阅读说明：本技术 结直肠癌标志物及其应用 (Colorectal cancer marker and application thereof ) 是由 李南南 揭著业 易吉 吴逵 朱师达 贾慧珏 仝欣 罗慧娟 陈小芳 于 2018-08-22 设计创作，主要内容包括：本发明涉及基因检测领域,具体涉及一种结直肠癌标志物及其应用。所述标志物包括选自下列中的至少一种：SEQ ID NO:1～SEQ ID NO:5；来源于具核梭杆菌且与所述SEQ ID NO:1～SEQ ID NO:5中的任意一种具有95％以上同源性的核酸序列。同时还提供了引物对、核酸探针、构建体以及试剂盒以及对待测者进行结直肠癌检测的系统。通过本发明提供的标志物可以实现结直肠癌的快速检测。(The invention relates to the field of gene detection, in particular to a colorectal cancer marker and application thereof. The marker comprises at least one selected from the group consisting of: 1 to 5 of SEQ ID NO; a nucleic acid sequence which is derived from fusobacterium nucleatum and has homology of more than 95 percent with any one of SEQ ID NO 1-SEQ ID NO 5. Also provides a primer pair, a nucleic acid probe, a construction body, a kit and a system for detecting the colorectal cancer of a person to be detected. The marker provided by the invention can realize the rapid detection of colorectal cancer.)

1. A colorectal cancer marker, wherein the marker comprises at least one selected from the group consisting of:

SEQ ID NO:1～SEQ ID NO:5；

a nucleic acid sequence which is derived from fusobacterium nucleatum and has homology of more than 95 percent with any one of SEQ ID NO 1-SEQ ID NO 5.

2. A set of primer pairs capable of specifically amplifying at least one of the markers of claim 1, said primer pairs being selected from any one of SEQ ID NOs 6 to 35, wherein each two adjacently numbered nucleic acid sequences of SEQ ID NOs 6 to 35 are a pair.

3. The primer pair of claim 2, wherein any one of SEQ ID NO 6 to SEQ ID NO 11 is capable of specifically amplifying SEQ ID NO 1;

any one of the primer pair SEQ ID NO 12-SEQ ID NO 19 can specifically amplify SEQ ID NO 2;

any one of the primer pair SEQ ID NO. 20-SEQ ID NO. 23 can specifically amplify SEQ ID NO. 3;

any one of the primer pair SEQ ID NO. 24-SEQ ID NO. 31 can specifically amplify SEQ ID NO. 4;

any one of the primer pair SEQ ID NO 32-SEQ ID NO 35 can specifically amplify SEQ ID NO 5.

4. A nucleic acid probe capable of specifically capturing a nucleic acid comprising the colorectal cancer marker of claim 1.

5. A construct comprising the colorectal cancer marker of claim 1.

6. The construct of claim 5, wherein the construct is a recombinant vector comprising the colorectal cancer marker of claim 1;

optionally, the vector is a plasmid.

7. A kit comprising reagents for detecting the colorectal cancer marker of claim 1;

optionally, the kit comprises at least one of:

the primer pair of claim 2 or 3;

the nucleic acid probe of claim 4;

the construct of claim 5 or 6.

8. A system for detecting colorectal cancer in a subject, comprising:

a quantification device for determining the amount of the target nucleic acid in the fecal DNA of the subject, the target nucleic acid being the marker of claim 1;

and the judging device is connected with the quantifying device and determines whether the person to be detected is the colorectal cancer patient or not based on the content of the target nucleic acid.

9. The system of claim 8, wherein the quantification device is capable of determining the content of the target nucleic acid in the fecal DNA of the subject according to a method comprising the steps of:

performing PCR amplification on the target nucleic acid, and determining the content of the target nucleic acid in the fecal DNA of the testee;

optionally, the PCR is a fluorescent quantitative PCR.

10. The system according to claim 8 or 9, wherein the judging means determines whether the subject is a colorectal cancer patient based on the following criteria:

if the content of the target nucleic acid is greater than or equal to a predetermined threshold value, the tested person is a colorectal cancer patient; if the content of the target nucleic acid is less than a predetermined threshold value, the subject is a non-colorectal cancer patient;

optionally, the predetermined threshold is determined by:

performing fluorescent quantitative PCR amplification on the target nucleic acid in the fecal DNA of each individual in a population consisting of colorectal cancer patients and non-colorectal cancer patients to obtain detection results;

drawing an ROC curve based on the detection result, and finding out a point with optimal sensitivity and specificity from the ROC curve;

based on the point with the best sensitivity and specificity, corresponding to the fluorescence quantitative PCR, obtaining the copy number of the target nucleic acid as the preset threshold value;

optionally, the predetermined threshold is 6.302898.

Technical Field

The invention relates to the field of gene detection, in particular to a colorectal cancer biomarker and application thereof, and particularly relates to a colorectal cancer marker, a primer pair, a nucleic acid probe, a kit and a colorectal cancer detection method.

Background

According to statistics, the incidence rate of colorectal cancer (CRC) in various cancers ranks third, the mortality rate ranks fourth, and nearly 55% of incidence cases occur in more developed countries. The incidence is lower in people under 40 years of age, but it also increases gradually with increasing age. Colorectal cancer is also one of the most preventable tumors at present, and the medical community considers that intestinal cancer is the most curable cancer if it is discovered early. The traditional colorectal cancer screening method mainly comprises colonoscopy, biopsy and cast-off cytology, and the examination methods are invasive, particularly the cast-off cytology examination has complicated material taking, is difficult to obtain satisfactory specimens, and has few clinical applications. Many people are not willing to go to early screening for colorectal cancer if the associated symptoms are not present.

Therefore, further improvements are needed for the early diagnosis and screening of colorectal cancer.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. Therefore, the invention aims to provide a colorectal cancer tumor marker, a primer pair, a kit and a colorectal cancer detection method. The colorectal cancer tumor marker provided by the invention can be used for early screening and diagnosis of colorectal cancer patients.

The present invention is obtained based on the following findings of the inventors:

the human intestinal microbiome contains a huge number of microorganisms, which participate in food and drug metabolism and human immune regulation and play an extremely important role in maintaining human health. There are a lot of literature reports that fusobacterium nucleatum has strong correlation with colorectal cancer, and the research on the change of the microbial composition in different stages of the disease can be used as a new means for cancer diagnosis and prognosis effect test: therefore, the invention aims to detect the occurrence and the development of the colorectal cancer early and intervene in the treatment in time by determining the specific gene of the fusobacterium nucleatum in the intestinal tract.

The invention carries out quantitative PCR (qPCR) detection on the specific genes of the fusobacterium nucleatum in the population with case-control, determines the amount of the genes, and analyzes and verifies the probability of the genes for predicting colorectal cancer. The kit is used for developing the intestinal microbial gene detection kit for colorectal cancer patients, and provides quick, simple and convenient effective measures for early screening and diagnosis of colon cancer for the majority of people. Provides basis for early warning, diagnosis and intervention of colorectal cancer.

Specifically, the invention provides the following technical scheme:

according to a first aspect of the present invention there is provided a marker for colorectal cancer, the marker comprising at least one selected from the group consisting of: 1-5, and a nucleic acid sequence which is derived from fusobacterium nucleatum and has homology of more than 95% with any one of the SEQ ID NO 1-5. The research shows that the fusobacterium nucleatum has strong correlation with the colorectal cancer, and the specific nucleic acid sequence with strong correlation with the colorectal cancer in the fusobacterium nucleatum is screened by utilizing the metagenome sequencing research, is used as the marker for detecting the colorectal cancer, and can be used for early diagnosis and screening of the colorectal cancer. The colorectal cancer marker can be any one of SEQ ID NO 1-SEQ ID NO 5, or can be a nucleic acid sequence which is derived from nucleic acid bacillus and has homology of more than 95% with any one of SEQ ID NO 1-SEQ ID NO 5, preferably more than 99%. These markers can be used to reflect the condition of colorectal cancer.

According to a second aspect of the invention, there is provided a set of primer pairs capable of specifically amplifying at least one of the markers of the first aspect of the invention, the primer pairs being selected from any one of SEQ ID NO 6 to SEQ ID NO 35. Wherein, every two adjacent numbered nucleic acid sequences in SEQ ID NO. 6-35 are a pair. Any one of SEQ ID NO 1-SEQ ID NO 5 can be specifically amplified by the primer pairs, so that the primer pairs can be used for early diagnosis and detection of colorectal cancer.

In some embodiments of the invention, the primer pair of any one of SEQ ID NO 6 to SEQ ID NO 11 is capable of specifically amplifying SEQ ID NO 1; any one of the primer pair SEQ ID NO 12-SEQ ID NO 19 can specifically amplify SEQ ID NO 2; any one of the primer pair SEQ ID NO. 20-SEQ ID NO. 23 can specifically amplify SEQ ID NO. 3; any one of the primer pair SEQ ID NO. 24-SEQ ID NO. 31 can specifically amplify SEQ ID NO. 4; any one of the primer pair SEQ ID NO 32-SEQ ID NO 35 can specifically amplify SEQ ID NO 5.

According to a third aspect of the present invention there is provided a nucleic acid probe capable of specifically capturing a marker for colorectal cancer according to the first aspect of the present invention. The colorectal cancer marker used by the invention can be captured by designing a nucleic acid probe, so that the colorectal cancer can be rapidly detected.

According to a fourth aspect of the invention there is provided a construct comprising a colorectal cancer marker according to the first aspect of the invention. By constructing colorectal cancer markers into vectors, a construct is formed that can be used as a positive standard to characterize the diagnosis of colorectal cancer.

In some embodiments of the invention, the construct is a recombinant vector comprising a colorectal cancer marker according to the first aspect of the invention.

In some embodiments of the invention, the vector is a plasmid. The colorectal cancer marker of the first aspect of the present invention is constructed into a plasmid, for example, using the plasmid pUC57, to form a recombinant plasmid, which can be used as a positive standard for characterizing colorectal cancer diagnosis.

According to a fifth aspect of the present invention there is provided a kit comprising reagents for detecting a marker for colorectal cancer according to the first aspect of the invention.

In some embodiments of the invention, the kit may comprise at least one of: the primer set according to the second aspect of the present invention, the nucleic acid probe according to the third aspect of the present invention and/or the construct according to the fourth aspect of the present invention.

In a sixth aspect of the present invention, there is provided a system for detecting colorectal cancer in a subject, comprising: a quantification device for determining the content of the target nucleic acid in the fecal DNA of the subject, the target nucleic acid being a marker according to the first aspect of the invention; and the judging device is connected with the quantifying device and determines whether the person to be detected is the colorectal cancer patient or not based on the content of the target nucleic acid. The 'system for detecting the colorectal cancer of the subject' can also be expressed as 'equipment for detecting the colorectal cancer of the subject' according to needs, and refers to a product capable of being used for detecting the colorectal cancer.

In some embodiments of the present invention, the above-mentioned system for detecting colorectal cancer of a subject may further have the following technical features:

in some embodiments of the present invention, the quantification apparatus is capable of determining the content of the target nucleic acid in the fecal DNA of the subject according to a method comprising: and carrying out PCR amplification on the target nucleic acid, and determining the content of the target nucleic acid in the fecal DNA of the testee.

In some embodiments of the invention, the PCR is a fluorescent quantitative PCR.

In some embodiments of the present invention, the judging means determines whether the subject is a colorectal cancer patient based on the following criteria: if the content of the target nucleic acid is greater than or equal to a predetermined threshold value, the tested person is a colorectal cancer patient; if the content of the target nucleic acid is less than the predetermined threshold value, the subject is a non-colorectal cancer patient.

In some embodiments of the invention, the predetermined threshold is determined by: performing fluorescent quantitative PCR amplification on the target nucleic acid in the fecal DNA of each individual in a population consisting of colorectal cancer patients and non-colorectal cancer patients (normal control) as research objects so as to obtain detection results; drawing an ROC curve based on the detection result, and finding out a point with optimal sensitivity and specificity from the ROC curve; and obtaining the copy number (absolute value) of the target nucleic acid, which is the preset threshold value, corresponding to the fluorescence quantitative PCR based on the point with the optimal sensitivity and specificity. According to an embodiment of the present invention, when the ROC curve is plotted using the detection result, the ROC curve is plotted according to a grouping manner of colorectal cancer patients and non-colorectal cancer patients.

Wherein the copy number (absolute value) of the target nucleic acid is obtained by a standard curve method using a standard with a known copy number.

In some embodiments of the present invention, the predetermined threshold is 6.302898.

Drawings

Fig. 1 is a box plot (boxplot) result provided according to one embodiment of the present invention.

Fig. 2 is a graph roc of a recipient operating characteristic curve provided according to one embodiment of the invention.

Fig. 3 is a schematic diagram of a system for colorectal cancer detection of a subject provided according to an embodiment of the present invention.

Detailed Description

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

The invention aims to solve the technical problem of realizing a noninvasive low-cost early screening or diagnosis technology by screening a new colorectal cancer tumor marker and then detecting the colorectal cancer tumor marker through the excrement DNA of a colorectal cancer patient. For example, the nucleic acid in the fecal DNA of the subject can be sequenced by high-throughput sequencing technology to obtain the content information of the colorectal cancer markers, thereby characterizing the diseased condition of the subject. And for example, the content information of the colorectal cancer markers can be obtained by characterizing the nucleic acid in the fecal DNA of the person to be detected by means of a fluorescent quantitative PCR technology, and the detection result can be conveniently and quickly obtained by means of the fluorescent quantitative PCR technology, so that the popularization and industrialization of colorectal cancer detection are facilitated.

To this end, according to one aspect of the present invention, there is provided a colorectal cancer marker selected from at least one of SEQ ID NO 1 to SEQ ID NO 5. The invention screens the specific sequence of the nucleic acid bacillus by a genome-Wide Association Study (MGWAS) method, and any one of the obtained SEQ ID NO 1-SEQ ID NO 5 can be used for representing the diseased condition of colorectal cancer. In a preferred embodiment of the invention, two, three, four or the species SEQ ID NO 1 to SEQ ID NO 5 are used to characterize the diseased condition of colorectal cancer. In another preferred embodiment of the present invention, SEQ ID NO 1 or SEQ ID NO 2 is used as colorectal cancer marker, preferably SEQ ID NO 1 and SEQ ID NO 2 are used as colorectal cancer markers.

In another aspect of the present invention, the present invention provides a method for detecting colorectal cancer in a subject, comprising: (1) determining the content of the target nucleic acid in the fecal DNA of the testee based on the feces of the testee, wherein the target nucleic acid is at least one of SEQ ID NO. 1-SEQ ID NO. 5; (2) determining whether the subject is a colorectal cancer patient based on the content of the target nucleic acid.

In some embodiments of the present invention, the method for detecting colorectal cancer in a subject as described above may further comprise the following technical features:

in some embodiments of the invention, in step (1), the content of the target nucleic acid in the subject's fecal DNA is determined based on PCR amplification of the target nucleic acid in the subject's fecal DNA.

In some embodiments of the invention, the PCR is a fluorescent quantitative PCR.

In some embodiments of the invention, if the content of the target nucleic acid is greater than or equal to a predetermined threshold, the subject is a colorectal cancer patient; if the amount of the target nucleic acid is less than a predetermined threshold, the subject is not a colorectal cancer patient.

In some embodiments of the invention, the predetermined threshold is determined by: performing fluorescent quantitative PCR amplification on the target nucleic acid in the fecal DNA of each individual in a population consisting of colorectal cancer patients and non-colorectal cancer patients to obtain detection results; based on the detection result, drawing an ROC curve according to a grouping mode of colorectal cancer patients and non-colorectal cancer patients, and finding out a point with the best sensitivity and specificity from the ROC curve; and obtaining the copy number (absolute value) of the target nucleic acid, which is the preset threshold value, corresponding to the fluorescence quantitative PCR based on the point with the optimal sensitivity and specificity.

In some embodiments of the present invention, the predetermined threshold is 6.302898.

According to another aspect of the present invention, there is provided a system for detecting colorectal cancer in a subject, as shown in fig. 3, comprising: the quantitative device is connected with the judging device and used for determining the content of target nucleic acid in the fecal DNA of the person to be detected by carrying out PCR amplification on the target nucleic acid in the fecal DNA of the person to be detected, wherein the target nucleic acid is at least one of SEQ ID NO 1-SEQ ID NO 5; the judging means determines whether the subject is a colorectal cancer patient based on the content of the target nucleic acid. According to an embodiment of the present invention, the judging means determines whether the subject is a colorectal cancer patient based on the following criteria: if the content of the target nucleic acid is greater than or equal to a predetermined threshold value, the tested person is a colorectal cancer patient; if the content of the target nucleic acid is less than a predetermined threshold value, the subject is a non-colorectal cancer patient.

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.