CN116121387A - Combined marker for colorectal cancer detection and application thereof - Google Patents

Abstract

The invention discloses a combined marker for colorectal cancer detection and application thereof, wherein the combined marker consists of an ALX4 gene, an SFRP2 gene, an SEPT9 gene, an SDC2 gene and a TFPI2 gene. Experiments prove that the ALX4 gene, the SFRP2 gene, the SEPT9 gene, the SDC2 gene and the TFPI2 gene are used for diagnosing early colorectal cancer in a combined mode, and the kit is simple to operate, short in time consumption, high in sensitivity and specificity, and meanwhile, the detection rate can be effectively improved, and false positives of results are reduced. The invention has important application value.

Description

Combined marker for colorectal cancer detection and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a combined marker for colorectal cancer detection and application thereof.

Background

Colorectal cancer is a common malignancy in the gastrointestinal tract, with early symptoms not apparent. With the increase of cancer, the symptoms of stool habit change, hematochezia, diarrhea and constipation alternation, local abdominal pain and the like are presented, and the symptoms of anemia, weight loss and the like appear in the late stage. Colorectal cancer grows slowly with a long latency, 93% of colorectal cancers originate from adenomas, and 5-7 years are required for progression from adenomas to carcinomas. Studies have shown that the annual occult blood test can reduce colorectal cancer mortality by 33%. Although colorectal neoplasms are treatable, the early diagnosis rate is only 10-15%. At present, diagnosis and treatment of colorectal cancer still face serious problems such as difficult early diagnosis, high recurrence and metastasis rate, chemo-radiotherapy tolerance, lack of effective treatment targets and the like. Early discovery, early diagnosis and early treatment of colorectal cancer can effectively improve the treatment effect of colorectal cancer.

Most colorectal cancers begin with growth of the inner wall of the colon or rectum, which is known as polyps. Common polyp types are proliferative polyps and colorectal adenomatous polyps, the latter likely developing into cancer as they progress. Adenomas are therefore known as cancerous diseases. Intestinal adenoma can be screened by an endoscope, but the technical requirement on the inspector is higher, and the omission rate of clinical endoscope screening is higher. Thus, gene screening for adenomas is of great importance for the diagnosis and screening of early colorectal cancer.

Colorectal cancer takes 5-10 years to progress from initial polyps to colorectal cancer. The treatment cost is low and the effect is good; however, once the stage is broken through, the disease course is prolonged, the disease speed is increased, the treatment cost is high, and the effect is extremely poor. Colorectal cancer has no specific clinical manifestation in early stages, lacks high sensitivity and specific early diagnosis techniques, and most patients are already in late stages of cancer at the time of diagnosis, so that the optimal treatment opportunity is lost. Thus, early diagnosis and treatment are the best methods for managing colorectal cancer. At present, diagnosis of cancer mainly depends on imaging CT and MRI detection, but the benign and malignant properties of the cancer cannot be judged, and long-term image follow-up examination is needed. Pathological examination requires tumor tissue, often already in the middle and late stages. Early diagnosis of colorectal cancer has been a clinical difficulty, and electronic colonoscopy is a gold standard for diagnosing colorectal cancer, which can resect and biopsy diseased parts, but is not suitable for early screening; CA199 may be used for colorectal cancer screening, but it has poor specificity and sensitivity. The tumor markers can be detected in body fluid or tissues, and can reflect the existence, differentiation degree, prognosis estimation, treatment effect judgment and the like of tumors. Compared with tissue biopsy, the colon lavage fluid, blood plasma, sputum and other samples are easy to obtain, the sample is not wounded to the testee, and can be continuously sampled for visit comparison. However, markers in serum such as CEA, CA125, CA199, etc. are currently not only low in sensitivity but also lack in specificity. Thus, there is an urgent need to develop new colorectal cancer early screening biomarkers.

It has been found that almost all tumors undergo DNA methylation, an early event of tumorigenesis, which can be detected prior to clinical diagnosis of the disease, is a potentially reliable early screening indicator for early diagnosis of the tumor, risk prediction, clinical course monitoring and efficacy evaluation, and is associated with the activation of the demethylation of cancerous early oncogenes and the inactivation of oncogenes due to methylation of genes in normal cells; the sensitivity is better than that of the existing protein serum marker, and the protein serum marker has more specificity than that of the serum marker. As a new molecular marker, DNA methylation has received increasing attention in tumor diagnosis, and its advantages include: (1) Promoter hypermethylation frequently occurs during neoplasia, even above gene mutation, where there are many important genes involved in neoplasia; (2) methylation is an important event in the early stages of tumorigenesis; (3) DNA methylation is stable and can be detected by PCR amplification effects. Therefore, methylation detection has potential application value in early diagnosis of tumors.

Common methods for detecting DNA methylation include: methylation-specific PCR (MSP), sulfite sequencing (Bisulfite sequencing PCR, BSP), high resolution melting curve (High Resolution Melting, HRM), direct genome sequencing, and the like.

Disclosure of Invention

The object of the present invention is to diagnose colorectal cancer early.

The invention firstly protects the application of the combined marker in preparing colorectal cancer detection kit;

the combined marker can consist of an ALX4 gene, an SFRP2 gene, an SEPT9 gene, an SDC2 gene and a TFPI2 gene;

the GeneBank of the ALX4 gene is 60529;

the GeneBank of the SFRP2 gene is 6423;

the GeneBank of the SEPT9 gene is 10801;

the GeneBank of the SDC2 gene is 6383;

the GeneBank of the TFPI2 gene is 7980.

The invention also provides a colorectal cancer detection kit, which comprises the detection reagent of the combined marker.

The kit can specifically consist of a detection reagent for the combined marker.

In any of the above kits, the detection reagent for the combination marker may comprise primers and/or probes for detecting the methylation level of each gene in the combination marker.

In any of the above kits, the detection reagent for the combined marker may specifically consist of a primer and/or a probe for detecting the methylation level of each gene in the combined marker.

The primers used to detect the methylation level of each gene in the combination marker described above may be SEQ ID NO:1, ALX4-F, SEQ ID NO:2, ALX4-R, SEQ ID NO:4, SERP2-F, SEQ ID NO:5, SERP2-R, SEQ ID NO:7, SEPTIN9-F, SEQ ID NO:8, SEPTIN9-R, SEQ ID NO:10, SDC2-F, SEQ ID NO:11, SDC2-R, SEQ ID NO:13 and TFPI2-F shown in SEQ ID NO:14 TFPI2-R.

The probe for detecting methylation level of each gene in the combined marker may be any one of SEQ ID NOs: 3, ALX4-P, SEQ ID NO:6, SERP2-P, SEQ ID NO:9, SEPTIN9-P, SEQ ID NO:12 and SDC2-P shown in SEQ ID NO:15 TFPI2-P.

Any of the above kits may further comprise a reference gene detection reagent. The reference gene may be an ACTB gene. The GeneBank of the ACTB gene is 60.

The kit can specifically comprise the detection reagent of any one of the combined markers and the reference gene detection reagent.

Any of the above-mentioned reference gene detection reagents includes a primer and/or a probe for detecting a reference gene.

Any of the above-mentioned reference gene detection reagents may specifically be composed of a primer and/or a probe for detecting a reference gene.

The primer for detecting the reference gene is respectively shown in SEQ ID NO:16 and ACTB-F shown in SEQ ID NO: ACTB-R as shown in FIG. 17.

Any of the probes for detecting the reference gene is SEQ ID NO:18 ACTB-P.

One end of any of the probes (such as a probe for detecting a reference gene and a probe for detecting the methylation level of each gene in the combined marker) is provided with a fluorescent label, and the other end is provided with a fluorescence quenching label.

The detection object of any of the above kits may specifically be blood plasma cfDNA, fecal cfDNA or genomic DNA of colon tissue.

Any of the above kits may further comprise a data processing system; the data processing system converts the methylation level of each gene in the combined marker to dCT of the subject _X For determining whether the subject is a colorectal cancer patient;

dCT of the subject _X The calculation method of (1) is as follows: chemically modifying blood plasma, fecal cfDNA or colon tissue genome DNA of a subject (such as bisulphite modification), performing fluorescent PCR amplification with the primers and probes as described above, collecting fluorescent signals, and respectively obtaining CT values of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB, which are sequentially recorded as CT _ALX4 、CT _SFRP2 、CT _SEPT9 、CT _SDC2 、CT _TFPI2 And CT _ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not of the "S" type or the CT value is blank, the CT value is marked as 45; further calculating dCT values of the respective genes ALX4, SFRP2, SEPT9, SDC2 or TFPI2, dCT _X ＝CT _x -CT _ACTB ；

The judging method comprises the following steps: if at least two of the ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes of the subject are methylated, the subject is a colorectal cancer patient; otherwise the subject is not a colorectal cancer patient; whether the gene is methylated or not is achieved by comparing the dCT and dCT critical values of the genes of the sample to be tested;

if dCT of the ALX4 gene, the SFRP2 gene, the SEPT9 gene, the SDC2 gene or the TFPI2 gene of the testee is less than or equal to dCT critical value, methylation is carried out on the basis of the genes of the testee;

the threshold value of each gene dCT is an average statistical value obtained by comparing dCT values of colon cancer tissues and paracancerous normal tissues, and can maximally distinguish one threshold value of tumor and non-tumor (for example, statistics that the intermediate value of all verified colorectal cancer positive and negative samples at the dCT value of each target gene is the dCT threshold value, the methylation region detection Ct value of the gene is less than or equal to the threshold value and is positive, and the methylation region detection Ct value of the gene is more than or equal to the threshold value and is negative).

Experiments prove that the ALX4 gene, the SFRP2 gene, the SEPT9 gene, the SDC2 gene and the TFPI2 gene in colon cancer tissues can be used for diagnosing early colorectal cancer, and have the advantages of simple operation, short time consumption, higher sensitivity and specificity, and capability of effectively improving the detection rate and reducing false positives of results. The invention has important application value.

Drawings

FIG. 1 shows the detection results of the ROC method in example 2.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

In the following examples, "sample to be tested" refers to a nucleic acid sample to be tested; specifically, the sample to be tested may be isolated blood cells, one or more of the cells isolated from blood, cell lines, tissue sections, biopsies, paraffin-embedded tissues, body fluids, feces, urine, plasma, serum, whole blood, and the like gDNA, cfDNA, and ctDNA.

In the examples below, the "target nucleic acid" refers to nucleic acid fragments of five colorectal cancer-associated genes, namely, methylated DNA-specific fragments of the human ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes.

In the following examples, a "probe" refers to a single-stranded nucleic acid having a known nucleotide sequence whose nucleotide sequence structure is substantially complementary to a target nucleic acid, and which can form a double strand with the "target nucleic acid". The 5 'end of the probe may carry a fluorophore and/or the 3' end may carry a quencher label. The binding of the primers and probes to methylation specific sequences in the sample DNA allows the molecular markers to detect disease colorectal cancer.

In the method of the present invention, chemical modification of the extracted DNA is required, and bisulphite, bisulfite or hydrazine salt modification can be applied to such chemical modification.

In view of the clinical testing needs, the liquid biopsy method can effectively reduce the damage to patients. When the real-time fluorescence PCR is used for detection, the probe is connected with a fluorescent group suitable for judging methylated DNA fragments of different genes. One end of the probe is marked with a fluorescent group, and the other end is marked with a quenching group; wherein the quenching group quenches fluorescence emitted by the fluorescent group. When the PCR amplification reaction is carried out, the forward exo-activity of the polymerase is utilized to cut off the base with the fluorescent group, the free fluorescent group is not influenced by the quenching group any more, and a fluorescent signal with a certain wavelength can be emitted under the action of the excitation light. As PCR products accumulate, the fluorescent signal increases continuously, so that the presence of specifically methylated DNA can be detected. As a preferred mode of the invention, 5 specific probes marked by 5 different fluorophores are added into the same reaction tube for detection, and the presence of 5 target methylated DNA fragments is indicated simultaneously in the same reaction tube, wherein the specific probes correspond to ALX4, SFRP2, SEPT9, SDC2, TFPI2 genes and internal reference gene ACTB respectively. As a preferred mode of the present invention, the fluorescent group labeled with the detection probe may be VIC, ROX, FAM, cy, cy5.5, VIC, HEX, TET, JOE, NED, TAMRA or the like; and the quenching group may be BHQ, MGB or Dabcy1. The invention is suitable for the commonly used multichannel PCR detection technology of the clinical detection at present, and realizes multichannel fluorescence detection in one reaction tube.

In the examples below, all patients had informed consent and the experiment was ethically approved.

In the examples below, a human whole-gene methylated DNA standard (EpiTect PCR Control DNA Set, qiagen CatNo./ID:59695, where methylated DNA is expressed as +ve ctr) was used as positive control DNA. Human whole-gene unmethylated DNA standard (EpiTect PCR Control DNA Set, qiagen CatNo./ID:59695, where unmethylated DNA is denoted by-ve ctr) or water was negative control DNA.

Example 1 obtaining markers for colorectal cancer detection

The inventor of the invention combines various databases and comprehensive clinical information, designs methylation sites of related genes by large-scale screening, uses operation tissues (proved by pathology, C) of colorectal cancer patients as tumor positive samples, and uses cancer side normal tissues (proved by pathology, A) as tumor negative samples during operation of the same case. Markers for colorectal cancer detection were obtained by comparison of differences in methylation levels. Markers for colorectal cancer detection consisted of ALX4 (GeneID: 60529), SFRP2 (GeneID: 6423), SEPT9 (GeneID: 10801), SDC2 (GeneID: 6383), TFPI2 (GeneID: 7980) and ACTB (GeneID: 60).

The primers and probes shown in Table 1 were designed and synthesized, respectively, from the nucleotide sequences of the above-mentioned respective genes by Nanjin Style biotechnology Co.

TABLE 1

Note that: the primer name contains "F" as the upstream primer, "R" as the downstream primer, and "P" as the probe; CY5.5 represents a CY5.5 label; VIC represents a VIC marker; ROX represents a ROX marker; FAM represents FAM markers; TAMRA represents a TAMRA marker; CY5 represents a CY5 label; MGB represents a fluorescence quenching label; ACTB (GeneID: 60) is a reference gene.

Example 2 detection of Gene methylation markers in colorectal cancer (A) and paracancerous Normal tissue (C)

1. The upstream primer, downstream primer and probe of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB in Table 1 were diluted with water, respectively.

2. Samples to be tested (20 cases of colorectal cancer tissues to be tested (represented by C) and paracancerous normal tissues (represented by A), 3 cases of human colorectal cancer cell lines (HT 29 cells (Shanghai Fu He Jib Co., FH 0024), HCT8 cells (Shanghai Fu He Co., FH 0025) and HCT116 cells (Shanghai Fu He Co., FH 0027)) were taken for homogenization, and then genomic DNA was extracted using a blood/cell/tissue genomic DNA extraction kit (Beijing Tiangen Biochemical technology (Beijing) Co., cat. # DP 304-03) to obtain genomic DNA of the samples to be tested.

3. Taking genomic DNA of a sample to be tested, and adopting EZ DNAMethylation-Direct ^TM KIT (ZYMO RESEARCH, D5001/D5002) is bisulphite modified to obtain the DNA transformed by the tester.

4. Preparing a reaction system (25. Mu.L in total) shown in Table 2, wherein the template is DNA transformed by a subject, positive control DNA, negative control DNA or blank control; fluorescent PCR amplification was then performed according to the reaction procedure. The reaction procedure is: the first stage: 3min at 95 ℃ for 1 cycle; and a second stage: 15sec at 95 ℃; 25sec at 62 ℃;4 cycles; and a third stage: 15sec at 95 ℃; 25sec at 58 ℃;41 cycles. Collecting fluorescence signals at 58 deg.C at the third stage to obtain CT values of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB, respectively, which are sequentially recorded as CT _ALX4 、CT _SFRP2 、CT _SEPT9 、CT _SDC2 、CT _TFPI2 And CT _ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not "S" shaped or the CT value is blank, the CT value is noted as 45.

TABLE 2

Note that: DNase was Accurate Tag HS DNA polymerase (CM 0008,5u/ul, AGL Bio Inc., china).

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

TABLE 3 Table 3

The CT values of the results are shown in Table 4, and dCT of each gene is further calculated and recorded as dCT _X (dCT _X ＝CT _X -CT _ACTB )。

TABLE 4-1

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

TABLE 4-2

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5. Gene methylation markers in colorectal cancer tissue DNA determination results

(1) Colorectal cancer tissue (C), human colorectal cancer cell lines HCT-8, HT-29 and HCT-116 were gene methylation positive DNA according to Table 4. The paracancerous normal tissue (A) is gene methylation negative DNA. The BS-treated DNA was amplified by five-gene fluorescent PCR. The CT value obtained by detection is the dCT value of the gene after the CT value of the internal control ACTB is subtracted. The positive sample group dCT values are formed by the colorectal cancer tissues and positive control DNA confirmed by pathology, and the negative sample group dCT is formed by the paracancerous normal tissues and negative control DNA.

(2) Whether the gene is methylated or not is determined by comparing the dCT values of the test samples ALX4, SFRP2, SEPTIN9, SDC2 and TFPI2 (dCT _X ) To realize: dCT _X ＝CT _x -CT _ACTB . Namely, the CT value obtained by the detection is subtracted from the CT value of ACTB, and if the dCT of the ALX4 gene, the SFRP2 gene, the SEPT9 gene, the SDC2 gene or the TFPI2 gene of the tested person is less than or equal to dCT critical value, methylation occurs to the tested person based on the gene. Whereas the threshold value of each gene dCT is a value of dCT which is an average statistic after dCT of colon cancer tissue and paracancerous normal tissue confirmed by a relatively large number of cases, i.e., a threshold value (threshold value), which is a threshold dCT value capable of maximally distinguishing between tumor and non-tumor.

The results showed that the dCT thresholds for ALX4, SFRP2, SEPT9, SDC2 and TFPI2 were 5, 5 and 5, respectively, under the composed PCR reaction conditions.

(3) Determination of whether the sample is from colorectal cancer patient is made by comparing dCT of each gene in the sample to the threshold comparison set and method C ₅ ² And (3) determining: namely, if the dCT value of at least two genes among the five genes of ALX4, SFRP2, SEPT9, SDC2 and TFPI2 of the sample to be tested is smaller than or equal to a threshold value, which indicates that the sample is methylated, the sample to be tested is positive, namely, the sample to be tested is from a colorectal cancer patient; otherwise, the sample to be tested is negative, i.e. not from colorectal cancer patients.

dCT the results are shown in Table 5, and 16 cases of methylation of at least two genes among ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes of colorectal cancer tissues of 20 colorectal cancer patients; whereas 19 of the paracancerous normal tissues in 20 colorectal patients were unmethylated. Thus, the detection rate of colorectal cancer can be improved by cooperatively detecting ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes in colon tissues.

TABLE 5

It follows that by detecting genomic DNA of colon tissue it is possible to identify whether the subject is a colorectal cancer patient or a non-colorectal cancer patient.

6. 79 colorectal cancer tissue samples and 79 normal colon tissue samples were used as samples to be tested according to the above method.

The detection results are shown in Table 6. The results showed that the single gene markers showed higher detection rates, SFRP2 (86%), SEPTIN9 (85%) and TFPI2 (84%) in sequence. The ALX4 gene showed the lowest detection rate but the highest specificity (98%). The methylation of the samples was determined in a combinatorial fashion as whether or not the methylation was from a tumor, with a detection rate of 91% and a specificity of 89%.

TABLE 6

The result analysis is carried out by the ROC method, and the detection results are shown in the graph 1, and the curve and the area under the curve (AUC) show that five gene methylation markers in colon cancer tissues have higher value for diagnosing early lung cancer, wherein the AUC of SEPTIN9 is as high as 0.905. The detection rate of the method was 91%, the specificity was 89% and the AUC was 0.89, as determined by combination. The 5 gene markers cover multiple mechanisms of canceration, so that the methylation positive rate or the detection rate of a detection sample is improved, and the five-choice 2 method in a combined mode reduces false positive and improves the detection authenticity.

Example 3 sensitivity experiment

The samples to be tested were 100% MetBisDNA (100% + ve ctr DNA), 10% MetBisDNA (10% + ve ctr DNA plus 90% -ve ctr DNA), 1% MetBisDNA (1% + ve ctr DNA plus 99% -ve ctr DNA), 0% MetBisDNA (100% -ve ctr DNA) and TE (no DNA, only TE buffer), respectively.

Each sample to be tested was subjected to the following experiment:

1. the upstream primer, downstream primer and probe of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB in Table 1 were diluted with water, respectively.

2. Taking a sample to be tested, adopting EZ DNAMethylation-Direct ^TM And (3) carrying out bisulphite modification on the KIT to obtain DNA converted by the sample to be detected.

3. Preparing a reaction system (total 25 mu L) shown in Table 2, wherein the template is DNA converted by a sample to be tested, positive control DNA or negative control DNA; PCR amplification was then performed according to the reaction procedure. The reaction procedure is: the first stage: 3min at 95 ℃ for 1 cycle; and a second stage: 15sec at 95 ℃; 25sec at 62 ℃;4 cycles; and a third stage: 15sec at 95 ℃; 25sec at 58 ℃;41 cycles. Collecting fluorescence signals at 58 deg.C at the third stage to obtain CT values of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB, respectively, which are sequentially recorded as CT _ALX4 、CT _SFRP2 、CT _SEPT9 、CT _SDC2 、CT _TFPI2 And CT _ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not "S" shaped or the CT value is blank, the CT value is noted as 45. Further calculation of the dCT value (indicated as dCT) of each gene ALX4, SFRP2, SEPT9, SDC2 or TFPI2 _X )，dCT _X ＝CT _x -CT _ACTB 。

The results of the 5 dilutions are shown in table 7.

TABLE 7

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4. According to step one 5 of example 2, it is determined whether 5 samples to be tested are positive or negative. The results of 5 samples to be tested are shown in Table 7. The results show that the method provided by the invention can detect whether methylation occurs or not, and the minimum detection limit is 10% of MetBisDNA.

Example 4 colorectal cancer detection Using blood as detection sample

1. The upstream primer, downstream primer and probe of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB in Table 1 were diluted with water, respectively.

2. 8ml whole blood from 5 rectal cancer patients (HX 21-NGS014, HX21-NGS366, HX21-NGS374, HX21-NGS398 and HX21-NGS540, respectively) were separately extracted from EDTA anticoagulation vacuum blood collection tubes, and subjected to two centrifugation cycles (first 1600g centrifugation for 15min and second 15000g centrifugation for 15 min) over 2 hours to obtain cell-free plasma.

3. And (3) respectively taking the cell-free plasma, and extracting free DNA by using a plasma free DNA centrifugation kit (D3182-03S, meyer' S patches) to obtain cfDNA of the plasma of the person to be tested.

4. Respectively taking cfDNA of blood plasma of a person to be tested, and applying EZ DNAMethylation-Direct ^TM KIT (Cat.no.D5002, zymo Research, USA) was bisulphite modified to obtain cfDNA transformed by the tester.

5. Preparing a reaction system (total 25 μl) shown in table 2, wherein the template is cfDNA, positive control DNA or negative control DNA transformed by the tester; PCR amplification was then performed according to the reaction procedure. The reaction procedure is: the first stage: 3min at 95 ℃ for 1 cycle; and a second stage: 15sec at 95 ℃; 25sec at 62 ℃;4 cycles; and a third stage: 15sec at 95 ℃; 25sec at 58 ℃;41 cycles. Collecting fluorescence signals at 58 deg.C at the third stage to obtain CT values of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB, respectively, which are sequentially recorded as CT _ALX4 、CT _SFRP2 、CT _SEPT9 、CT _SDC2 、CT _TFPI2 And CT _ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not "S" shaped or the CT value is blank, the CT value is noted as 45. Further calculate the dCT value of ALX4, SFRP2, SEPT9, SDC2 or TFPI2 (noted dCT _X )：dCT _X ＝CT _x -CT _ACTB I.e. the CT value obtained by the detection is subtracted from the CT value of the internal control ACTB.

The detection results are shown in Table 8.

TABLE 8

6. The cfDNA sample of plasma was detected in fluorescent quantitative PCR.

(1) Five-gene fluorescent PCR amplification of peripheral blood DNA, positive control DNA and negative control DNA from colorectal cancer patients according to Table 6 resulted in CT values to form each dCT. .

(2) Whether the gene is methylated or not is determined by comparing the dCT values of the test samples ALX4, SFRP2, SEPTIN9, SDC2 and TFPI2 (dCT _X ) To realize: dCT _X ＝CT _x -CT _ACTB . Whereas the gene dCT threshold is a value of dCT which is an average statistic after dCT of colon cancer tissue and paracancerous normal tissue confirmed by a relatively large number of cases, i.e., a threshold value (threshold value), which is a threshold dCT value capable of maximally distinguishing between tumor and non-tumor. A dCT threshold (i.e., threshold) was counted in a sample group consisting of peripheral blood from pathologically confirmed colorectal cancer. If dCT.ltoreq. dCT of the sample gene to be tested (ALX 4 gene, SFRP2 gene, SEPT9 gene, SDC2 gene or TFPI2 gene) indicates that methylation of the gene occurs in the sample. The dCT thresholds for ALX4, SFRP2, SEPT9, SDC2 and TFPI2 are 5, 5 and 5, respectively.

(3) The dCT and dCT threshold values obtained by each gene of the peripheral blood DNA of colorectal cancer patients are obtained by using a mathematical combination mode C ₅ ² To determine if the peripheral blood sample is from a colorectal cancer patient. The judgment criteria are as follows: if at least two genes among the ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes of the sample to be tested are methylated, the sample to be tested is positive, namely the sample is from colorectal cancer patients; otherwise, the sample to be tested is negative, i.e. not from colorectal cancer patients.

The test results are shown in Table 8, and the methylation of at least two genes among ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes in peripheral blood of 5 colorectal cancer patients is 4, namely 80% sensitivity is achieved. Thus, the detection rate of colorectal cancer can be improved by cooperatively detecting ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes in peripheral blood.

It follows that by detection of the plasma cfDNA it is possible to identify whether the subject is a colorectal cancer patient or a non-colorectal cancer patient.

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Claims (10)

1. The application of the combined marker in preparing a colorectal cancer detection kit;

the combined marker consists of an ALX4 gene, an SFRP2 gene, an SEPT9 gene, an SDC2 gene and a TFPI2 gene;

the GeneBank of the ALX4 gene is 60529;

the GeneBank of the SFRP2 gene is 6423;

the GeneBank of the SEPT9 gene is 10801;

the GeneBank of the SDC2 gene is 6383;

the GeneBank of the TFPI2 gene is 7980.

2. A colorectal cancer detection kit comprising the detection reagent of the combination marker of claim 1.

3. The kit of claim 2, wherein: the detection reagent of the combined marker comprises a primer and/or a probe for detecting the methylation level of each gene in the combined marker.

4. A kit according to claim 3, wherein: the primers for detecting the methylation level of each gene in the combined marker are SEQ ID NO:1, ALX4-F, SEQ ID NO:2, ALX4-R, SEQ ID NO:4, SERP2-F, SEQ ID NO:5, SERP2-R, SEQ ID NO:7, SEPTIN9-F, SEQ ID NO:8, SEPTIN9-R, SEQ ID NO:10, SDC2-F, SEQ ID NO:11, SDC2-R, SEQ ID NO:13 and TFPI2-F shown in SEQ ID NO: TFPI2-R shown at 14;

the methylation level probes for detecting the genes in the combined marker are SEQ ID NO:3, ALX4-P, SEQ ID NO:6, SERP2-P, SEQ ID NO:9, SEPTIN9-P, SEQ ID NO:12 and SDC2-P shown in SEQ ID NO:15 TFPI2-P.

5. The kit of claim 2, wherein: the kit also comprises an internal reference gene detection reagent.

6. The kit of claim 5, wherein: the reference gene is ACTB gene; the GeneBank of the ACTB gene is 60.

7. The kit of claim 5, wherein: the reference gene detection reagent comprises a primer and/or a probe for detecting a reference gene;

the primers for detecting the reference genes are SEQ ID NO:16 and ACTB-F shown in SEQ ID NO: ACTB-R shown in 17;

the probe for detecting the reference gene is SEQ ID NO:18 ACTB-P.

8. The kit of claim 4 or 7, wherein: one end of the probe is provided with a fluorescent label, and the other end is provided with a fluorescence quenching label.

9. The kit of claim 2, wherein: the detection object of the kit is blood plasma cfDNA, fecal cfDNA or colon tissue genome DNA.

10. According to claimKit according to any one of claims 2 to 9, characterized in that: the kit further comprises a data processing system; the data processing system converts the methylation level of each gene in the combined marker to dCT of the subject _X For determining whether the subject is a colorectal cancer patient;

dCT of the subject _X The calculation method of (1) is as follows: chemically modifying the plasma, fecal cfDNA or genomic DNA of colon tissue of a subject, performing fluorescent PCR amplification using the primers and probes of claim 3 or 7 as templates, collecting fluorescent signals to obtain CT values of ALX4, SFRP2, SEPT9, SDC2, TFPI2 and ACTB, respectively, and sequentially recording as CT _ALX4 、CT _SFRP2 、CT _SEPT9 、CT _SDC2 、CT _TFPI2 And CT _ACTB The method comprises the steps of carrying out a first treatment on the surface of the If the amplification curve is not of the "S" type or the CT value is blank, the CT value is marked as 45; further calculating dCT values of the respective genes ALX4, SFRP2, SEPT9, SDC2 or TFPI2, dCT _X ＝CT _x -CT _ACTB ；

The judging method comprises the following steps: if at least two of the ALX4, SFRP2, SEPT9, SDC2 and TFPI2 genes of the subject are methylated, the subject is a colorectal cancer patient; otherwise the subject is not a colorectal cancer patient; whether the gene is methylated or not is achieved by comparing the dCT and dCT critical values of the genes of the sample to be tested;

if dCT of the ALX4 gene, the SFRP2 gene, the SEPT9 gene, the SDC2 gene or the TFPI2 gene of the testee is less than or equal to dCT critical value, methylation is carried out on the basis of the genes of the testee;

the threshold value of each gene dCT is an average statistical value obtained by comparing dCT values of colon cancer tissue and paracancerous normal tissue, and is a threshold value capable of maximally distinguishing between tumor and non-tumor.

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