CN112538529B

CN112538529B - Molecular marker for colorectal cancer in blood and detection kit and method thereof

Info

Publication number: CN112538529B
Application number: CN201911114321.3A
Authority: CN
Inventors: 许林浩; 王洪; 陈志伟; 范建兵
Original assignee: AnchorDx Medical Co Ltd
Current assignee: AnchorDx Medical Co Ltd
Priority date: 2019-11-14
Filing date: 2019-11-14
Publication date: 2021-11-30
Anticipated expiration: 2039-11-14
Also published as: CN112538529A

Abstract

The invention relates to a molecular marker for detecting colorectal cancer, a kit and a detection method, wherein the kit comprises a primer and a probe for detecting methylation of at least one gene in 20 genes, namely three pairs of the primer and three probes are respectively designed for the molecular markers ST8SIA4, ZNF132, TAF3, Twist1, VAV3-AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12, THBD, ZNF304, EVC, S1PR1, ZFHX4-AS1, EMID1, FAM19A4 and NEUROD 1. The detection method comprises multiplex PCR and qPCR quantitative detection. The sensitivity and specificity of the kit and the detection method of the invention meet the clinical requirements, and for the detection schemes of the plurality of markers, the colorectal cancer diagnosis at stage 1 achieves 90% of specificity and 83% of sensitivity.

Description

Molecular marker for colorectal cancer in blood and detection kit and method thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a molecular marker for colorectal cancer in blood, and a detection kit and a detection method thereof.

Background

There are currently several techniques in common use for colorectal cancer screening. One is fecal occult blood test. Usually, the development of intestinal cancer is not accompanied by any signs in the early stage, cancer cells can grow on the wall of large intestine for decades and then metastasize to other parts, and before any symptoms, the hyperplastic tissue can exude a small amount of blood, which enters the stool and is excreted. The fecal occult blood test is to detect blood components (hemoglobin is the detection object) in the stool, and if multiple and continuous positive reactions suggest gastrointestinal hemorrhage, further examination should be made to alert the occurrence of intestinal tumor. The main advantages of the fecal occult blood test are that the result is displayed quickly and clearly, and the result can be semi-quantitative. It has a series of disadvantages: first, it has poor specificity and is greatly affected by diet. Foods and drugs containing ferrous ions interfere with the outcome with a false positive rate of 30%. Before occult blood examination, the patient is instructed to avoid taking iron, animal blood, liver, lean meat and a large amount of green vegetables for 3 days, if gum bleeding exists, bloody saliva is swallowed by mistake, so as to prevent the excrement occult blood examination from being false positive; secondly, the sensitivity is low, and the bleeding amount is more than 90 mug/ml for detection; again, this methodology is subject to multiple constraints: the preparation time of a patient is long in advance, and because the color development is judged differently due to different material taking parts and different reaction times, errors can be generated in the test of the same method, so that the patient is required to adopt excrement samples of different days for continuous measurement in common test.

Another method is enteroscopy. Enteroscopy is currently the most effective and reliable diagnostic method for the diagnosis of intestinal lesions. The vast majority of early bowel cancer patients can be discovered and diagnosed by endoscopic examination. It can be examined by insertion through the anus into the rectum, sigmoid colon, descending colon, transverse colon, ascending colon and cecum, as well as a small segment of small intestine (ileocecal end) connected to the large intestine. Not only intestinal lesions can be clearly found through the mirror, but also partial intestinal lesions can be treated, such as: directly remove benign lesions such as large intestine polyp under the microscope, stop bleeding of intestinal tract bleeding under the microscope, and remove foreign matters in the large intestine. The enteroscopy technology is a main means which cannot be replaced by other diagnosis and treatment means at present. Enteroscopy is currently the most effective and reliable diagnostic method for the diagnosis of intestinal lesions. Enteroscopy, although the current gold standard for colorectal cancer, has its own drawbacks: first, it requires preparation before examination, enteroscopy requires starting with a liquid or low-sediment semi-liquid diet 3 days before examination, starting with a cathartic such as mannitol the day before examination to cleanse the intestine, usually up to 2L, on the morning of the examination, and cleansing the enema to ensure the cleanliness of the intestine, after which no food can be taken. During examination, a doctor injects a certain amount of gas into the intestinal cavity through the enteroscope to expand the intestinal tract so as to facilitate observation. Due to the tortuous colon structure, the examined person will have different degrees of distending pain or traction feeling during the examination. In subjects with excessive stress or high intestinal spasticity, sedatives or spasmolytic drugs are required. For uncooperative children, it is necessary to perform under anesthesia. Secondly, it has very high requirement to the operation proficiency level of doctor, and the beginner easily misses the pathological change position under the intestines mirror and causes the missed diagnosis. The need to inject gas during the examination increases the pressure in the intestine, which tends to cause perforation. Thirdly, the patient is laboursome, time-consuming and painful in the examination process. And the inspection cost is high, and the large-scale popularization is difficult.

In addition to the above two methods, molecular diagnostic methods are cancer diagnostic methods that have been rapidly developed in recent years. At present, a large number of molecular markers for colorectal cancer have been studied and reported in tissue and blood samples. Related products based on molecular detection are applied to clinic, and the products are mainly divided into two types according to different samples at present, wherein one type is based on DNA in excrement for detection, for example, Cologuard in foreign countries and Colosafe in domestic countries are both aimed at screening and auxiliary diagnosis of colorectal cancer of DNA in excrement. Another class is based on DNA in blood, for example Epicolon from Epigenomics, abroad.

The colorectal cancer screening and diagnosing product based on molecular level firstly has the most important point for patients to be detected non-invasively and has very high safety, and the low compliance of gold standard enteroscopy is a very good auxiliary scheme; the other characteristic is that the screening diagnosis product based on molecular level has good performance in both sensitivity and specificity, and the fecal occult blood detection is good in this respect; in addition, it is important that the molecular level-based screening and diagnosis product basically has no more requirements for patients, does not need special diet contraindications, does not need special preparation during detection, and is very convenient for the patients. However, these methods also have problems: in the case of a fecal sample, a plurality of enzymes exist in the feces, which can digest and degrade DNA in the feces, and in addition, components in the feces are complex and too many interfering components, which puts high requirements on sample treatment, and improper sample treatment easily affects downstream detection. For the detection of blood samples, the extraction of DNA and the purity of DNA are guaranteed to a certain extent, but the content of cfDNA (Cell-free DNA) in blood plasma is far lower than that of human DNA in feces, and particularly for early tumor samples, the content of cfDNA is lower, on the other hand, compared with the DNA of exfoliated cells in feces, the integrity of cfDNA in blood plasma is worse, and the development of corresponding detection is more difficult. With respect to such characteristics of cfDNA, the only product (Epicolon) on the market today detects only one marker (marker), but there is room for improvement in sensitivity and specificity of a single marker, especially for detection of early stage tumors. On the other hand, for the characteristics of the cfDNA, the second-generation sequencing platform is more utilized for cfDNA detection, the second-generation sequencing can sensitively detect trace cfDNA, and meanwhile, the second-generation sequencing is a high-throughput detection scheme, no matter for the marker or the number of samples. However, products developed based on the second-generation sequencing platform also have certain limitations, namely, the first is that the detection period is long, and the time period is long from the extraction of sample DNA to the on-machine sequencing and the report analysis of data. On the other hand, the cost of the second generation sequencing platform is high. The two points have certain limitations for the popularization and application of products, particularly on the screening and diagnosis of early tumors. Therefore, the development of a noninvasive detection product for the plasma cfDNA has the characteristics of high sensitivity, rapidness and low cost, and is of great significance for early clinical screening and diagnosis.

However, the cfDNA content is lower for early tumor samples, which is a great challenge for developing a detection method that satisfies the multi-markers flux and requires a detection means with sufficiently good sensitivity, and there is a certain technical challenge in terms of system optimization and stability.

Disclosure of Invention

It is an object of the present invention to provide a class of markers for detecting colorectal cancer in blood.

The technical scheme for achieving the purpose is as follows.

A molecular marker related to colorectal cancer in blood comprises any one or a combination of more than two of FBN1, SFMBT2, FAM72C, ITGA4, ZNF132 and KCNJ 12.

In one embodiment, FBN1, SFMBT2, FAM72C, and ITGA4 are included.

In one embodiment, the kit further comprises at least one of the following molecular markers: ST8SIA4, TAF3, TWIST1, VAV3-AS1, C9orf50, KCNQ5, THBD, ZNF304, EVC, S1PR1, ZFH 4-AS1, EMID1, FAM19A4, NEUROD.

In one embodiment, the molecular marker for detecting colorectal cancer in blood is a combination of TWIST1, VAV3-AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12, ZNF 132.

In one embodiment, the molecular marker is a combination of TWIST1, C9orf50, KCNJ12, ZNF 132.

In one embodiment, the molecular marker is a combination of FBN1, SFMBT2, FAM72C, ITGA4, ZNF132, KCNJ12, ST8SIA4, TAF3, TWIST1, VAV3-AS1, C9orf50, KCNQ5, THBD, ZNF304, EVC, S1PR1, zf 4-AS1, EMID1, FAM19a4, neuod.

In another aspect of the present invention, there is provided the use of the above molecular marker in the preparation of a kit for detecting colorectal cancer in blood.

In another aspect of the invention, a kit is provided that can be used to detect colorectal cancer.

A kit for detecting colorectal cancer, which comprises a reagent for detecting any one or a combination of more than two molecular markers of ST8SIA4, ZNF132, TAF3, TWIST1, VAV3-AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12, THBD, ZNF304, EVC, S1PR1, ZF HX4-AS1, EMID1, FAM19A4 and NEUROD 1.

In some embodiments, primers and probes for detecting methylation of at least one gene of 20 molecular markers are included, the primers and probes comprising:

at least one of the following groups directed to the ST8SIA4 molecular markers: SEQ ID NO.1, SEQ ID NO.4, and SEQ ID NO. 7; SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO. 8; SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO. 9;

at least one of the following groups for ZNF132 molecular markers: SEQ ID NO.10, SEQ ID NO.13, and SEQ ID NO. 16; SEQ ID NO.11, SEQ ID NO.14, and SEQ ID NO. 17; SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO. 18;

at least one of the following groups for the TAF3 molecular markers: SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO. 25; SEQ ID NO.20, SEQ ID NO.23, and SEQ ID NO. 26; SEQ ID NO.21, SEQ ID NO.24, and SEQ ID NO. 27;

at least one of the following groups for TWIST1 molecular markers: SEQ ID NO.28, SEQ ID NO.31, and SEQ ID NO. 34; SEQ ID NO.29, SEQ ID NO.32, and SEQ ID NO. 35; SEQ ID No.30, SEQ ID No.33, and SEQ ID No. 36;

at least one of the following for the VAV3-AS1 molecular markers: SEQ ID NO.37, SEQ ID NO.40, and SEQ ID NO. 43; SEQ ID NO.38, SEQ ID NO.41, and SEQ ID NO. 44; SEQ ID NO.39, SEQ ID NO.42, and SEQ ID NO. 45;

at least one of the following groups for FBN1 molecular markers: SEQ ID No.46, SEQ ID No.49, and SEQ ID No. 52; SEQ ID No.47, SEQ ID No.50, and SEQ ID No. 53; SEQ ID No.48, SEQ ID No.51, and SEQ ID No. 54;

at least one of the following for the C9orf50 molecular marker: SEQ ID NO.55, SEQ ID NO.58, and SEQ ID NO. 61; SEQ ID NO.56, SEQ ID NO.59, and SEQ ID NO. 62; SEQ ID NO.57, SEQ ID NO60, and SEQ ID NO. 63;

at least one of the following groups for the SFMBT2 molecular markers: SEQ ID NO.64, SEQ ID NO67, and SEQ ID NO. 70; SEQ ID NO.65, SEQ ID NO68, and SEQ ID NO 71; SEQ ID NO.66, SEQ ID NO69, and SEQ ID NO. 72;

at least one of the following for the molecular markers KCNQ 5: SEQ ID NO.73, SEQ ID NO76, and SEQ ID NO. 79; SEQ ID NO.74, SEQ ID NO77, and SEQ ID NO. 80; SEQ ID NO.75, SEQ ID NO78, and SEQ ID NO. 81;

at least one of the following groups for FAM72C molecular markers: SEQ ID No.82, SEQ ID No.85, and SEQ ID No. 88; SEQ ID No.83, SEQ ID No.86, and SEQ ID No. 89; SEQ ID No.84, SEQ ID No.87, and SEQ ID No. 90;

at least one of the following for the ITGA4 molecular markers: SEQ ID No.91, SEQ ID No.94, and SEQ ID No. 97; SEQ ID No.92, SEQ ID No.95, and SEQ ID No. 98; SEQ ID NO.93, SEQ ID NO.96, and SEQ ID NO. 99; at least one of the following for KCNJ12 molecular markers: SEQ ID No.100, SEQ ID No.103, and SEQ ID No. 106; SEQ ID NO.101, SEQ ID NO.104, and SEQ ID NO. 107; SEQ ID No.102, SEQ ID No.105, and SEQ ID No. 108;

at least one of the following groups for THBD molecular markers: SEQ ID No.109, SEQ ID No.112, and SEQ ID No. 115; SEQ ID No.110, SEQ ID No.113, and SEQ ID No. 116; SEQ ID No.111, SEQ ID No.114, and SEQ ID No. 117;

at least one of the following groups for ZNF304 molecular markers: SEQ ID No.118, SEQ ID No.121, and SEQ ID No. 124; SEQ ID No.119, SEQ ID No.122, and SEQ ID No. 125; SEQ ID No.120, SEQ ID No.123, and SEQ ID No. 126;

at least one set of the following for EVC molecular markers: SEQ ID No.127, SEQ ID No.130, and SEQ ID No. 133; SEQ ID No.128, SEQ ID No.131, and SEQ ID No. 134; SEQ ID NO.129, SEQ ID NO.132 and SEQ ID NO.135,

at least one of the following groups for the S1PR1 molecular markers: SEQ ID No.136, SEQ ID No.139, and SEQ ID No. 142; SEQ ID No.137, SEQ ID No.140, and SEQ ID No. 143; SEQ ID No.138, SEQ ID No.141, and SEQ ID No. 144;

at least one of the following groups for ZFHX4-AS1 molecular markers: SEQ ID No.145, SEQ ID No.148, and SEQ ID No. 151; SEQ ID No.146, SEQ ID No.149, and SEQ ID No. 152; SEQ ID No.147, SEQ ID No.149, and SEQ ID No. 153;

at least one of the following groups for EMID1 molecular markers: SEQ ID No.154, SEQ ID No.157, and SEQ ID No. 160; SEQ ID No.155, SEQ ID No.158, and SEQ ID No. 161; SEQ ID No.156, SEQ ID No.159, and SEQ ID No. 162;

at least one of the following groups for FAM19a4 molecular markers: SEQ ID No.163, SEQ ID No.166, and SEQ ID No. 169; SEQ ID No.164, SEQ ID No.167, and SEQ ID No. 170; SEQ ID No.165, SEQ ID No.168, and SEQ ID No. 171;

at least one of the following groups for the NEUROD molecular markers: SEQ ID No.172, SEQ ID No.175, and SEQ ID No. 178; SEQ ID No.173, SEQ ID No.176, and SEQ ID No. 179; SEQ ID NO.174, SEQ ID NO.177, and SEQ ID NO. 180.

In some of these embodiments, primers and probes for the control gene are also included, SEQ ID NO.181-SEQ ID NO. 183.

Another object of the present invention is to provide a method for detecting the molecular marker.

The detection method of the molecular marker comprises the following steps:

1) extracting a cfDNA sample from blood to be detected;

2) performing bisulfite treatment on the cfDNA sample to obtain a converted cfDNA sample;

3) performing multiple PCR reaction on the transformed cfDNA sample by using an amplification primer to obtain a PCR amplification product;

4) taking PCR amplification products, diluting the PCR amplification products, and then carrying out fluorescence PCR reaction by using primers and probes.

In some of these embodiments, the multiplex PCR reaction conditions are as follows: pre-denaturation, 30s at 98 ℃ for 15-35 cycles: denaturation at 98 deg.C for 15s, annealing at 58-66 deg.C for 15-30s, and elongation at 72 deg.C for 15-30 s.

In some of these embodiments, the fluorescent PCR reaction system is: pre-denaturation, 30s at 95 ℃ for 35-50 cycles: denaturation at 95 ℃ for 15s, annealing at 60-64 ℃ for 20 s.

The invention has the following beneficial effects:

1) the invention finds molecular markers related to colorectal cancer in blood, and methylation of the molecular markers is highly related to colorectal cancer, and particularly can be used in combination for detecting colorectal cancer with high specificity.

2) Aiming at the requirement that the amount of the cfDNA of the blood plasma is small and the detection of multiple markers is required, a method for quantitative detection of multiple PCR and qPCR is developed, for trace cfDNA, the detection sensitivity and specificity of the system can meet the clinical requirement, particularly for the detection schemes of the multiple markers, the colorectal cancer diagnosis at the stage 1 can achieve 90% of specificity, and meanwhile 83% of sensitivity is achieved, and the performance is far better than that of the product in the existing market which only detects one marker in the blood plasma.

3) Aiming at the common second-generation sequencing platform in the blood plasma cfDNA detection method, the PCR-based method is far superior to the second-generation sequencing method in time efficiency and cost performance.

Drawings

FIG. 1 is a ROC plot of 10 molecular markers from example 2.

FIG. 2 is a ROC plot of the 4 molecular markers of example 3.

FIG. 3 is a graph showing the results of detection sensitivity of ITGA4 and FBN1 in example 4.

FIG. 4 is a graph of the detected amplification of ITGA4 in example 4.

FIG. 5 is a graph of the detected amplification of FBN1 in example 4.

FIG. 6 is a graph of 20 molecular markers ROC set in example 5.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

In one aspect, the invention relates to novel molecular markers (markers) for diagnosing colorectal cancer in blood, comprising any one or a combination of more than two of ST8SIA4, ZNF132, TAF3, TWIST1, VAV3-AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12, THBD, ZNF304, EVC, S1PR1, ZFHX4-AS1, EMID1, FAM19A4 and NEUROD 1. In some of these embodiments, these molecular markers can be combined in any combination to achieve novel molecular markers for diagnosing colorectal cancer in the blood.

In one aspect, the invention relates to a kit for detecting colorectal cancer by using the molecular marker.

In one aspect, the invention relates to a kit for detecting colorectal cancer, which comprises a reagent for detecting any one or a combination of more than two molecular markers of ST8SIA4, ZNF132, TAF3, TWIST1, VAV3-AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12, THBD, ZNF304, EVC, S1PR1, ZFHX4-AS1, EMID1, FAM19A4 and NEUROD 1. For example, PCR amplification, fluorescence PCR diagnosis, digital PCR (digital PCR), or a detection platform such as a detection chip can be applied, and a platform capable of realizing high-throughput detection is preferable.

The invention designs primers and probes aiming at specific methylation regions of the markers, utilizes the primers to perform multiplex amplification on a small amount of cfDNA after bisulfite treatment, fully amplifies signals of the markers, and then utilizes qPCR to perform high-specificity detection on the signals of the markers, and has the main advantages that for a cfDNA sample with little content and weak signals of early tumors, the detection of the early tumors can be only enhanced, namely, a plurality of markers related to the tumors are jointly detected, if the qPCR method is directly adopted for detection, the markers which can be detected by each sample at each time are limited, in addition, the detected signals fall in a comparison signal interval, and the method adopted by people firstly amplifies the signals of the plurality of markers simultaneously by the multiplex PCR, thereby realizing the simultaneous detection of the plurality of markers and also in the subsequent qPCR detection, a result with high specificity and reasonable signal interval is obtained. This is very critical for non-invasive detection of colorectal cancer using cfDNA.

Example 1

A method of detecting a molecular marker for colorectal cancer in blood, comprising the steps of:

1. whole blood processing

1.1 Using EDTAK2 anticoagulated vacuum blood collection tube (BD, Cat #367525), 10mL whole blood was collected, mixed well,

avoid hemolysis and the whole blood is processed for plasma separation within 4-6 hours.

1.2 the whole blood is centrifuged at 4 ℃ and 1600g for 15min in a low-speed centrifuge, the upper plasma is carefully absorbed to avoid absorbing the middle leucocyte layer, and the obtained plasma is centrifuged at 4 ℃ and 16000g for 10min in a high-speed centrifuge again to obtain the required sample plasma.

2. Extraction of cfDNA from blood plasma

The specific method comprises the following steps: the specific procedures for plasma DNA extraction were carried out according to MagMAX of Thermo Fisher corporation^TMCell-Free DNA Isolation Kit instructions.

3. Sulfite conversion of extracted cfDNA

The extracted DNA is subjected to bisulfite conversion, so that unmethylated cytosine in the DNA is deaminated and converted into uracil, and methylated cytosine remains unchanged, thereby obtaining bisulfite converted DNA, wherein the specific conversion operation is performed according to protocol of Zymo DNA Methylation-Direct MagPrep, wherein the range of the input cfDNA is 5-20ng, preferably 10ng in the embodiment. The products of the bisulfite conversion were all used to perform the multiplex methylation amplification.

4. Multiplex methylation amplification of specific multiple Markers for transformed cfDNA

The converted products were all subjected to multiplex methylation amplification, wherein the reaction components were primer sets of 20 markers at a concentration of 300nM, a magnesium ion concentration of 2-5mM, preferably 4mM in this example, a dNTP mix concentration of 200-600uM, preferably 500uM in this example, Phusion U (Thermo Fisher, Cat # F555L) as an enzyme, and the number of units in one reaction was 1-3U, preferably 1.5U in this example. The specific reaction conditions are as follows: pre-denaturation, 30s at 98 ℃ for 15-35 cycles (denaturation, 15s at 98 ℃, annealing, 58-66 ℃, preferably 60 ℃,15 s in this example, elongation 72 ℃, 15-30s, preferably 15s in this example), preferably 28 cycles.

The multiple reaction system is prepared as follows:

5. fluorometric detection of specific methylation probes on multiplex methylation amplification products

Performing 1-1000 dilution, preferably 10-fold dilution, on the multiplex product, wherein the concentration of the primer in the fluorescent PCR reaction component is 200

nM, probe concentration at 100nM, dNTP mix concentration at 150-200uM, preferably at 150uM, ROX concentration at 1X, enzyme used as Epimark (NEB, Cat # M0490L), and unit number of one reaction at 0.5U/20ul PCR. The specific reaction conditions are as follows: pre-denaturation, 30s at 95 ℃ for 35-50 cycles (denaturation, 15s at 95 ℃, annealing, 60-64 ℃, preferably 60 ℃, 20s, signal collection), preferably 40 cycles.

The fluorescent PCR reaction system is prepared as follows:

components	Volume (ul)	Final concentration
			5X Epimark PCR Buffer	4	1X
10mM dNTP mix(10mM each)	0.3	0.15mM each
			10μM forward primer	0.4	0.2μM
10μM reverse primer	0.4	0.2μM
			Probe	0.2	0.1μM
ROX	0.4	1x
			Epimark DNA Polymerase	0.1	0.5U
Template DNA
		1
DEPC Water				up to 20ul

6. According to the result of the fluorescence measurement, when the Ct of the detected reference gene ACTB falls within the range of 10-20, the sample is judged to be a valid sample, namely, a sufficient amount of cfDNA is detected.

7. And under the condition that the sample is judged to be an effective sample, performing primary judgment on each marker, namely judging that the marker is positive if the Ct is less than 35 and judging that the marker is negative if the Ct is more than 35.

8. The kit comprises a plurality of specific molecular markers, corresponding primers and probes, and specifically comprises the following components:

a kit for detecting colorectal cancer is designed aiming at 20 cfDNA markers ST8SIA4, ZNF132, TAF3, TWIST1, VAV3-AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12, THBD, ZNF304, EVC, S1PR1, ZFHX4-AS1, EMID1, FAM19A4 and NEUROD1 for diagnosing the colorectal cancer, wherein three pairs of primers and three probes are respectively designed and respectively marked AS a primer and a probe set 1, 2 and 3. See in particular the following table. Wherein, the selection primer and

probe

1, 2, 3 group of a locus can be arbitrarily selected to be combined with other primers and probes 1, 2, 3 of the locus for further combination on the same platform for detection.

In this and the following examples, the combination of primers and probes used were: ST8SIA4 combination 1, ZNF132 combination 2, TAF3 combination 3, Twist1 combination 2, VAV3-AS1 combination 2, FBN1 combination 2, C9orf50 combination 2, SFMBT2 combination 2, KCNQ5 combination 3, FAM72C combination 2, ITGA4 combination 1, KCNJ12 combination 2, THBD combination 3, ZNF304 combination 1, EVC combination 3, S1PR1 combination 2, hxzf 4-AS1 combination 1, EMID1 combination 1, FAM19a4 combination 2, FAM19a4 combination 1.

Designing and preparing an internal standard gene primer and a probe, wherein the primer and the probe are designed aiming at the ACTB gene and are specifically shown as SEQ ID 181-SEQ ID 183. The internal standard gene primer and the probe are respectively prepared into 10 mu M mother liquor for storage, and are prepared into 1.15 mu M working solution for later use according to the detection requirement.

Example 2

Plasma samples of 46 enteroscopy colorectal cancer-free normal persons and 175 colorectal cancer patients were tested by the test method of example 1, the specific test kit and test method and data judgment treatment were the same as example 1, and the combination of primers and probes was also preferably used in example 1. 10markers (TWIST1, VAV3-AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12 and ZNF132) are selected for modeling analysis by using a logistic regression model, and the method comprises the following steps of: 4, 100 repetitions are carried out, and under the specificity of 90%, the detection sensitivity of the test set at stage 1 is 78 +/-12%, the detection sensitivity of stage 2 is 77 +/-10%, the detection sensitivity of stage 3 is 82 +/-11%, the detection sensitivity of stage 4 is 88 +/-5%, and the overall detection sensitivity of all colorectal cancer samples is 85 +/-5%. The overall AUC was 0.91. + -. 0.03. These markers are shown to be highly correlated with colorectal cancer and by this protocol method colorectal cancer, including early stage colorectal cancer, can be detected with great sensitivity. The results are shown in FIG. 1.

Example 3

Plasma samples of 46 enteroscopy normal persons without colorectal cancer and 175 colorectal cancer patients were tested using the test kit and test method of example 1, the specific test and data judgment treatment were in accordance with example 1, and the combination of primers and probes was also preferably used in example 1. 4markers (TWIST1, C9orf50, KCNJ12 and ZNF132) are selected from the raw materials, and are subjected to modeling analysis by using a logistic regression model according to the ratio of 6: 4, 100 repetitions are carried out, and under the specificity of 90%, the detection sensitivity of the test set at stage 1 is 78 +/-12%, the detection sensitivity of stage 2 is 76 +/-10%, the detection sensitivity of stage 3 is 81 +/-11%, the detection sensitivity of stage 4 is 88 +/-5%, and the overall detection sensitivity of all colorectal cancer samples is 82 +/-5%. The overall AUC was 0.91. + -. 0.03. These markers are shown to be highly correlated with colorectal cancer and by this protocol method colorectal cancer, including early stage colorectal cancer, can be detected with great sensitivity. See FIG. 2 for results.

Example 4 sensitive and specific detection of molecular markers

By using

NA12878 DNA was treated with Single Cell Kit (Qiagen, Cat #150343) and Mung Bean nucleic (NEB, Cat # M0250L) to prepare 0% methylated standards, and the prepared 0% methylated standards were treated with CpG methyl ransferase (m.sssi) to give 100% methylated standards. Mix the 0% and 100% methylation standards according to the desired methylation ratio gradient to obtain 1%, 0.5%, 0.2% methylation standards. 10ng of DNA is put into each methylation proportion sample for each detection, and each methylation proportion sample is heavyThe test was repeated 4 times in order to detect the sensitivity of the molecular marker. Meanwhile, the amplification specificity of the sample is detected by using a sample with the methylation ratio of 0%. The specific steps of each detection are as follows:

1. bisulfite conversion of standard DNA at different methylation ratios

And (3) carrying out bisulfite conversion on standard DNA with different Methylation ratios to deaminate cytosine which is not methylated in the DNA into uracil, wherein the methylated cytosine is kept unchanged to obtain DNA after bisulfite conversion, and the specific conversion operation is carried out according to protocol of Zymo DNA Methylation-Direct MagPrep, wherein the input cfDNA is 10 ng. The bisulfite converted DNA products were all used for multiplex methylation amplification.

2. Performing multiple methylation amplification of multiple Markers on the converted standard DNA

The transformed DNA products were all subjected to multiplex methylation amplification, wherein the reaction components were primer sets for each specific Marker (20 molecular markers, the preferred primer set in example 1 + primer set for reference gene, the same in the following examples), in which the concentration was 300nM, the concentration of magnesium ion was 2-5mM, in this example 4mM, the concentration of dNTP mix was 200-600uM, in this example 500uM, the enzyme used was Phusion U, and the number of units in one reaction was 1-3U, in this example 1.5U. The specific reaction conditions are as follows: pre-denaturation, 30s at 98 ℃ for 15-35 cycles (denaturation, 15s at 98 ℃, annealing, 58-66 ℃, preferably 60 ℃,15 s in this example, elongation 72 ℃, 15-30s, preferably 15s in this example), preferably 28 cycles. The multiple reaction system is prepared as follows:

3. fluorometric detection of specific methylation probes on multiplex methylation amplification products

Performing 1-1000 dilution, preferably 10-fold dilution on the multiplex product, wherein the fluorescent PCR reaction component is a primer

4. The concentration was 200nM, the probe concentration was 100nM, the dNTP mix concentration was 150-200uM, preferably 150uM, the ROX concentration was 1X, the enzyme used was Epimark (NEB, Cat # M0490L), and the number of units for one reaction was 0.5U/20ul PCR. The specific reaction conditions are as follows: pre-denaturation, 30s at 95 ℃ for 35-50 cycles (denaturation, 15s at 95 ℃, annealing, 60-64 ℃, preferably 60 ℃, 20s, signal collection.), preferably 40 cycles.

In this example, the detection sensitivity of ITGA4, FBN1, and VAV3-AS1, all primers and primers were determined AS described in example 1, wherein the primer set of ITGA4 was SEQ NO.91, the primer set of SEQ NO.94 probe was SEQ NO.97, the primer set of FBN1 was SEQ NO.47, SEQ NO.50, the probe was SEQ NO.53, and the primer set of VAV3-AS1 was SEQ NO.38, SEQ NO.41, and the probe was SEQ NO. 44.

The fluorescent PCR reaction system is prepared as follows:

5. according to the result of the fluorescence measurement, when the Ct of the detected reference gene ACTB falls within the range of 10-20

And (4) judging the sample to be a valid sample, namely, a sufficient amount of cfDNA is detected.

6. And under the condition that the sample is judged to be an effective sample, performing primary judgment on each marker, namely judging that the marker is positive if the Ct is less than 35 and judging that the marker is negative if the Ct is more than 35.

In 4 repeated detections, the detection rates of 3 markers under samples with different methylation ratios are obtained according to the results, the detection sensitivity of the scheme of multiplex PCR plus single qPCR on the molecular markers is preliminarily obtained, and the results are shown in FIG. 3. It can be seen that, the scheme of detecting each molecular marker by using multiplex PCR and then performing separate qPCR can detect multiple molecular markers of one sample simultaneously, and the separate detection sensitivity of each molecular marker is very high, the detection rate of ITAG4 in the sample with the methylation ratio as low as 0.2% is 100%, while that of FBN1 in the sample with the methylation ratio of 0.5% is 100%, and that in the sample with the methylation ratio of 0.2%, the detection rate is 75%, that is, in 4 times of repeated detection, positive signals can be obtained. While for a sample with a negative result, for example, a sample with a 0.2% methylation ratio, in which the detection signal of FBN1 in this example is negative, the detection rate of ITGA4 in this sample is positive, this complementary advantage is very great for a sample with very weak signal detection, such as a plasma sample, using multiple molecular markers in combination.

7. The amplification curves for the 0% methylation ratio samples are shown in FIGS. 4 and 5:

it can be seen that primers and probes for ITGA4 and FBN1 and the detection method were specifically detected using unmethylated standard DNA, and that the primer and probe design and the detection protocol involved in this example were very specific.

Example 5 correlation of molecular markers with colorectal cancer

1. Extraction of DNA from paraffin tissue of colorectal cancer

The specific method comprises the following steps: the specific procedures for DNA extraction from paraffin tissues were performed according to the instructions of the ALLPrep DNA/RNA FFPE Kit from Qiagen.

2. Sulfite conversion of extracted 20ng DNA

As described in example 4.

3. Performing multiple methylation amplification of specific multiple Markers on the transformed DNA

As described in example 4.

4. Fluorometric detection of specific methylation probes on multiplex methylation amplification products

As described in example 4.

And in the inner space, the sample is judged to be a valid sample, namely, a sufficient amount of DNA is detected.

7. In 37 samples of tissues without abnormality in enteroscopy and 58 samples of tissues with colorectal cancer or canceration as a result of enteroscopy, 20 molecular markers were detected, and the results are shown in FIG. 6. Wherein, single molecular markers are detected for a plurality of molecular markers of ITGA4, FBN1, C9orf50, FAM72C, KCNQ5, SFMBT2b, TWIST1, VAV3-AS1, ZNF132, THBD and EVC. The results are as follows:

wherein ITGA4 is used as a molecular marker for colorectal cancer diagnosis, and AUC is as high as 0.942. Wherein, when the specificity is 90%, the sensitivity can reach 89.7%, and when the specificity is 95%, the sensitivity is 70.7%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

Wherein FBN1 is used as a molecular marker alone for colorectal cancer diagnosis, and the AUC is as high as 0.922. Wherein, when the specificity is 90%, the sensitivity can reach 79.3%, and when the specificity is 95%, the sensitivity is 70.7%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

Wherein C9orf50 is used as a molecular marker for colorectal cancer diagnosis alone, and the AUC is as high as 0.887. Wherein, when the specificity is 90%, the sensitivity can reach 72.4%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

The FAM72C is used as a molecular marker for colorectal cancer diagnosis, and the AUC is as high as 0.895. Wherein, when the specificity is 90%, the sensitivity can reach 81%, and when the specificity is 95%, the sensitivity is 74.1%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

Wherein KCNQ5 is used alone as a molecular marker for colorectal cancer diagnosis, and AUC is as high as 0.932. Wherein, when the specificity is 90%, the sensitivity can reach 81%, and when the specificity is 95%, the sensitivity is 65.5%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

Wherein SFMBT2 is used as a molecular marker for colorectal cancer diagnosis, and AUC is as high as 0.944. Wherein, when the specificity is 90%, the sensitivity can reach 88%, and when the specificity is 95%, the sensitivity is also 79.3%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

Wherein TWIST1 is used as a molecular marker alone for colorectal cancer diagnosis, and AUC is as high as 0.853. Wherein, when the specificity is 90%, the sensitivity can reach 70.7%, and when the specificity is 95%, the sensitivity is 67.2%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

Wherein VAV3-AS1 is used AS a molecular marker for colorectal cancer diagnosis, and AUC is AS high AS 0.944. Wherein, when the specificity is 90%, the sensitivity can reach 83.6%, and when the specificity is 95%, the sensitivity is 64.7%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

Wherein ZNF132 is used as a molecular marker for colorectal cancer diagnosis, and the AUC is up to 0.916. Wherein, when the specificity is 90%, the sensitivity can reach 82.8%, and when the specificity is 95%, the sensitivity is 75.9%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

Wherein THBD is used as a molecular marker for colorectal cancer diagnosis alone, and AUC is as high as 0.913. Wherein, when the specificity is 90%, the sensitivity can reach 81%, and when the specificity is 95%, the sensitivity is 74.1%.

Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

Wherein EVC is used as a molecular marker for colorectal cancer diagnosis alone, and AUC is as high as 0.863. Wherein, when the specificity is 90%, the sensitivity can reach 67.2%, and when the specificity is 95%, the sensitivity is also 60.3%. Indicating that the methylation level of the molecular marker is highly related to the canceration of the colorectal cancer.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Sequence listing

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<212> DNA

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<212> DNA

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<210> 29

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<212> DNA

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gtagaggaag tcgatgtatt tggtc 25

<210> 30

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<212> DNA

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<400> 30

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<210> 31

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<212> DNA

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<212> DNA

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<400> 32

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<400> 33

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<210> 34

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<212> DNA

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<400> 34

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<210> 35

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<212> DNA

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<400> 35

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<210> 36

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<212> DNA

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<400> 36

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<210> 37

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<212> DNA

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<212> DNA

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<210> 40

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<400> 40

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<210> 41

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<400> 41

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<210> 43

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<210> 44

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<400> 44

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<210> 45

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<210> 46

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<400> 46

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<212> DNA

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<400> 47

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<210> 48

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<212> DNA

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<400> 48

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<210> 49

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<212> DNA

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<400> 49

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<210> 50

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<212> DNA

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<400> 50

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<210> 51

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<212> DNA

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<400> 51

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<210> 52

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<212> DNA

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<400> 52

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<210> 53

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<400> 53

catacgtcga aaacgtctac 20

<210> 54

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<400> 54

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<210> 55

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<400> 55

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<210> 56

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<212> DNA

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<400> 56

ggggttttat ttagtggttt cggt 24

<210> 57

<211> 22

<212> DNA

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<400> 57

cggggtttta tttagtggtt tcg 23

<210> 58

<211> 17

<212> DNA

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<400> 58

gacgcgtccc gaaaatc 17

<210> 59

<211> 24

<212> DNA

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<400> 59

cgacttctta aacgccttcc taaa 24

<210> 60

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<212> DNA

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<400> 60

ccgacttctt aaacgccttc cta 23

<210> 61

<211> 19

<212> DNA

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<400> 61

aaaacgcgaa cgcccccga 19

<210> 62

<211> 30

<212> DNA

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<400> 62

ccccaacaaa ttccgtaaat tcctacacca 30

<210> 63

<211> 30

<212> DNA

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<400> 63

ccaacaaatt ccgtaaattc ctacaccaac 30

<210> 64

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<212> DNA

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<400> 64

aaaacgtttt taggtatttg gtcg 24

<210> 65

<211> 29

<212> DNA

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<400> 65

cgttgttgcg ttaattataa atttggtta 29

<210> 66

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<400> 66

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<210> 67

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<400> 67

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<210> 68

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<212> DNA

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<400> 68

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<210> 69

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<400> 69

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<210> 70

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<400> 70

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<210> 71

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<400> 71

ctcctccgaa cccgcgaacc taatct 26

<210> 72

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<400> 72

acctcctccg aacccgcgaa cctaat 26

<210> 73

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<400> 73

tcggatttag aattcgaagt taaaggt 27

<210> 74

<211> 21

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<400> 74

ctctacaacc cctcctttcc c 21

<210> 75

<211> 27

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<400> 75

gttcggattt agaattcgaa gttaaag 27

<210> 76

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<400> 76

aaaacctcta aaccgaaaac gaaaa 25

<210> 77

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<212> DNA

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<400> 77

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<210> 78

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<400> 78

aaacctctaa accgaaaacg aaaaa 25

<210> 79

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<400> 79

acaaccaaca aaccgcccga acttc 25

<210> 80

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<400> 80

ccccgaaacc cgaacgattt actaactacc 30

<210> 81

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<400> 81

aaccaacaaa ccgcccgaac ttcga 25

<210> 82

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<400> 82

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<210> 83

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<400> 83

tagtaacggt gttgagatta attcgg 26

<210> 84

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<400> 84

ttggtcggtt gagtttggtt tc 22

<210> 85

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<400> 85

cgtaccttac cactttcact aaaacta 27

<210> 86

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<400> 86

cgtaccttac cactttcact aaaactaaa 29

<210> 87

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<400> 87

cactttcact aaaactaaaa caaaatttac cg 32

<210> 88

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<400> 88

taccgaaacc tcccgaaacc gaccaata 28

<210> 89

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<400> 89

ttaccgaaac ctcccgaaac cgaccaataa 30

<210> 90

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<400> 90

aacctcccga aaccgaccaa taaaatcgaa 30

<210> 91

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<400> 91

ttcgttcggt tttattttcg gt 22

<210> 92

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<400> 92

cggttttttg tcgttagtcg gga 23

<210> 93

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<400> 93

tgcggaggcg tagggtc 17

<210> 94

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<400> 94

ccacgcccga aaccccaccc aacg 24

<210> 95

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<400> 95

caaccgcgcg taaacaaaaa 20

<210> 96

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<400> 96

caaccgaaat tccccaacg 19

<210> 97

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<400> 97

acccgaaaaa caacgcgaac accc 24

<210> 98

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<400> 98

cctacaaccg cgcgtaaaca aaaacg 26

<210> 99

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<400> 99

cgcaccacgc ccgaaacc 18

<210> 100

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<400> 100

cgtaggaagt aagtaggagg gttt 24

<210> 101

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<400> 101

gtaggagggt ttcgtgtata ttagg 25

<210> 102

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<400> 102

aaaaacaaaa actacctaaa tttacaacgc 30

<210> 103

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<400> 103

cctaaattta caacgcgaaa ccaaa 25

<210> 104

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aaactaccta aatttacaac gcgaaa 26

<210> 105

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gatttgtttg cgcgttcgag 20

<210> 106

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<400> 106

cttccctaaa actcgacgac cgccctata 29

<210> 107

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<400> 107

aacttcccta aaactcgacg accgcccta 29

<210> 108

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 108

aacttcccta aaactcgacg accgcccta 29

<210> 109

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 109

gtggttttag tagcggagat agcg 24

<210> 110

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 110

gttgtttttc gtaatttatt ggaagtc 27

<210> 111

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 111

cgagtcgtgt ttatttgtta tagttggt 28

<210> 112

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 112

gacgcctcta aatcgaccta caac 24

<210> 113

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 113

cctctaaatc gacctacaac tacca 25

<210> 114

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 114

gacgcctcta aatcgaccta caacta 26

<210> 115

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 115

ccacccgact acgacgaccc caaac 25

<210> 116

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 116

cgacgacccc aaacgcctcg 20

<210> 117

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 117

acccgactac gacgacccca aacgc 25

<210> 118

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 118

ttttgtagtt tagagtagta ggaaacg 27

<210> 119

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 119

ctccgaaata tcgttttccc aatc 24

<210> 120

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 120

ttgtagttta gagtagtagg aaacgt 26

<210> 121

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 121

attaaataca aaatacaaac tacgttaccc 30

<210> 122

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 122

cgtagtttgt attttgtatt taattaggta acg 33

<210> 123

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 123

taattaaata caaaatacaa actacgttac c 31

<210> 124

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 124

acccaactca ttattaaccg acgtacaacc 30

<210> 125

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 125

ccctccctac acgacgcctc ttaacaaac 29

<210> 126

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 126

aacccaactc attattaacc gacgtacaac ca 32

<210> 127

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 127

aagttttgag cggtgattta ggt 23

<210> 128

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 128

aaagttttga gcggtgattt aggt 24

<210> 129

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 129

agttttgagc ggtgatttag gttt 24

<210> 130

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 130

ctatacaatc tcccgactcc tacttc 26

<210> 131

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 131

atacaatctc ccgactccta cttc 24

<210> 132

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 132

cctatacaat ctcccgactc ctac 24

<210> 133

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 133

cccttcccta cttcgccgaa ccg 23

<210> 134

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 134

cccttcccta cttcgccgaa ccga 24

<210> 135

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 135

tccccttccc tacttcgccg aacc 24

<210> 136

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 136

cgtagtaggt agggaattgg tcg 23

<210> 137

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 137

atttcgcgta gtaggtaggg aatt 24

<210> 138

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 138

gatttcgcgt agtaggtagg gaat 24

<210> 139

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 139

gtaatactct cgcaaactta ctcgaatc 28

<210> 140

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 140

gcaaacttac tcgaatcgaa atcc 24

<210> 141

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 141

tcgcaaactt actcgaatcg aaat 24

<210> 142

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 142

cctccctccg caacgaaaca ctccaata 28

<210> 143

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 143

ctccctccgc aacgaaacac tccaat 26

<210> 144

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 144

tccctccgca acgaaacact ccaataac 28

<210> 145

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 145

tcgtttagtg tgcggggttg gc 22

<210> 146

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 146

ttcgaaggtc gtttagtgtg cg 22

<210> 147

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 147

aggttcgggg gttcgtga 18

<210> 148

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 148

cccgctccga taaacgaa 18

<210> 149

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 149

caacgttcac cccaacctct ta 22

<210> 150

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 150

ccgcgcccgc tccgataaac 20

<210> 151

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 151

accccaaccc caacg 15

<210> 152

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 152

cccctcccca aaactcacga acccc 25

<210> 153

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 153

aaccccaacg ttcaccc 17

<210> 154

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 154

gcggtgaggt gtttaatata cgtg 24

<210> 155

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 155

ggttggattt cgtaaaggtg tcg 23

<210> 156

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 156

ggtgaggtgt ttaatatacg tggc 24

<210> 157

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 157

aacgaataat atccgaaact catcctt 27

<210> 158

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 158

aaaactaaaa cccgaaccac gat 23

<210> 159

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 159

acgaataata tccgaaactc atcctttt 28

<210> 160

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 160

cgacaccttt acgaaatcca accctccaa 29

<210> 161

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 161

ccaaacgcaa ccgaaacccg aacc 24

<210> 162

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 162

gacaccttta cgaaatccaa ccctccaaaa 30

<210> 163

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 163

gcacccctac tcaaacgacg 20

<210> 164

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 164

ttggtcgggt cgttcggtt 19

<210> 165

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 165

gtttacgtgg gtcggt 16

<210> 166

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 166

gagtggtggt tatagcgagg c 21

<210> 167

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 167

aaaaaccgac ccacgtaaac 20

<210> 168

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 168

aaaaccgaca ataaaa 16

<210> 169

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 169

ccactaccga cctccaaacg cctct 25

<210> 170

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 170

tggtttcgcg gtttacgtgg 20

<210> 171

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 171

tttaggcggc gcgttttta 19

<210> 172

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 172

cgttaggcgt atagatttgt tagttt 26

<210> 173

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 173

gttttttgcg tgggcgaat 19

<210> 174

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 174

ggcgttaggc gtatagattt gtta 24

<210> 175

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 175

cgaacgctca aattatataa cccaat 26

<210> 176

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 176

ccgcgcttaa catcactaac taaa 24

<210> 177

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 177

gcgaacgctc aaattatata accca 25

<210> 178

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 178

atactaaacg cgaacgaaac cgctaactaa 30

<210> 179

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 179

cgcgcgacca cgacacgaaa 20

<210> 180

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 180

taatactaaa cgcgaacgaa accgctaact 30

<210> 181

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 181

gtgatggagg aggtttagta agtt 24

<210> 182

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 182

ccaataaaac ctactcctcc cttaa 25

<210> 183

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 183

accaccaccc aacacacaat aacaaacaca 30

Claims

1. A molecular marker associated with colorectal cancer in human blood, wherein the molecular marker is a combination of TWIST1, VAV3-AS1, FBN1, C9orf50, SFMBT2, KCNQ5, FAM72C, ITGA4, KCNJ12 and ZNF132, and the human colorectal cancer is diagnosed by detecting the methylation level of the molecular marker in human blood.

2. A human blood colorectal cancer related molecular marker, wherein the molecular marker is a combination of TWIST1, C9orf50, KCNJ12 and ZNF132, and the human colorectal cancer is diagnosed by detecting the methylation level of the molecular marker in human blood.

3. A molecular marker related to colorectal cancer in human blood is characterized in that the molecular marker is a combination of FBN1, SFMBT2, FAM72C, ITGA4, ZNF132, KCNJ12, ST8SIA4, TAF3, TWIST1, VAV3-AS1, C9orf50, KCNQ5, THBD, ZNF304, EVC, S1PR1, ZFHX4-AS1, EMID1, FAM19A4 and NEUROD, and the colorectal cancer of the human is diagnosed by detecting the methylation level of the molecular marker in the human blood.

4. Use of a reagent for detecting the methylation level of a molecular marker according to any one of claims 1 to 3 in human blood for the manufacture of a kit for detecting colorectal cancer in humans.

5. A kit for detecting colorectal cancer, comprising a reagent for detecting the methylation level of the molecular marker of any one of claims 1 to 3, wherein the reagent comprises a primer and a probe for detecting the molecular marker;

the primers and probes include those selected from the group consisting of:

at least one of the following groups for the ST8SIA4 gene: SEQ ID NO.1, SEQ ID NO.4, and SEQ ID NO. 7; SEQ ID NO.2, SEQ ID NO.5, and SEQ ID NO. 8; SEQ ID NO.3, SEQ ID NO.6, and SEQ ID NO. 9;

at least one of the following groups for the ZNF132 gene: SEQ ID NO.10, SEQ ID NO.13, and SEQ ID NO. 16; SEQ ID NO.11, SEQ ID NO.14, and SEQ ID NO. 17; SEQ ID NO.12, SEQ ID NO.15, and SEQ ID NO. 18;

at least one of the following groups for the TAF3 gene: SEQ ID NO.19, SEQ ID NO.22, and SEQ ID NO. 25; SEQ ID NO.20, SEQ ID NO.23, and SEQ ID NO. 26; SEQ ID NO.21, SEQ ID NO.24, and SEQ ID NO. 27;

at least one of the following groups for the TWIST1 gene: SEQ ID NO.28, SEQ ID NO.31, and SEQ ID NO. 34; SEQ ID NO.29, SEQ ID NO.32, and SEQ ID NO. 35; SEQ ID No.30, SEQ ID No.33, and SEQ ID No. 36;

at least one of the following groups for the VAV3-AS1 gene: SEQ ID NO.37, SEQ ID NO.40, and SEQ ID NO. 43; SEQ ID NO.38, SEQ ID NO.41, and SEQ ID NO. 44; SEQ ID NO.39, SEQ ID NO.42, and SEQ ID NO. 45;

at least one of the following groups for the FBN1 gene: SEQ ID No.46, SEQ ID No.49, and SEQ ID No. 52; SEQ ID No.47, SEQ ID No.50, and SEQ ID No. 53; SEQ ID No.48, SEQ ID No.51, and SEQ ID No. 54;

at least one of the following groups for the C9orf50 gene: SEQ ID NO.55, SEQ ID NO.58, and SEQ ID NO. 61; SEQ ID NO.56, SEQ ID NO.59, and SEQ ID NO. 62; SEQ ID NO.57, SEQ ID NO60, and SEQ ID NO. 63;

at least one of the following for the SFMBT2 gene: SEQ ID NO.64, SEQ ID NO67, and SEQ ID NO. 70; SEQ ID NO.65, SEQ ID NO68, and SEQ ID NO 71; SEQ ID NO.66, SEQ ID NO69, and SEQ ID NO. 72;

at least one set of the following for the KCNQ5 gene: SEQ ID NO.73, SEQ ID NO76, and SEQ ID NO. 79; SEQ ID NO.74, SEQ ID NO77, and SEQ ID NO. 80; SEQ ID NO.75, SEQ ID NO78, and SEQ ID number 81;

at least one of the following groups for the FAM72C gene: SEQ ID No.82, SEQ ID No.85, and SEQ ID No. 88; SEQ ID No.83, SEQ ID No.86, and SEQ ID No. 89; SEQ ID No.84, SEQ ID No.87, and SEQ ID No. 90;

at least one of the following for the ITGA4 gene: SEQ ID No.91, SEQ ID No.94, and SEQ ID No. 97; SEQ ID No.92, SEQ ID No.95, and SEQ ID No. 98; SEQ ID NO.93, SEQ ID NO.96, and SEQ ID NO. 99;

at least one set of the following for the KCNJ12 gene: SEQ ID No.100, SEQ ID No.103, and SEQ ID No. 106; SEQ ID NO.101, SEQ ID NO.104, and SEQ ID NO. 107; SEQ ID No.102, SEQ ID No.105, and SEQ ID No. 108;

at least one of the following groups for the THBD gene: SEQ ID No.109, SEQ ID No.112, and SEQ ID No. 115; SEQ ID No.110, SEQ ID No.113, and SEQ ID No. 116; SEQ ID No.111, SEQ ID No.114, and SEQ ID No. 117;

at least one of the following groups for the ZNF304 gene: SEQ ID No.118, SEQ ID No.121, and SEQ ID No. 124; SEQ ID No.119, SEQ ID No.122, and SEQ ID No. 125; SEQ ID No.120, SEQ ID No.123, and SEQ ID No. 126;

at least one of the following groups for EVC genes: SEQ ID No.127, SEQ ID No.130, and SEQ ID No. 133; SEQ ID No.128, SEQ ID No.131, and SEQ ID No. 134; SEQ ID No.129, SEQ ID No.132, and SEQ ID No. 135;

at least one of the following groups for the S1PR1 gene: SEQ ID No.136, SEQ ID No.139, and SEQ ID No. 142; SEQ ID No.137, SEQ ID No.140, and SEQ ID No. 143; SEQ ID No.138, SEQ ID No.141, and SEQ ID No. 144;

at least one set of the following for the ZFHX4-AS1 gene: SEQ ID No.145, SEQ ID No.148, and SEQ ID No. 151; SEQ ID No.146, SEQ ID No.149, and SEQ ID No. 152; SEQ ID No.147, SEQ ID No.149, and SEQ ID No. 153;

at least one set of the following for the EMID1 gene: SEQ ID No.154, SEQ ID No.157, and SEQ ID No. 160; SEQ ID No.155, SEQ ID No.158, and SEQ ID No. 161; SEQ ID No.156, SEQ ID No.159, and SEQ ID No. 162;

at least one of the following groups for the FAM19a4 gene: SEQ ID No.163, SEQ ID No.166, and SEQ ID No. 169; SEQ ID No.164, SEQ ID No.167, and SEQ ID No. 170; SEQ ID No.165, SEQ ID No.168, and SEQ ID No. 171;

at least one of the following groups for the NEUROD gene: SEQ ID No.172, SEQ ID No.175, and SEQ ID No. 178; SEQ ID No.173, SEQ ID No.176, and SEQ ID No. 179; SEQ ID NO.174, SEQ ID NO.177, and SEQ ID NO. 180.

6. The kit for detecting colorectal cancer according to claim 5, further comprising internal standard gene primers and probes, wherein the internal standard gene primers and probes are SEQ ID No.181-SEQ ID No. 183.

7. The kit for detecting colorectal cancer according to claim 5 or 6, wherein the primers and probes for detecting molecular markers include the following:

for the ST8SIA4 gene: SEQ ID NO.1, SEQ ID NO.4, and SEQ ID NO. 7;

for the ZNF132 gene: SEQ ID NO.11, SEQ ID NO.14, and SEQ ID NO. 17;

for the TAF3 gene: SEQ ID NO.21, SEQ ID NO.24, and SEQ ID NO. 27;

for the TWIST1 gene: SEQ ID NO.29, SEQ ID NO.32, and SEQ ID NO. 35;

for the VAV3-AS1 gene: SEQ ID NO.38, SEQ ID NO.41, and SEQ ID NO. 44;

of the genes for FBN 1: SEQ ID No.47, SEQ ID No.50, and SEQ ID No. 53;

for the C9orf50 gene: SEQ ID NO.56, SEQ ID NO.59, and SEQ ID NO. 62;

against the SFMBT2 gene: SEQ ID NO.65, SEQ ID NO68, and SEQ ID NO. 71;

for KCNQ5 gene: SEQ ID NO.75, SEQ ID NO78, and SEQ ID NO. 81;

against FAM72C gene: SEQ ID No.83, SEQ ID No.86, and SEQ ID No. 89;

for the ITGA4 gene: SEQ ID No.91, SEQ ID No.94, and SEQ ID No. 97;

for KCNJ12 gene: SEQ ID NO.101, SEQ ID NO.104, and SEQ ID NO. 107;

for the THBD gene: SEQ ID No.111, SEQ ID No.114, and SEQ ID No. 117;

for the ZNF304 gene: SEQ ID No.118, SEQ ID No.121, and SEQ ID No. 124;

for the EVC gene: SEQ ID No.129, SEQ ID No.132, and SEQ ID No. 135;

for the S1PR1 gene: SEQ ID No.137, SEQ ID No.140, and SEQ ID No. 143;

for the ZFHX4-AS1 gene: SEQ ID No.145, SEQ ID No.148, and SEQ ID No. 151;

for the EMID1 gene: SEQ ID No.154, SEQ ID No.157, and SEQ ID No. 160;

against FAM19a4 gene: SEQ ID No.164, SEQ ID No.167, and SEQ ID No. 170;

for the NEUROD gene: SEQ ID NO.172, SEQ ID NO.175, and SEQ ID NO. 178.