695, wherein the non-methylation DNA is indicated by -ve ctr) or water is the negative control DNA.

[0045] Example 1, obtaining markers for colorectal cancer detection

[0046] The inventors of the present application combine various databases and comprehensive clinical information, design probes to enrich methylation sites of related genes through large-scale screening, use surgical tissues of colorectal cancer patients (confirmed by pathology, C) as tumor positive samples, and normal tissues adjacent to the cancer of the same case (confirmed by pathology, A) as tumor negative samples. By comparing the differences in methylation levels, a marker for detecting colorectal cancer is obtained. The marker for detecting colorectal cancer consists of ALX4 (GeneID: 60529), SFRP2 (GeneID: 6423), SEPT9 (GeneID: 10801), SDC2 (GeneID: 6383), TFPI2 (GeneID: 7980) and ACTB (GeneID: 60).

[0047] According to the nucleotide sequences of each of the above genes, the primers and probes shown in Table 1 were designed and synthesized by Nanjing Kingsriver Biotech Co., Ltd.

[0048] Table 1

[0049]

[0050]

[0051] Note: "F" in the primer name indicates an upstream primer, "R" indicates a downstream primer, and "P" indicates a probe; CY5.5 indicates CY5.5 labeling; VIC indicates VIC labeling; ROX indicates ROX labeling; FAM indicates FAM labeling; TAMRA indicates TAMRA labeling; CY5 indicates CY5 labeling; MGB indicates fluorescence quenching labeling; ACTB (GeneID: 60) is an internal reference gene.

[0052] Example 2, Detection of Gene Methylation Markers in Colorectal Cancer (A) and Normal Tissues Adjacent to Cancer (C)

[0053] 1. Dilute the upstream primers, downstream primers and probes of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB in Table 1 with water, respectively.

[0054] 2. Homogenize the samples to be tested (20 samples of colorectal cancer tissues (indicated by C) and normal tissues adjacent to cancer (indicated by A), and 3 samples of human colorectal cancer cell lines (HT29 cells (Shanghai Fuheng Company, FH0024), HCT8 cells (Shanghai Fuheng Company, FH0025) and HCT116 cells (Shanghai Fuheng Company, FH0027), respectively)) and then extract genomic DNA using a blood / cell / tissue genomic DNA extraction kit (Beijing Tiangeng Biochemical Technology (Beijing) Co., Ltd., Cat. # DP304-03) to obtain the genomic DNA of the samples to be tested.

[0055] 3. Obtain genomic DNA from the sample to be tested using EZ DNAMethylation-Direct. TM The DNA of the test subject was modified with bisulfite using KIT (ZYMORESEARCH, D5001 / D5002) to obtain the transformed DNA.

[0056] 4. Prepare the reaction system shown in Table 2 (total 25 μL), where the template is the DNA transformed from the test subject, positive control DNA, negative control DNA, or blank control; then perform fluorescent PCR amplification according to the reaction program. The reaction program is as follows: Stage 1: 95℃ for 3 min, 1 cycle; Stage 2: 95℃ for 15 sec; 62℃ for 25 sec; 4 cycles; Stage 3: 95℃ for 15 sec; 58℃ for 25 sec; 41 cycles. In the third stage at 58℃, collect the fluorescence signal and obtain the CT values ​​of ALX4, SFRP2, SEPT9, SDC2, TFPI2, and ACTB, respectively, and record them as CT. ALX4 CT scan SFRP2 CT scan SEPT9 CT scan SDC2 CT scan TFPI2 and CT ACTB If the amplification curve is not "S"-shaped or the CT value is blank, the CT value is recorded as 45.

[0057] Table 2

[0058]

[0059]

[0060] Note: The DNA enzyme is Accurate Tag HS DNA polymerase (CM0008, 5u / ul, AGL Biosciences, China).

[0061] The DNA sequences of the amplified regions of ALX4, SFRP2, SEPT9, SDC2, TFPI2, and ACTB after sulfite conversion are shown in Table 3.

[0062] Table 3

[0063]

[0064] The CT values ​​of the detection results are shown in Table 4. Further calculation of the dCT for each gene is denoted as dCT. X (dCT X =CT X -CT ACTB ).

[0065] Table 4-1

[0066]

[0067] Note: +ve ctr is positive control DNA, i.e. human whole genome methylated DNA standard; -ve ctr is negative control DNA, i.e. human whole genome unmethylated DNA standard; TE is 10 mM Tris HCl-EDTA (10 mM / 1 mM) buffer as blank control; dCT Th is dCT threshold value.

[0068] Table 4-2

[0069]

[0070]

[0071] 5. Results of determination of gene methylation markers in colorectal cancer tissue DNA

[0072] (1) According to Table 4, colorectal cancer tissue (C), human colorectal cancer cell lines HCT-8, HT-29 and HCT-116 are gene methylation positive DNA. The para-cancer normal tissue (A) is gene methylation negative DNA. The DNA treated by BS is subjected to five-gene fluorescent PCR amplification. The CT value obtained after deducting the CT value of the internal control ACTB is the dCT value of the gene. The dCT values of the positive sample group composed of the colorectal cancer tissue confirmed by pathology and the positive control DNA, and the dCT values of the negative sample group composed of the para-cancer normal tissue and the negative control DNA.

[0073] (2) Whether the gene is methylated or not is realized by comparing the dCT values (denoted as dCT X ) of the ALX4, SFRP2, SEPTIN9, SDC2 and TFPI2 genes of the sample to be tested: dCT X = CT x - CT ACTB . That is, the CT value obtained after deducting the CT value of ACTB, if the dCT of the ALX4 gene, SFRP2 gene, SEPT9 gene, SDC2 gene or TFPI2 gene of the sample to be tested is ≤ dCT threshold value, then the sample to be tested is based on the methylation of the gene. The dCT threshold value of each gene is an average statistical dCT value obtained by comparing the dCT values of a large number of cases of colorectal cancer tissue and para-cancer normal tissue, i.e. the threshold value, which is a critical dCT value that can best distinguish tumors and non-tumors.

[0074] The results show that under the PCR reaction conditions, the dCT threshold values of ALX4, SFRP2, SEPT9, SDC2 and TFPI2 are 5, 5, 5, 5 and 5, respectively.

[0075] (3) Determine whether the sample is from a colorectal cancer patient by comparing the dCT of each gene in the sample with the cutoff value using the panel and method C5 2 Decision: If the dCT of at least two of the five genes ALX4, SFRP2, SEPT9, SDC2 and TFPI2 in the sample to be tested is less than or equal to the threshold value, it means that the sample is methylated, and the sample to be tested is positive, i.e. from a colorectal cancer patient; otherwise, the sample to be tested is negative, i.e. not from a colorectal cancer patient.

[0076] The dCT detection results are shown in Table 5. At least two of the genes ALX4, SFRP2, SEPT9, SDC2 and TFPI2 in the colorectal cancer tissues of 16 of the 20 colorectal cancer patients were methylated; while 19 of the 20 colorectal cancer patients' normal tissues adjacent to the cancer were not methylated. Thus, the detection rate of colorectal cancer can be improved by detecting the genes ALX4, SFRP2, SEPT9, SDC2 and TFPI2 in the colon tissue.

[0077] Table 5

[0078]

[0079] Thus, by detecting the genomic DNA of the colon tissue, it can be determined whether the subject to be tested is a colorectal cancer patient or not.

[0080] 6. 79 colorectal cancer tissue samples and 79 normal colon tissue samples were detected according to the above method.

[0081] The detection results are shown in Table 6. The results show that the individual gene markers with the highest detection rates are SFRP2 (86%), SEPTIN9 (85%) and TFPI2 (84%) in turn. The ALX4 gene shows the lowest detection rate, but has the highest specificity (98%). Using the combined method to determine whether the sample is methylated from a tumor, the detection rate of the method is 91% and the specificity is 89%.

[0082] Table 6

[0083]

[0084] The results were analyzed using the ROC method, and the detection results are shown in Figure 1The curves and area under the curve (AUC) showed that the five gene methylation markers in the colon cancer tissue had high values for diagnosing early lung cancer, and the AUC of SEPTIN9 was as high as 0.905. The detection rate of the method was 91%, the specificity was 89%, and the AUC was 0.89 when measured in a combined manner. It is shown that the 5-gene marker covers multiple mechanisms of carcinogenesis, improves the positive rate of methylation detection or the detection rate of the sample, and the five-to-two method of the combined manner reduces false positives and improves the authenticity of detection.

[0085] Example 3, Sensitivity Experiment

[0086] The samples to be tested were 100% MetBis DNA (100% +ve ctr DNA), 10% MetBis DNA (10% +ve ctr DNA added to 90% -ve ctr DNA), 1% MetBis DNA (1% +ve ctr DNA added to 99% -ve ctr DNA), 0% MetBis DNA (100% -ve ctr DNA), and TE (no DNA, only TE buffer).

[0087] Each sample to be tested was subjected to the following experiment:

[0088] 1. The upstream primers, downstream primers, and probes of ALX4, SFRP2, SEPT9, SDC2, TFPI2, and ACTB in Table 1 were diluted with water, respectively.

[0089] 2. The sample to be tested was taken, and EZ DNA Methylation-Direct KIT was used for bisulfite modification to obtain the converted DNA of the sample to be tested. TM

[0090] 3. The reaction system shown in Table 2 (25 μL in total) was prepared, in which the template was the converted DNA of the sample to be tested, the positive control DNA, or the negative control DNA; and then PCR amplification was performed according to the reaction program. The reaction program was as follows: first stage: 95°C for 3 min, 1 cycle; second stage: 95°C for 15 sec; 62°C for 25 sec, 4 cycles; third stage: 95°C for 15 sec; 58°C for 25 sec, 41 cycles. The fluorescence signal was collected at 58°C in the third stage, and the CT values of ALX4, SFRP2, SEPT9, SDC2, TFPI2, and ACTB were obtained, which were recorded as CT ALX4 , CT SFRP2 , CT SEPT9 , CT SDC2 , CT TFPI2 , and CT ACTB ​; if the amplification curve is not "S" type or CT value is blank, the CT value is recorded as 45. Further calculate the dCT value of each gene (ALX4, SFRP2, SEPT9, SDC2 or TFPI2) (recorded as dCT X ), dCT X = CT x - CT ACTB .

[0091] The detection results of the 5 dilution samples are shown in Table 7.

[0092] Table 7

[0093]

[0094]

[0095] 4、According to the judgment of 5 in step one of example 2, determine whether the 5 test samples are positive or negative. The results of the 5 test samples are shown in Table 7. The results show that the method provided by the application can detect whether methylation occurs, and the minimum detection limit is 10% MetBisDNA.

[0096] Example 4, blood as a detection sample for colorectal cancer detection

[0097] 1、Dilute the upstream primers, downstream primers and probes of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB in Table 1 with water respectively.

[0098] 2、Extract 8ml of whole blood of 5 colorectal cancer patients (numbered as HX21-NGS014, HX21-NGS366, HX21-NGS374, HX21-NGS398 and HX21-NGS540) respectively into EDTA anticoagulant vacuum blood collection tubes, and centrifuge twice within 2h (first time 1600g for 15min, second time 15000g for 15min) to obtain cell-free plasma.

[0099] 3、Take the cell-free plasma respectively, and use the plasma free DNA centrifugation kit (D3182-03S, Megabiosciences, Guangzhou) to extract free DNA to obtain the cfDNA of the testee's plasma.

[0100] 4、Take the testee's plasma cfDNA respectively, and apply EZ DNA Methylation-Direct TM KIT (Cat. no. D5002, Zymo Research, USA) for bisulfite modification to obtain the converted cfDNA of the testee.

[0101] 5. Prepare the reaction system shown in Table 2 (total 25 μL), where the template is the transformed cfDNA from the test subject, positive control DNA, or negative control DNA; then perform PCR amplification according to the reaction program. The reaction program is as follows: Stage 1: 95℃ for 3 min, 1 cycle; Stage 2: 95℃ for 15 sec; 62℃ for 25 sec; 4 cycles; Stage 3: 95℃ for 15 sec; 58℃ for 25 sec; 41 cycles. During the third stage at 58℃, collect fluorescence signals and obtain the CT values ​​of ALX4, SFRP2, SEPT9, SDC2, TFPI2, and ACTB, respectively, and record them as CT. ALX4 CT scan SFRP2 CT scan SEPT9 CT scan SDC2 CT scan TFPI2 and CT ACTB If the amplification curve is not S-shaped or the CT value is blank, the CT value is recorded as 45. Further calculate the dCT values ​​(denoted as dCT) of ALX4, SFRP2, SEPT9, SDC2, or TFPI2. X ): dCT X =CT x -CT ACTB That is, the CT value obtained by subtracting the CT value of the internal control ACTB from the detected CT value.

[0102] The test results are shown in Table 8.

[0103] Table 8

[0104]

[0105] 6. Results of plasma cfDNA sample detection by real-time PCR.

[0106] (1) Based on the peripheral blood DNA, positive control DNA, and negative control DNA from colorectal cancer patients in Table 6, five-gene fluorescent PCR amplification was performed to generate CT values, which were then used to form various dCTs.

[0107] (2) Whether gene methylation has occurred is determined by comparing the dCT values ​​(denoted as dCT) of ALX4, SFRP2, SEPTIN9, SDC2, and TFPI2 in the test sample. X To achieve: dCT X =CT x -CT ACTBThe dCT critical value is an average statistical dCT value obtained by comparing the dCT values of a large number of cases of colon cancer tissues and cancer-adjacent normal tissues, i.e. a critical dCT value that can best distinguish tumors from non-tumors. The dCT critical value (i.e. threshold value) is statistically obtained from a sample group of peripheral blood of patients with pathologically confirmed colorectal cancer. If the dCT of the gene (ALX4 gene, SFRP2 gene, SEPT9 gene, SDC2 gene or TFPI2 gene) to be detected is ≤ the dCT critical value, it indicates that methylation of the gene occurs in the sample. The dCT critical values of ALX4, SFRP2, SEPT9, SDC2 and TFPI2 are 5, 5, 5, 5 and 5, respectively.

[0108] (3) The dCT of each gene obtained from the peripheral blood DNA of colorectal cancer patients and the dCT critical value are used to determine whether the peripheral blood sample is from a colorectal cancer patient by using a mathematical combination mode C5 2 The judgment criteria are as follows: if at least two of the ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes of the sample to be detected are methylated, the sample to be detected is positive, i.e. from a colorectal cancer patient; otherwise, the sample to be detected is negative, i.e. not from a colorectal cancer patient.

[0109] The detection results are shown in Table 8. At least two of the ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes of the peripheral blood of 5 colorectal cancer patients are methylated in 4 cases, i.e. the sensitivity reaches 80%. It can be seen that the detection of the ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes in peripheral blood can improve the detection rate of colorectal cancer.

[0110] It can be seen that the detection of plasma cfDNA can identify whether the subject to be detected is a colorectal cancer patient or a non-colorectal cancer patient.

[0111] The above describes the present application in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In summary, according to the principle of the present application, the present application intends to include any changes, uses or improvements of the present application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Application of combined biomarker detection reagents in the preparation of a multichannel fluorescence detection kit for colorectal cancer blood; The combined biomarker consists of the ALX4 gene, SFRP2 gene, SEPT9 gene, SDC2 gene, and TFPI2 gene. The ALX4 gene has a GeneBank entry number of 60529; The SFRP2 gene has a GeneBank address of 6423. The SEPT9 gene has a GeneBank address of 10801; The SDC2 gene has 6383 entries in GeneBank; The GeneBank index for the TFPI2 gene is 7980. The detection reagent for the combined biomarker includes primers and probes for detecting the methylation level of each gene in the combined biomarker; The primers used to detect the methylation level of each gene in the combined biomarker are ALX4-F shown in SEQ ID NO: 1, ALX4-R shown in SEQ ID NO: 2, SERP2-F shown in SEQ ID NO: 4, SERP2-R shown in SEQ ID NO: 5, SEPTIN9-F shown in SEQ ID NO: 7, SEPTIN9-R shown in SEQ ID NO: 8, SDC2-F shown in SEQ ID NO: 10, SDC2-R shown in SEQ ID NO: 11, TFPI2-F shown in SEQ ID NO: 13, and TFPI2-R shown in SEQ ID NO:

14. The probes used to detect the methylation level of each gene in the combined biomarker are ALX4-P shown in SEQ ID NO: 3, SERP2-P shown in SEQ ID NO: 6, SEPTIN9-P shown in SEQ ID NO: 9, SDC2-P shown in SEQ ID NO: 12 and TFPI2-P shown in SEQ ID NO:

15. The colorectal cancer blood multichannel fluorescence detection kit also includes an internal reference gene detection reagent; the internal reference gene is the ACTB gene; the GeneBank address of the ACTB gene is 60; the internal reference gene detection reagent includes primers and probes for detecting the internal reference gene; The kit also includes a data processing system; the data processing system converts the methylation levels of each gene in the combined biomarkers into the subject's dCT. X It is used to determine whether a person being tested has colorectal cancer. The subject's dCT X The calculation method is as follows: Genomic DNA from the plasma of the test subject is chemically modified, and then used as a template for fluorescent PCR amplification with primers and probes. Fluorescent signals are collected, and the CT values ​​of ALX4, SFRP2, SEPT9, SDC2, TFPI2, and ACTB are obtained, respectively, and denoted as CT. ALX4 CT scan SFRP2 CT scan SEPT9 CT scan SDC2 CT scan TFPI2 and CT ACTB If the amplification curve is not "S"-shaped or the CT value is blank, the CT value is recorded as 45; further calculate the dCT values ​​of each gene ALX4, SFRP2, SEPT9, SDC2, or TFPI2. X =CT x -CT ACTB ; The method for determination is as follows: if at least two of the ALX4, SFRP2, SEPT9, SDC2, and TFPI2 genes of the test subject are methylated, then the test subject is a colorectal cancer patient; otherwise, the test subject is not a colorectal cancer patient; whether a gene is methylated is determined by comparing the dCT and dCT threshold values ​​of the genes in the test sample. If the dCT of the ALX4, SFRP2, SEPT9, SDC2, or TFPI2 genes in the subject is less than or equal to the dCT threshold, then the subject has undergone methylation based on that gene. Under the given PCR reaction conditions, the dCT critical values ​​for ALX4, SFRP2, SEPT9, SDC2, and TFPI2 were 5, 5, 5, 5, and 5, respectively.

2. A multichannel fluorescence detection kit for colorectal cancer, comprising a detection reagent for the combined biomarker described in claim 1; the kit is used to detect plasma cfDNA or genomic DNA from colon tissue.

3. The reagent kit according to claim 2, characterized in that: The kit also includes an internal reference gene detection reagent.

4. The reagent kit according to claim 3, characterized in that: The internal reference gene is the ACTB gene; the GeneBank address of the ACTB gene is 60.

5. The reagent kit according to claim 3, characterized in that: The internal reference gene detection reagent includes primers and probes for detecting the internal reference gene; The primers for detecting the internal reference gene are ACTB-F (SEQ ID NO: 16) and ACTB-R (SEQ ID NO: 17), respectively. The probe for detecting the internal reference gene is ACTB-P, as shown in SEQ ID NO:

18.

6. The kit according to claim 2 or 5, characterized in that: Each probe has a fluorescent label at one end and a fluorescent quenching label at the other end.

7. The reagent kit according to claim 2, characterized in that: The kit also includes a data processing system; the data processing system converts the methylation levels of each gene in the combined biomarkers into the subject's dCT. X It is used to determine whether a person being tested has colorectal cancer. The subject's dCT X The calculation method is as follows: Genomic DNA from the plasma or colon tissue of the test subject is chemically modified, and then used as a template for fluorescent PCR amplification using the primers and probes described in claim 5. Fluorescent signals are collected, and the CT values ​​of ALX4, SFRP2, SEPT9, SDC2, TFPI2, and ACTB are obtained, respectively, and denoted as CT. ALX4 CT scan SFRP2 CT scan SEPT9 CT scan SDC2 CT scan TFPI2 and CT ACTB If the amplification curve is not "S"-shaped or the CT value is blank, the CT value is recorded as 45; further calculate the dCT values ​​of each gene ALX4, SFRP2, SEPT9, SDC2, or TFPI2. X =CT x -CT ACTB ; The method for determination is as follows: if at least two of the ALX4, SFRP2, SEPT9, SDC2, and TFPI2 genes of the test subject are methylated, then the test subject is a colorectal cancer patient; otherwise, the test subject is not a colorectal cancer patient; whether a gene is methylated is determined by comparing the dCT and dCT threshold values ​​of the genes in the test sample. If the dCT of the ALX4, SFRP2, SEPT9, SDC2, or TFPI2 genes in the subject is less than or equal to the dCT threshold, then the subject has undergone methylation based on that gene. The dCT threshold value for each gene is the average statistical value obtained by comparing the dCT values ​​of colon cancer tissue and adjacent normal tissue. It is a threshold value that can best distinguish between tumors and non-tumors.

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