CN114959024A - Biological target for DNA methylation detection of fecal exfoliated cells, primer probe composition, application and kit and use method thereof - Google Patents
Biological target for DNA methylation detection of fecal exfoliated cells, primer probe composition, application and kit and use method thereof Download PDFInfo
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Abstract
A biological target for DNA methylation detection of fecal exfoliated cells, a primer probe composition, application, a kit and a use method thereof relate to the technical field of in vitro detection; the application of the biological target in the biological target for DNA methylation detection of the fecal cast-off cells comprises SFRP2, NPY, PDX1, APC, COL4A2, FGF5 and SDC2 genes. The primer probe composition can realize methylation specific amplification detection of SFRP2, NPY, PDX1, APC, COL4A2, FGF5 and SDC2 genes, and methylation conditions of the gene combination can be used as molecular biological markers for colorectal cancer malignancy and prognosis evaluation, so that colorectal cancer screening is realized, and the accuracy is high.
Description
Technical Field
The invention belongs to the technical field of in-vitro detection, and particularly relates to a biological target for DNA methylation detection of fecal exfoliated cells, a primer probe composition, application, a kit and a use method thereof.
Background
Colorectal cancer is one of the most common human malignancies, the third most common of which is the worldwide malignancy. The survival rate of colorectal cancer patients is closely related to the stage of tumor, the survival rate of colorectal cancer patients in early stage after operation for 5 years exceeds 90%, but most of the colorectal cancer patients are in the advanced stage when visiting. 93% of colorectal cancers are derived from adenomas, no specific symptoms exist in the early stage, and 3-17 years are needed for the development of the adenomas to the carcinoma, so that a time basis is provided for early screening, the precancerous lesion can be timely found through effective early screening, and the survival rate of patients is improved by adopting corresponding treatment measures.
At present, colorectal cancer screening methods are mainly divided into two categories, namely fecal detection and colon structural detection. Fecal detection includes Fecal Occult Blood Test (FOBT) and fecal DNA test (sDNA); colon structural detection includes sigmoidoscopy (FSIG), Colonoscopy (CS), double barium colonography (DCBE), and CT colonography (CTC). The fecal occult blood detection FOBT is a simple and rapid colorectal cancer screening method, and is used for detecting trace hemoglobin in feces. FOBT detection is simple, convenient, cheap and noninvasive, can be used for large-scale screening research, but has more influencing factors, positive results can be detected only under the condition of ulcerated bleeding after tumor formation, bleeding of polyp or cancer tissues is intermittent and cannot be contained in any stool specimen, so that the specificity is not high, and missed diagnosis is easy to occur. Sigmoidoscopy (FSIG) is a method for directly examining the distal colon by using an endoscope technology, has simpler operation, lower requirement on intestinal tract preparation, does not need sedation during operation, can carry out biopsy and treatment on pathological changes, and is a relatively cheap, simple and safe screening method; however, the site of FSIG examination is limited to the distal large intestine (bounded by splenic flexure), and if FSIG detects a distal large intestine tumor before performing colonoscopy, 72.0% of the proximal large intestine tumors will be missed. Colonoscopy (CS) is the current gold standard for diagnosis and treatment of large bowel cancer, allowing a complete view of the entire colon and the ability to perform tissue biopsies as well as the removal of polyps found. Although CS has the advantages of good curative effect, high accuracy and the like, CS also has certain limitations: (1) CS is an invasive procedure with a higher risk of complications, possibly leading to colonic perforation (0.3%) and even death (1/5000); (2) long time consuming, requiring adequate bowel preparation and sedation prior to examination; (3) more expensive, more expensive if a tissue biopsy is required and the polyps found are resected; (4) the missed diagnosis rate of the colonoscope on colorectal cancer is 5%. CT colonography (CTC) is a non-invasive examination procedure that allows tomographic scanning at a thickness of 1-2mm to examine intestinal lesions in two and three dimensional images. CTC has the advantages of rapidness, accuracy, low complication and the like, and the screening efficiency of CTC on larger polyps and colorectal cancer is close to that of CS. However, as a conventional screening method, CTCs have significant disadvantages: in order to obtain satisfactory images, 2-4 rads of X-rays are still required to be released to the abdomen of the patient during the CTC examination process, so that the risk of cancer is increased; air or carbon dioxide is usually injected into the intestinal cavity to expand the intestinal tract, so that a patient feels certain discomfort during examination; in addition, high inspection costs impose a heavy economic burden on both individuals and society.
Fecal exfoliated cells and gene detection (sDNA) thereof are the leading technology of colorectal cancer screening at present, and are a non-invasive screening method, compared with normal large intestine mucous membranes, the turnover metabolism speed of the colorectal cancer cells is obviously increased, the number of the exfoliated cells is increased, and partial colorectal cancer can be effectively screened by quantitatively analyzing the DNA of the fecal exfoliated cells. The method generally comprises two types of detection: one is the detection of fecal cast-off cytology: the method is to carry out routine pathological examination on the intestinal epithelial cells exfoliated from the feces so as to find out the tumor cells in the intestinal epithelial cells, thereby achieving the purpose of clearly diagnosing the intestinal tumor. The second type is the detection of DNA from exfoliated cells of feces: as shown by the research, the mutant gene in the tumor cell shed from the excrement of the patient has high consistency with the mutant gene of the tumor tissue. If the genes can be detected from the tumor cells dropped from the feces, the diagnosis of the intestinal tumor can be also definite, the method also has higher specificity, and the existence of the lesion can be basically confirmed by a positive result; the dietary habits of the screened population do not need to be changed; there is no need to restrict the use of the drug, etc.
At present, the existing methods for detecting DNA methylation of exfoliated cells of feces all adopt a single gene target, the method is easily influenced by eating habits, living environment and other chronic diseases of human bodies, and meanwhile, the accuracy of colorectal cancer screening is improved by adopting a plurality of gene targets, but the research on the correlation between the gene targets and colorectal cancer is rarely reported.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a biological target for DNA methylation detection of fecal cast-off cells, which can improve the accuracy of DNA methylation detection of the fecal cast-off cells.
The invention also aims to provide a primer probe composition for DNA methylation detection of the fecal cast-off cells, which can realize methylation specific amplification detection of SFRP2, NPY, PDX1, APC, COL4A2, FGF5 and SDC2 genes and improve the accuracy of analysis of colorectal cancer.
The invention also aims to provide application of the primer probe composition for DNA methylation detection of the exfoliated cells of the feces.
The fourth purpose of the invention is to provide a kit for detecting colorectal cancer based on the DNA methylation state of the exfoliated cells of the feces, which can realize early screening and auxiliary diagnosis of colorectal cancer and has the advantages of high sensitivity, strong specificity and high accuracy.
The fifth object of the present invention is to provide a method for using the above-mentioned kit for detecting colon cancer based on the DNA methylation state of exfoliated fecal cells.
One of the purposes of the invention is realized by adopting the following technical scheme:
a biological target for DNA methylation detection of fecal exfoliated cells, the biological target comprising SFRP2, NPY, PDX1, APC, COL4a2, FGF5 and SDC2 genes.
The second purpose of the invention is realized by adopting the following technical scheme:
a primer probe composition for DNA methylation detection of a fecal cast-off cell specifically comprises: a first forward primer, a first probe and a first reverse primer for methylation specific amplification of the SFRP2 gene; a second forward primer, a second probe and a second reverse primer for methylation specific amplification of the NPY gene; a third forward primer, a third probe and a third reverse primer for methylation specific amplification of the PDX1 gene; a fourth forward primer, a fourth probe and a fourth reverse primer for methylation specific amplification of the APC gene; a fifth forward primer, a fifth probe and a fifth reverse primer for methylation specific amplification of COL4A2 gene; a sixth forward primer, a sixth probe and a sixth reverse primer for methylation specific amplification of the FGF5 gene; a seventh forward primer, a seventh probe, and a seventh reverse primer for methylation specific amplification of the SDC2 gene;
the nucleotide sequence of the first forward primer is shown as SEQ ID NO. 1;
the nucleotide sequence of the first probe is shown as SEQ ID NO. 2;
the nucleotide sequence of the first reverse primer is shown as SEQ ID NO. 3;
the nucleotide sequence of the second forward primer is shown as SEQ ID NO. 4;
the nucleotide sequence of the second probe is shown as SEQ ID NO. 5;
the nucleotide sequence of the second reverse primer is shown as SEQ ID NO. 6;
the nucleotide sequence of the third forward primer is shown as SEQ ID NO. 7;
the nucleotide sequence of the third probe is shown as SEQ ID NO. 8;
the nucleotide sequence of the third reverse primer is shown as SEQ ID NO. 9;
the nucleotide sequence of the fourth forward primer is shown as SEQ ID NO. 10;
the nucleotide sequence of the fourth probe is shown as SEQ ID NO. 11;
the nucleotide sequence of the fourth reverse primer is shown as SEQ ID NO. 12;
the nucleotide sequence of the fifth forward primer is shown as SEQ ID NO. 13;
the nucleotide sequence of the fifth probe is shown as SEQ ID NO. 14;
the nucleotide sequence of the fifth reverse primer is shown as SEQ ID NO. 15;
the nucleotide sequence of the sixth forward primer is shown as SEQ ID NO. 16;
the nucleotide sequence of the sixth probe is shown as SEQ ID NO. 17;
the nucleotide sequence of the sixth reverse primer is shown as SEQ ID NO. 18;
the nucleotide sequence of the seventh forward primer is shown as SEQ ID NO. 19;
the nucleotide sequence of the seventh probe is shown as SEQ ID NO. 20;
the nucleotide sequence of the seventh reverse primer is shown as SEQ ID NO. 21.
Further, the 5' ends of the first probe, the second probe, the third probe, the fourth probe, the fifth probe, the sixth probe and the seventh probe are respectively marked with any one of FAM, VIC, ROX and HEX fluorescent groups; the 3' end of the compound is respectively marked with any one of MGB and BHQ quenching groups.
The third purpose of the invention is realized by adopting the following technical scheme:
an application of a primer probe composition for DNA methylation detection of a fecal exfoliated cell in preparation of a reagent for detecting colorectal cancer.
The fourth purpose of the invention is realized by adopting the following technical scheme:
a kit for detecting colorectal cancer based on the DNA methylation state of a fecal exfoliative cell comprises a fluorescent quantitative reaction solution, wherein the fluorescent quantitative reaction solution comprises a primer probe composition for the DNA methylation detection of the fecal exfoliative cell.
Further, the fluorescent quantitative reaction solution comprises a first fluorescent quantitative reaction solution and a second fluorescent quantitative reaction solution;
the first fluorescent quantitative reaction solution includes: a first forward primer, a first probe, a first reverse primer, a second forward primer, a second probe, a second reverse primer, a third forward primer, a third probe, a third reverse primer, a fourth forward primer, a fourth probe, and a fourth reverse primer;
the second fluorescent quantitative reaction solution includes: a fifth forward primer, a fifth probe, a fifth reverse primer, a sixth forward primer, a sixth probe, a sixth reverse primer, a seventh forward primer, a seventh probe, and a seventh reverse primer.
Further, the kit also comprises lysis solution, primers and probes of internal reference genes and bisulfite solution.
Furthermore, the reference gene is GAPDH, and a forward primer, a probe and a reverse primer of the reference gene are respectively shown as SEQ ID NO. 22-24.
The fifth purpose of the invention is realized by adopting the following technical scheme:
a use method of a kit for detecting colorectal cancer based on DNA methylation state of exfoliated cells of feces comprises the following steps:
1) collecting a sample of exfoliated cells from the feces;
2) extracting genomic DNA from the fecal exfoliated cell sample;
3) carrying out methylation conversion on the genome DNA to convert cytosine of the genome DNA into uracil to obtain bis-DNA;
4) the bis-DNA was PCR amplified.
Further, in step 4), the fluorescence program for PCR amplification is as follows: the first step is that the temperature is 93-97 ℃, the time is 4-6min, and the circulation is carried out for 1 time; the second step is that the temperature is 93-97 ℃, the temperature is 63-65 ℃ after the time is 14-16s, the time is 28-32s, and the circulation is carried out for 18-22 times; the third step is that the temperature is 93-97 ℃ and the time is 9-11s, after circulation is carried out for 38-42 times, the temperature is 58-62 ℃ and the time is 30-32s, and fluorescence is collected.
Compared with the prior art, the invention has the beneficial effects that:
the primer probe composition for DNA methylation detection of the fecal exfoliated cells can realize methylation specificity amplification detection of SFRP2, NPY, PDX1, APC, COL4A2, FGF5 and SDC2 genes, and the methylation condition of the gene combination can be used as a molecular biological marker for malignancy and prognosis evaluation of colorectal cancer, so that screening of colorectal cancer is realized, and the accuracy is high.
The kit for detecting colorectal cancer based on the DNA methylation state of the exfoliated cells of the feces has the advantages of simple operation, no pain and no wound, capability of realizing early screening and auxiliary diagnosis of colorectal cancer, high detection efficiency, high sensitivity, strong specificity and high accuracy.
Drawings
FIG. 1 is a graph showing the amplification curve of a normal sample in a first PCR reaction system according to the present invention; wherein, 1 is a gene curve containing CY5 fluorophore, 2 is a gene curve containing VIC fluorophore, 3 is a gene curve containing FAM fluorophore, and 4 is a gene curve containing ROX fluorophore.
FIG. 2 is a graph showing the amplification curve of a normal sample in a second PCR reaction system according to the present invention; wherein, 1 is a gene curve containing CY5 fluorophore, 2 is a gene curve containing HEX fluorophore, 3 is a gene curve containing FAM fluorophore, and 4 is a gene curve containing ROX fluorophore.
FIG. 3 is a graph showing the amplification of a colon cancer sample in the first PCR reaction system of the present invention; wherein, 1 is a gene curve containing CY5 fluorophore, 2 is a gene curve containing VIC fluorophore, 3 is a gene curve containing FAM fluorophore, and 4 is a gene curve containing ROX fluorophore.
FIG. 4 is a graph showing the amplification of a colon cancer sample in a second PCR reaction system according to the present invention; wherein, 1 is a gene curve containing CY5 fluorophore, 2 is a gene curve containing HEX fluorophore, 3 is a gene curve containing FAM fluorophore, and 4 is a gene curve containing ROX fluorophore.
FIG. 5 is a ROC diagram in the first PCR reaction system of the present invention.
FIG. 6 is a ROC diagram in the second PCR reaction system of the present invention.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
Example 1
A primer probe composition for DNA methylation detection of fecal exfoliated cells, wherein the primer probe composition is used for methylation detection of SFRP2, NPY, PDX1, APC, COL4A2, FGF5 and SDC2 genes.
In this example, multiple sets of primers and probes were designed based on the human whole genome sequences SFRP2, NPY, PDX1, APC, COL4A2, FGF5 and SDC2 gene sequences disclosed in the National Center for Biotechnology Information (NCBI). The primer sequences with optimal specificity and sensitivity effects are obtained by screening as follows:
a fourth forward primer, a fourth probe and a fourth reverse primer for methylation specific amplification of the APC gene; a fifth forward primer, a fifth probe and a fifth reverse primer for methylation specific amplification of COL4A2 gene; a sixth forward primer, a sixth probe and a sixth reverse primer for methylation specific amplification of the FGF5 gene; a seventh forward primer, a seventh probe, and a seventh reverse primer for methylation specific amplification of the SDC2 gene;
primer probe composition for methylation specific amplification of the SFRP2 gene:
the first forward primer is marked as SFRP2-Me-PF, and the nucleotide sequence is shown as SEQ ID NO. 1;
the Probe is marked as SFRP2-Me Probe (FAM), wherein the 5 'end is respectively marked with FAM fluorescent groups (FAM, VIC, ROX, CY5 and HEX in the name of the Probe are fluorescent groups), the 3' end is respectively marked with MGB quenching groups, and the nucleotide sequence is shown as SEQ ID NO. 2;
the first reverse primer is marked as SFRP2-Me-PR, and the nucleotide sequence is shown as SEQ ID NO. 3;
primer probe composition for methylation specific amplification of NPY gene:
the second forward primer is marked as NPY-Me-PF, and the nucleotide sequence is shown as SEQ ID NO. 4;
a second Probe, which is marked as NPY-Me Probe (VIC), and the nucleotide sequence is shown as SEQ ID NO. 5;
the second reverse primer is marked as NPY-Me-PR, and the nucleotide sequence is shown as SEQ ID NO. 6;
primer probe composition for methylation specific amplification of PDX1 gene:
a third forward primer, which is marked as PDX1-Me-PF, and the nucleotide sequence is shown as SEQ ID NO. 7;
a third Probe, which is marked as PDX1-Me Probe (ROX), and the nucleotide sequence is shown as SEQ ID NO. 8;
a third reverse primer, which is marked as PDX1-Me-PR, and the nucleotide sequence is shown as SEQ ID NO. 9;
primer probe composition for methylation specific amplification of APC genes:
the fourth forward primer is recorded as APC-Me-PF, and the nucleotide sequence is shown as SEQ ID NO. 10;
the fourth Probe is marked as APC-Me Probe (ROX), and the nucleotide sequence is shown as SEQ ID NO. 11;
a fourth reverse primer, which is marked as APC-Me-PR, and the nucleotide sequence is shown as SEQ ID NO. 12;
primer probe composition for methylation specific amplification of COL4a2 gene:
a fifth forward primer, which is marked as COL4A2-1-Me-PF, and the nucleotide sequence is shown in SEQ ID NO. 13;
a fifth Probe, which is marked as COL4A2-1 Me Probe (FAM), and the nucleotide sequence is shown in SEQ ID NO: 14;
the fifth reverse primer is marked as COL4A2-1-Me-PR, and the nucleotide sequence is shown as SEQ ID NO. 15;
primer probe composition for methylation specific amplification of FGF5 gene:
a sixth forward primer, which is marked as FGF5-Me-PF, and the nucleotide sequence is shown as SEQ ID NO. 16;
a sixth Probe, which is marked as FGF5-Me Probe (ROX), and the nucleotide sequence is shown as SEQ ID NO. 17;
a sixth reverse primer, which is marked as FGF5-Me-PR, and the nucleotide sequence is shown as SEQ ID NO. 18;
primer probe compositions for methylation specific amplification of SDC2 gene:
the seventh forward primer is recorded as SDC2-Me-PF, and the nucleotide sequence is shown as SEQ ID NO. 19;
a seventh Probe, which is marked as SDC2-Me Probe (HEX), and the nucleotide sequence is shown as SEQ ID NO: 20;
the seventh reverse primer, is marked as SDC2-Me-PR, and has the nucleotide sequence shown in SEQ ID NO. 21.
Primer probe composition for methylation specific amplification of the reference gene GAPDH:
the forward primer (GAPDH-Me-PR), the Probe (GAPDH-Me Probe (CY5)) and the reverse primer (GAPDH-Me-PF) of the reference gene are shown in SEQ ID NO: 22-24 respectively.
The nucleotide sequence of the primer probe composition of this example is specifically shown in Table 1.
TABLE 1 nucleotide sequence
Example 2
A kit for detecting colorectal cancer based on the DNA methylation state of a fecal exfoliative cell comprises a fluorescent quantitative reaction solution, a lysis solution, primers and probes of an internal reference gene and a bisulfite solution, wherein the fluorescent quantitative reaction solution comprises the primer probe composition for detecting the DNA methylation state of the fecal exfoliative cell.
The fluorescent quantitative reaction liquid comprises a first fluorescent quantitative reaction liquid and a second fluorescent quantitative reaction liquid;
the first fluorescent quantitative reaction solution includes: a first forward primer, a first probe, a first reverse primer, a second forward primer, a second probe, a second reverse primer, a third forward primer, a third probe, a third reverse primer, a fourth forward primer, a fourth probe, and a fourth reverse primer;
the second fluorescent quantitative reaction solution comprises: a fifth forward primer, a fifth probe, a fifth reverse primer, a sixth forward primer, a sixth probe, a sixth reverse primer, a seventh forward primer, a seventh probe, and a seventh reverse primer.
Example 3
A use method of a kit for detecting colorectal cancer based on DNA methylation state of exfoliated cells of feces comprises the following steps:
1. collecting a fecal exfoliated cell sample and a normal sample;
2. extracting genomic DNA from the fecal exfoliated cell sample;
the method specifically comprises the following steps: (1) weighing 200mg solid excrement sample or about 200 μ L liquid excrement sample, placing in a 1.5mL centrifuge tube, adding 1mL lysate, then oscillating for 1min to fully mix excrement at the bottom of the tube, and then placing the 1.5mL centrifuge tube in a water bath kettle at 70 ℃ for 5 min; taking out, centrifuging for 5min, slowly sucking 300 μ L supernatant, adding 200 μ L lysate and 20 μ L proteinase K, shaking, and placing in 70 deg.C water bath for 10min to obtain DNA lysis solution.
(2) And adding the DNA lysis solution into 200 mu L of isopropanol, stirring uniformly, adding into an adsorbent, centrifuging, discarding waste liquid, and washing to obtain the DNA of the fecal exfoliated cells.
3. Carrying out methylation conversion on the genomic DNA through a bisulfite solution to convert cytosine of the genomic DNA into uracil to obtain bis-DNA;
the method specifically comprises the following steps: (1) transferring all the DNA of the fecal exfoliated cells into a 0.2mL centrifuge tube, adding 150 mu L of bisulfite solution, shaking up, and placing in a temperature changer, wherein the setting conditions are as follows:
①98℃，9min；
②60℃，60min；
keeping the temperature at 4 ℃ (not more than 20 h).
(2) And after the conversion reaction is finished, moving the product to an adsorption column, washing, centrifuging and collecting to obtain bis-DNA.
And (4) gently blowing and beating the mixture by a flick centrifugal tube or a liquid transfer device, and centrifuging the mixture for a short time after the mixture is uniformly mixed.
4. The bis-DNA was subjected to PCR amplification.
The method specifically comprises the following steps: (1) a first PCR reaction system and a second PCR reaction system were prepared according to tables 2 and 3, respectively.
TABLE 2 first PCR reaction System
TABLE 3 second PCR reaction System
(2) The first PCR reaction system and the second PCR reaction system were sent to a PCR instrument for reaction, respectively, under the reaction conditions shown in Table 4.
TABLE 4 PCR reaction Condition settings
(3) And respectively reading Ct values of the first PCR reaction system and the second PCR reaction system.
Example 4
In this example, a normal sample was PCR-amplified by the first PCR reaction system and the second PCR reaction system of example 3, and the amplification curve is shown in fig. 1 and fig. 2.
Fig. 3 and 4 show amplification curves obtained by subjecting a colon cancer sample to PCR amplification in the first PCR reaction system and the second PCR reaction system of example 3.
Referring to FIGS. 1 to 4, in a normal sample, the reference gene GAPDH was labeled with a probe containing CY5 fluorophore (line 1), and the Ct value thereof increased with the increase of the number of PCR cycles, and the contents of the genes of interest (SFRP2, NPY, PDX1, APC, COL4A2, FGF5, and SDC2 genes) were almost unchanged; in the colorectal cancer sample, the Ct values of the GAPDH, SFRP2, NPY, PDX1, APC, COL4A2, FGF5 and SDC2 genes are increased along with the increase of the number of PCR cycles, and the increment rate is greater than that of an internal reference gene, so that methylation specific amplification detection of SFRP2, NPY, PDX1, APC, COL4A2, FGF5 and SDC2 genes which can be realized by the primer probe composition is shown, the methylation condition of the gene combination can be used as a molecular biological marker for evaluating the malignancy degree and prognosis of colorectal cancer, the screening of colorectal cancer is realized, and the accuracy is high.
Referring to fig. 5 to 6, which are ROC curves drawn by PCR results of the first and second PCR reaction systems, the detection method of the present invention has a good diagnostic effect, and has excellent sensitivity and specificity.
Example 5
Taking 23 groups of normal samples to carry out fluorescence quantitative PCR detection, and respectively marking the samples as N1-N23; and (3) performing fluorescent quantitative PCR detection on 9 groups of colorectal cancer samples, wherein the samples are respectively marked as C1-C9. The results are shown in Table 5, which were converted into methylation values (EI) based on the detected CT values.
The calculation method comprises the following steps:
the total EI value is the sum of EI values of all target genes;
the CT value of the target gene-the CT value of the reference gene;
conversion of CT value to EI value
(1) The target gene CT is 0; EI is 0;
the target gene delta CT is less than or equal to 0, and EI is 5;
(2) when the target gene is less than or equal to 20,
△CT＝1～5，EI＝3；△CT＝6～10，EI＝2；△CT>10，EI＝1；
(3) the CT of the target gene is 20< CT and is less than or equal to 25,
△CT＝1～10，EI＝2；△CT>10，EI＝1；
(4) the CT value of the target gene is more than 25,
△CT＝1～10，EI＝1；△CT>10，EI＝0。
TABLE 5
When cut-off value of the EI is 20, the sensitivity is 100% (9/9), and the specificity is 95.6% (22/23), which indicates that the methylation condition of the gene combination can be used as a molecular biological marker for assessing malignancy degree and prognosis of colorectal cancer; the primer probe composition can realize methylation specific amplification detection of SFRP2, NPY, PDX1, APC, COL4A2, FGF5 and SDC2 genes, and has high sensitivity and strong specificity.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
SEQUENCE LISTING
<110> Erlijian Biotechnology Ltd, Guangzhou
<120> biological target for DNA methylation detection of fecal exfoliated cells, primer probe composition, application, kit and use method thereof
<130> 2022
<160> 24
<170> PatentIn version 3.3
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Claims (10)
1. A biological target for DNA methylation detection of fecal exfoliated cells, wherein the biological target comprises SFRP2, NPY, PDX1, APC, COL4a2, FGF5 and SDC2 genes.
2. A primer probe composition for DNA methylation detection of a fecal cast-off cell is characterized by specifically comprising: a first forward primer, a first probe and a first reverse primer for methylation specific amplification of the SFRP2 gene; a second forward primer, a second probe and a second reverse primer for methylation specific amplification of the NPY gene; a third forward primer, a third probe and a third reverse primer for methylation specific amplification of the PDX1 gene; a fourth forward primer, a fourth probe and a fourth reverse primer for methylation specific amplification of the APC gene; a fifth forward primer, a fifth probe and a fifth reverse primer for methylation specific amplification of COL4A2 gene; a sixth forward primer, a sixth probe and a sixth reverse primer for methylation specific amplification of the FGF5 gene; a seventh forward primer, a seventh probe, and a seventh reverse primer for methylation specific amplification of the SDC2 gene;
the nucleotide sequence of the first forward primer is shown as SEQ ID NO. 1;
the nucleotide sequence of the first probe is shown as SEQ ID NO. 2;
the nucleotide sequence of the first reverse primer is shown as SEQ ID NO. 3;
the nucleotide sequence of the second forward primer is shown as SEQ ID NO. 4;
the nucleotide sequence of the second probe is shown as SEQ ID NO. 5;
the nucleotide sequence of the second reverse primer is shown as SEQ ID NO. 6;
the nucleotide sequence of the third forward primer is shown as SEQ ID NO. 7;
the nucleotide sequence of the third probe is shown as SEQ ID NO. 8;
the nucleotide sequence of the third reverse primer is shown as SEQ ID NO. 9;
the nucleotide sequence of the fourth forward primer is shown as SEQ ID NO. 10;
the nucleotide sequence of the fourth probe is shown as SEQ ID NO. 11;
the nucleotide sequence of the fourth reverse primer is shown as SEQ ID NO. 12;
the nucleotide sequence of the fifth forward primer is shown as SEQ ID NO. 13;
the nucleotide sequence of the fifth probe is shown as SEQ ID NO. 14;
the nucleotide sequence of the fifth reverse primer is shown as SEQ ID NO. 15;
the nucleotide sequence of the sixth forward primer is shown as SEQ ID NO. 16;
the nucleotide sequence of the sixth probe is shown as SEQ ID NO. 17;
the nucleotide sequence of the sixth reverse primer is shown as SEQ ID NO. 18;
the nucleotide sequence of the seventh forward primer is shown as SEQ ID NO. 19;
the nucleotide sequence of the seventh probe is shown as SEQ ID NO. 20;
the nucleotide sequence of the seventh reverse primer is shown as SEQ ID NO. 21.
3. The primer-probe composition for DNA methylation detection of fecal exfoliated cells of claim 2, wherein: the 5' ends of the first probe, the second probe, the third probe, the fourth probe, the fifth probe, the sixth probe and the seventh probe are respectively marked with any one of FAM, VIC, ROX, CY5 and HEX fluorescent groups; the 3' end of the compound is respectively marked with any one of MGB and BHQ quenching groups.
4. Use of the primer probe composition for detecting DNA methylation of exfoliated fecal cells as claimed in claim 2 or 3 in the preparation of a reagent for detecting colorectal cancer.
5. A kit for detecting colorectal cancer based on DNA methylation state of a fecal exfoliated cell is characterized in that: comprises a fluorescent quantitative reaction solution which comprises the primer probe composition for DNA methylation detection of the fecal exfoliated cells according to claim 2 or 3.
6. The kit of claim 5, wherein: the fluorescent quantitative reaction liquid comprises a first fluorescent quantitative reaction liquid and a second fluorescent quantitative reaction liquid;
the first fluorescent quantitative reaction solution includes: a first forward primer, a first probe, a first reverse primer, a second forward primer, a second probe, a second reverse primer, a third forward primer, a third probe, a third reverse primer, a fourth forward primer, a fourth probe, and a fourth reverse primer;
the second fluorescent quantitative reaction solution comprises: a fifth forward primer, a fifth probe, a fifth reverse primer, a sixth forward primer, a sixth probe, a sixth reverse primer, a seventh forward primer, a seventh probe, and a seventh reverse primer.
7. The kit of claim 5 or 6, wherein: also comprises lysis solution, primers and probes of reference genes and bisulfite solution.
8. The kit of claim 7, wherein: the reference gene is GAPDH, and a forward primer, a probe and a reverse primer of the reference gene are respectively shown as SEQ ID NO. 22-24.
9. A method of using the kit for detecting colon cancer based on DNA methylation status of exfoliated fecal cells according to any one of claims 5 to 8, comprising the steps of:
1) collecting a sample of exfoliated cells from the feces;
2) extracting genomic DNA from the fecal exfoliated cell sample;
3) carrying out methylation conversion on the genome DNA to convert cytosine of the genome DNA into uracil to obtain bis-DNA;
4) the bis-DNA was subjected to PCR amplification.
10. The method of using a kit for detecting colon cancer based on the methylation status of DNA of exfoliated fecal cells according to claim 9, wherein: in the step 4), the PCR amplification fluorescence program is as follows: the first step is that the temperature is 93-97 ℃, the time is 4-6min, and the circulation is carried out for 1 time; the second step is that the temperature is 93-97 ℃, the temperature is 63-65 ℃ after the time is 14-16s, the time is 28-32s, and the circulation is carried out for 18-22 times; the third step is that the temperature is 93-97 ℃ and the time is 9-11s, after circulation is carried out for 38-42 times, the temperature is 58-62 ℃ and the time is 30-32s, and fluorescence is collected.
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