Colorectal cancer testing composition, reagent kit and application

Abstract

The present invention provides a composition, a reagent kit and an application for testing colorectal cancer. The composition provided includes a reagent containing the methylation status of the ADHFE1 gene, the PPP2R5C gene and the SDC2 gene, and can be used to test for colorectal cancer, with high sensitivity and specificity.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, particularly to a composition, reagent kit and application for testing colorectal cancer. [Background technology]

[0002] Colorectal cancer, which develops in the colon and rectum of the lower gastrointestinal tract, is a common malignant tumor. According to the latest statistics, colorectal cancer ranks third in new cases among men and second among women worldwide, and fourth in mortality rates among men and third among women (Bray et al., 2018). China's colorectal cancer prevention and control situation is also very poor, ranking third in new cases (388,000) and fifth in mortality rates (187,000) among malignant tumors (Zheng Rongshou et al., 2019). Colorectal cancer progresses slowly, generally progressing through polyps, adenomas, and intestinal cancer. It takes 5–10 years for adenomas to develop into intestinal cancer. Early intervention in the progression of colorectal cancer can significantly reduce mortality rates. The five-year survival rate for patients with stage I colorectal cancer can reach over 90%, while the five-year survival rate for patients with stage IV colorectal cancer is lower than 20% (Marzieh Araghi et al., 2020).

[0003] Traditional early screening techniques for colorectal cancer primarily include colonoscopy, fecal occult blood testing, and tumor markers CEA and CA19-9. However, these techniques currently have certain limitations. Colonoscopy is the gold standard for colon cancer diagnosis. It involves inserting a lens and light source through the anus, sequentially passing through the rectum, ileum, and other areas, and then transmitting images in real time to a monitor for observation by the operating physician. Colonoscopy produces clear, intuitive images, allowing for the detection of various lesions, including cancer, polyps, ulcers, and bleeding. It also allows for the removal of polyps and other lesions. However, it is an invasive examination, requires complex preparation, dietary restrictions, and bowel cleansing, resulting in low patient compliance. Furthermore, it requires equipment and personnel, requires the assistance of hospital specialists and anesthesiologists, and carries the risk of discomfort and complications for some patients (3-5 cases per 1,000 patients). The fecal occult blood test is a non-invasive method that detects the presence or absence of gastrointestinal bleeding by detecting blood components (hemoglobin) in the stool, allowing doctors to assess the risk of colorectal cancer. This method is quick and simple, with high patient compliance. However, this test is susceptible to the influence of dietary sources such as liver, blood products, and green leafy vegetables, resulting in false positives. At the same time, it has low sensitivity for patients with early-stage colorectal cancer who experience minimal bleeding, making it easy to miss a diagnosis. Broad-spectrum tumor markers such as CEA have low specificity, frequent false positives, and limited sensitivity. Therefore, there is a need for an accurate, simple, and economical method for early colorectal cancer screening.

[0004] DNA methylation is an important gene expression regulatory mechanism that can regulate gene expression and repression, significantly impacting tumor development and progression. Abnormal methylation of cancer-related genes is often observed early in cancer development, so DNA methylation signals are considered a potential early tumor screening marker. Human fecal samples contain intestinal cell DNA carrying methylation information, and testing and analyzing this information can predict a subject's risk of developing colon cancer. Some genes, such as SFRP2, SEPT9, NDRG4, and SDC2 (Hannes M. Mueller et al., 2004; Jie Chen et al., 2019), have been shown to be useful as fecal methylation markers for colon cancer screening, but their performance does not fully meet clinical needs. For example, the sensitivity and specificity of SFRP2 for colon cancer detection is only 77% (Hannes M. Mueller et al., 2004). Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a colorectal cancer testing composition, a reagent kit, and applications thereof, which solves to a certain extent one of the technical problems in the related art. The provided composition can be used to test for colorectal cancer, and its specificity and sensitivity are both high, which can assist clinical testing and enrich the diagnosis and testing of colorectal cancer. [Means for solving the problem]

[0006] Specifically, the present invention provides the following technical solutions:

[0007] In a first aspect, the present invention provides a composition for testing colorectal cancer, which comprises reagents for testing the methylation status of the ADHFE1 gene, the PPP2R5C gene, and the SDC2 gene.

[0008] In a second aspect of the present invention, the present invention provides a reagent kit for diagnosing colorectal cancer, the reagent kit comprising the colorectal cancer testing composition according to the first aspect.

[0009] In a third aspect of the present invention, there is provided a method for determining the methylation status of genes in a sample, wherein the genes comprise SDC2 gene, ADHFE1 and PPP2R5C gene, the method comprising: (1) obtaining genomic DNA from the sample; (2) methylating and transforming the genomic DNA to obtain a transformation product, wherein the genomic DNA is methylated and exhibits a difference between methylated and unmethylated sites; (3) performing a fluorescent quantitative PCR test on the transformation product using a primer set and a probe to determine the methylation status of genes in the sample.

[0010] In a fourth aspect of the present invention, the present invention provides an isolated nucleic acid sequence, the isolated nucleic acid sequence comprising a primer set and a probe, the primer set comprising: (a) at least one pair of sequences selected from the sequences set forth in SEQ ID NO:3 to SEQ ID NO:40; (b) at least one pair of sequences set forth in SEQ ID NO: 41 to SEQ ID NO: 60; and (c) comprising at least one pair of sequences set forth in SEQ ID NO: 61 to SEQ ID NO: 98; The probe is At least one of the sequences shown in SEQ ID NO: 100 to SEQ ID NO: 112; At least one of the sequences set forth in SEQ ID NO: 113 to SEQ ID NO: 127, and It contains at least one of the sequences shown in SEQ ID NO:128 to SEQ ID NO:142.

[0011] In a fifth aspect, the present invention provides a method for assessing the colorectal cancer disease status of a subject, the method comprising: a) providing a sample from the subject, the sample comprising target nucleic acids of the subject, the target nucleic acids comprising ADHFE1 gene, PPP2R5C gene and SDC2 gene; b) assessing the methylation status of the target nucleic acids; and c) assessing the colorectal cancer disease status of the subject based on the methylation status of the target nucleic acids.

[0012] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. [Brief explanation of the drawings]

[0013] The above and / or additional aspects and advantages of the present invention will become apparent and easier to understand from the following description of the embodiments taken in conjunction with the drawings. [Figure 1] FIG. 1 is a schematic representation of methylation levels of colorectal cancer and non-colon cancer samples in the TCGA database according to an embodiment of the present invention. [Figure 2] 1 is a fluorescent quantitative PCR amplification graph of a positive standard in Example 2 of the present invention. [Figure 3] 1 is a fluorescent quantitative PCR amplification graph of a negative standard in Example 2 of the present invention. [Figure 4] 1 is a test flow chart of the colorectal cancer diagnostic reagent kit provided by Example 3 of the present invention. [Figure 5] 1 is an ROC graph of each gene-tested colorectal cancer sample in Example 3 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, the embodiments of the present invention will be described in detail, and the described embodiments are merely illustrative and are used only to interpret the present invention, and are not intended to limit the present invention. In the process of explaining the means provided by the present invention, the relevant terms will be interpreted and explained in this specification, but these interpretations and explanations are intended to facilitate understanding of the means, and are not considered to limit the protective means of the present invention.

[0015] In one aspect, the present invention provides a composition for detecting colorectal cancer, comprising reagents for detecting the ADHFE1 gene, the PPP2R5C gene, and the SDC2 gene. The composition referred to herein does not necessarily need to be in a form in which two or more substances are mixed and in contact, and the composition referred to means that it is applied in the same environment when used. For example, when detecting colorectal cancer, the methylation status of the ADHFE1 gene, the PPP2R5C gene, and the SDC2 gene are simultaneously detected.

[0016] Methylation modifications of the ADHFE1, PPP2R5C, and SDC2 genes mentioned herein are closely related to the development and progression of cancer and show significant differences between normal and cancer-affected tissues. For example, the SDC2, ADHFE1, and PPP2R5C genes show high methylation levels in cancer tissues. The ADHFE1 gene encodes a transferase, and its abnormal regulation by hypermethylation can shorten the colon cancer cell cycle and promote cancer cell proliferation (Hu YH et al., 2019). Compared to normal colon tissues, the ADHFE1 gene shows high methylation levels in both cancer tissues and advanced adenoma tissues (Tae CH et al., 2013). The methylation levels of the ADHFE1 gene differ significantly between normal, colon cancer, and adenoma tissues, making it an ideal marker for colon cancer testing (Naumov VA et al., 2013; Fan, J et al., 2020). The PPP2R5C gene encodes a regulatory subunit of PP2A phosphatase, a negative regulator of cell growth and proliferation (Veerle Janssen et al., 2001). There is evidence that its encoded protein regulates the dephosphorylation of p53 in response to DNA damage and suppresses the growth of intestinal cancer cells (Li HH et al., 2007). The hypermethylation status of the PPP2R5C gene is closely associated with the development and progression of intestinal cancer and can be used as a potential marker for intestinal cancer screening (Galamb O et al., 2016). The SDC2 gene encodes an integral membrane protein with important biological functions in processes such as cell division and migration (Kim JH et al., 2018). Many studies have compared the methylation status of cancer tissues and cancer-adjacent tissues in colorectal cancer patients and found that the methylation level of the SDC2 gene differed significantly between cancer tissues and cancer-adjacent tissues, and the detection rate in cancer tissues, adenoma tissues, polyp tissues, and normal tissues tended to decrease significantly, making it an effective marker for colon cancer screening (Oh TJ et al., 2017).

[0017] Currently, the SDC2 gene has become a relatively reliable standard for the diagnosis and testing of colorectal cancer. The present specification describes a method for combining the SDC2 gene and the ADHFE1 gene with the PPP2R5C gene, which provides higher test sensitivity and specificity than the SDC2 gene alone. Furthermore, the combination of the SDC2 gene, the ADHFE1 gene, and the PPP2R5C gene for diagnosing colorectal cancer has higher specificity and sensitivity than other genes, such as NDRG4 and BMP3. For example, the sensitivity can be 85% or more, the specificity can be 90% or more, and even 87% or more, or even 90% or more, and the specificity can be 90% or more, or even 94% or more. Furthermore, it can show better differentiation between colorectal cancer samples and normal samples.

[0018] In at least some embodiments, the reagents include a primer set and a probe. As referred to herein, a "primer" refers to a short nucleic acid sequence with a free 3' hydroxyl group that can base-pair with a complementary template and serve as a starting point for template strand replication. Under appropriate buffer and temperature conditions, primers can initiate DNA synthesis in the presence of different nucleoside triphosphates and a polymerization reagent (e.g., DNA polymerase or reverse transcriptase).

[0019] The term "probe" refers to a polynucleotide fragment, e.g., RNA or DNA, that can specifically bind to the mRNA or complementary DNA (cDNA) of a particular gene and has a length ranging from a few to several hundred base pairs. Because probes are labeled, they can be used to test for the presence or expression level of the target mRNA or cDNA to which the probe binds.

[0020] In some implementations, the primer set comprises: (a-1) at least one of the sequences shown in SEQ ID NO: 3 to SEQ ID NO: 40 (e.g., at least one pair of the sequences shown), (b-1) at least one of the sequences shown in SEQ ID NO: 41 to SEQ ID NO: 60 (e.g., at least one pair of the sequences shown), and (c-1) At least one of the sequences shown in SEQ ID NO: 61 to SEQ ID NO: 98 (e.g., at least one pair of the sequences shown), The probe is (a-2) at least one of the sequences shown in SEQ ID NO: 100 to SEQ ID NO: 112; (b-2) at least one of the sequences shown in SEQ ID NO: 113 to SEQ ID NO: 127, and (c-2) Contains at least one of the sequences shown in SEQ ID NO: 128 to SEQ ID NO: 142.

[0021] In at least some embodiments, the primer set includes (a-1) at least one pair of sequences set forth in SEQ ID NO:3 to SEQ ID NO:40, (b-1) at least one pair of sequences set forth in SEQ ID NO:41 to SEQ ID NO:60, and (c-1) at least one pair of sequences set forth in SEQ ID NO:61 to SEQ ID NO:98, and the probe includes (a-2) at least one of sequences set forth in SEQ ID NO:100 to SEQ ID NO:112, (b-2) at least one of sequences set forth in SEQ ID NO:113 to SEQ ID NO:127, and (c-2) at least one of sequences set forth in SEQ ID NO:128 to SEQ ID NO:142.

[0022] "At least one pair" among the sequences mentioned refers to a pair of primers consisting of any forward primer and any reverse primer for the same gene (i.e., the ADHFE1 gene, PPP2R5C gene, or SDC2 gene mentioned herein). Furthermore, when a primer and a probe for the same gene are used in combination, any forward primer and a reverse primer located after the forward primer, as well as a probe located between the forward and reverse primers, for the same gene can be used in combination to achieve amplification and testing of colorectal cancer-related genes. Those skilled in the art can select and use primer and probe combinations based on the sequences provided herein by comparing the sequences to genomes. For example, a forward primer numbered SEQ ID NO:7 or SEQ ID NO:11 can be used in combination with a reverse primer set numbered SEQ ID NO:8 or SEQ ID NO:24, respectively, as a pair of primers, and can be used in combination with a probe numbered SEQ ID NO:115, SEQ ID NO:114, SEQ ID NO:125, or SEQ ID NO:126, etc., as needed.

[0023] In some embodiments, the primer set further includes the sequences shown in SEQ ID NO:1 to SEQ ID NO:2, and the probe further includes the sequence shown in SEQ ID NO:99, the sequences shown in SEQ ID NO:1 to SEQ ID NO:2, and the sequence shown in SEQ ID NO:99, and can be used for PCR amplification and testing of the internal reference gene, GAPDH gene.

[0024] The sequences are listed in Table 1. These primer sets and probes were designed for the methylated sequences of the related genes ADHFE1, PPP2R5C, and SDC2, and can specifically identify methylated sequences. When the GAPDH gene is used as an internal reference, amplification of the internal reference gene is unaffected by methylation, allowing for quantification of the number of templates in a sample. The degree of methylation in a sample can be determined by calculating the difference between the Ct values ​​of the target gene and the internal reference gene, which is useful for colorectal cancer testing. The 5' end of the probes can be linked to a FAM, VIC, ROX, or CY5 fluorescent group, and the 3' end can be linked to a quencher group such as MGB. The fluorescent and quencher groups used are commonly used in this field.

[0025] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5]

[0026] In some embodiments, the reagent for testing the ADHFE1 gene comprises the sequences set forth in SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:127; the reagent for testing the PPP2R5C gene comprises the sequences set forth in SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:139; the reagent for testing the SDC2 gene comprises the sequences set forth in SEQ ID NO:89, SEQ ID NO:90, and SEQ ID NO:101; and the composition further comprises a reagent for testing the GAPDH gene, wherein the reagent for testing the GAPDH gene comprises the sequences set forth in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:99.

[0027] The primers or probes referred to can be artificially synthesized, for example, using phosphoramidite solid supports, or can be chemically synthesized using other methods commonly used in the art.

[0028] In at least some embodiments, the composition includes at least one selected from a PCR buffer, salt ions, dNTPs, DNA polymerase, and a target nucleic acid. The target nucleic acid can be extracted from a sample using commercially available reagent kits. The target nucleic acid is a nucleic acid sequence containing genes derived from ADHFE1, PPP2R5C, and SDC2.

[0029] In at least some embodiments, the fluorescent quantitative PCR test conditions mentioned include 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds.

[0030] In at least some embodiments, the mass ratio of the primer set to the probe is 1:1:1:1, which can provide ideal test results.

[0031] In another aspect of the present invention, a reagent kit for diagnosing colorectal cancer is provided, the reagent kit comprising the composition described above. The reagent kit provided by the present invention can specifically test the methylation status of the ADHFE1, PPP2R5C, and SDC2 genes in a sample, thereby assessing the risk of a subject developing colorectal cancer. The present invention simultaneously tests the methylation status of the three genes, ADHFE1, PPP2R5C, and SDC2, thereby effectively improving the test performance compared to single-gene methylation tests.

[0032] In at least some embodiments, the reagent kit further includes at least one of a DNA extraction reagent, a DNA methylation transformation reagent, and a nucleic acid purification reagent. The DNA extraction reagent can be used to extract DNA from a sample. The DNA methylation transformation reagent can convert cytosine in DNA to uracil. The nucleic acid purification reagent can extract purified nucleic acids for use in subsequent reactions.

[0033] The mentioned reagent kit can also be used to determine the gene methylation status in a sample or to assess the prevalence of colorectal cancer in a subject in the same manner as described below. Therefore, the present invention further provides a use of the reagent in the preparation of a reagent kit, which is used to determine the gene methylation status in a sample. The present invention further provides a use of the reagent in the preparation of a reagent kit, which is used to assess the prevalence of colorectal cancer in a subject.

[0034] In a further aspect of the invention, there is provided a method for determining the methylation status of genes in a sample, wherein said genes comprise the ADHFE1 gene, the PPP2R5C gene and the SDC2 gene, The method comprises: (1) obtaining genomic DNA from the sample; (2) methylating and transforming the genomic DNA to obtain a transformation product; (3) performing a fluorescent quantitative PCR test on the transformation product using a primer set and a probe to determine the methylation status of genes in the sample.

[0035] Methylation status generally refers to the presence or absence of 5-methylcytosine at one or more CpG dinucleotides in a DNA sequence. The present invention provides a method for determining the gene methylation status in a sample, which can be used as an accurate, simple, and economical early screening tool for colorectal cancer, which can increase the detection rate of colorectal cancer (especially early-stage colorectal cancer) in high-risk groups and general health checkup subjects, further improve the survival rate of colorectal cancer patients, save a large amount of medical expenses, and reduce the medical burden.

[0036] The sample is not particularly limited and may be a biological sample containing target nucleic acid, and these biological samples are in vitro. They include, but are not limited to, at least one of a tissue sample, a blood sample, a saliva sample, a secretion, or an excrement. The excrement may be a urine sample, a sweat sample, or a tear sample. In some preferred embodiments, the excrement is a fecal sample. Since fecal samples contain exfoliated intestinal cells and testing using fecal samples is non-invasive, fecal samples can be used as a preferred test subject.

[0037] The methylation transformation treatment mentioned above can be performed using several chemical reagents, such as sodium bisulfite. For example, several commercially available reagent kits can be used to perform the methylation transformation treatment, such as the EZ DNA Methylation-Gold Kit, which performs bisulfite transformation on DNA. The resulting product can then be purified using a column and redissolved using a commercially available reagent kit to obtain the purified product.

[0038] In another aspect of the invention, there is provided an isolatable nucleic acid sequence, said isolatable nucleic acid sequence comprising a primer set and a probe, said primer set comprising: (a) at least one pair of sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 40; (b) at least one pair of sequences set forth in SEQ ID NO: 41 to SEQ ID NO: 60; and (c) comprising at least one pair of sequences set forth in SEQ ID NO: 61 to SEQ ID NO: 98; The probe is At least one of the sequences shown in SEQ ID NO: 100 to SEQ ID NO: 112; At least one of the sequences set forth in SEQ ID NO: 113 to SEQ ID NO: 127, and It contains at least one of the sequences shown in SEQ ID NO:128 to SEQ ID NO:142.

[0039] The present invention further provides a method for assessing a subject's colorectal cancer status, the method comprising: (a) providing a sample from the subject, the sample containing target nucleic acids from the subject, the target nucleic acids including nucleic acids derived from the ADHFE1 gene, the PPP2R5C gene, and the SDC2 gene; (b) assessing the methylation status of the target nucleic acid; and (c) assessing the subject's colorectal cancer status based on the methylation status of the target nucleic acid. The reagent for assessing the methylation status of the target nucleic acid can be, for example, a chemical reagent such as sulfite, bisulfite, or bisulfite, or sodium bisulfite. After treatment with these chemical reagents, unmethylated cytosines in the target nucleic acid are converted to uracil, while methylated cytosines are not. The sequence differences after transformation can then be analyzed by various means, such as sequencing, PCR, high-resolution melting curve analysis, and particularly fluorescent quantitative PCR, allowing for rapid and easy differentiation. Of course, the reagent for assessing the methylation status of the target nucleic acid may be any biological reagent, such as a peptide or an enzyme, such as any methylation-sensitive restriction enzyme.

[0040] The subject referred to herein is a human being. For example, it may be a patient requiring diagnosis. The type of biological sample from the subject includes, but is not limited to, at least one of secretions or excrements, such as a tissue sample, a blood sample, or a saliva sample. The excrement may be a urine sample, a sweat sample, or a tear sample. Moreover, since fecal samples contain exfoliated intestinal cells and testing using fecal samples is non-invasive, fecal samples can be used as a preferred test subject.

[0041] In at least some embodiments, the subject is a mammal. In at least some embodiments, in step b), the methylation status of the target nucleic acid is assessed using the primer set and probes mentioned above.

[0042] The method further comprises the step of isolating the target nucleic acid from the sample. The target nucleic acid can be isolated from the sample using several commercially available reagent kits in the art.

[0043] The method further includes amplifying the target nucleic acid. Several common reagents for nucleic acid amplification can be used. Reagents for nucleic acid amplification can include enzymes such as those used in polynucleotide amplification reactions and quantitative fluorescent polymerase reaction (qPCR).

[0044] The method further includes assessing the methylation status of the target nucleic acid to obtain a methylation index, comparing the methylation index with a reference value, and determining the methylation status of the sample. In some implementations, the method includes assessing the methylation status of the target nucleic acid using a chemical reagent. According to preferred implementations of the present invention, the chemical reagent includes bisulfite or sulfite. In some preferred embodiments, the reference value is from an internal reference gene, GAPDH, and the methylation status of the sample gene is determined based on a threshold value by comparing the amplification Ct values ​​of the ADHFE1, PPP2R5C, and SDC2 genes with the amplification Ct value of the internal reference GAPDH gene. The threshold value can be set based on the analysis of a large number of clinical samples with known pathological information. Of course, the methylation index can also be other common methylation indexes, such as methylation frequency, methylation load, etc.

[0045] In at least some embodiments, the method further includes using an internal reference gene to determine whether the DNA content satisfies the test, and determining the methylation status using the difference (ΔCt) between the target gene Ct value and the internal reference gene Ct value.

[0046] In at least some embodiments, the method includes assessing the colorectal cancer status of the subject by fluorescent quantitative PCR using GAPDH as an internal reference gene; determining that the quality control has failed if the Ct value of GAPDH is greater than 37; determining the result as negative if the ADHFE1, PPP2R5C, and SDC2 genes are greater than 37; If the Ct values ​​of the ADHFE1, PPP2R5C, and SDC2 genes are less than or equal to 37, the difference ΔCt between the amplified Ct values ​​of the ADHFE1, PPP2R5C, and SDC2 genes and the amplified Ct value of the internal reference GAPDH gene is calculated, and the colorectal cancer susceptibility status of the subject is assessed based on the ROC curve results and AUC results of the ADHFE1, PPP2R5C, and SDC2 genes ΔCt.

[0047] The method further comprises the step of determining the result as positive if ΔCt(ADHFE1)≦8, ΔCt(PPP2R5C)≦5, and ΔCt(SDC2)≦12.

[0048] The method further includes assessing the likelihood of colorectal cancer based on the subject's methylation status, achieving purposes such as diagnostic assistance and tumor screening, and providing the subject with treatment as soon as possible. In at least some embodiments, the treatment includes at least one of chemotherapy, radiation therapy, immunotherapy, cell therapy, and surgery.

[0049] The following examples are used in combination to illustrate the present invention. Those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If specific techniques or conditions are not shown in the examples, they should be carried out according to the techniques or conditions described in the literature in the field or according to the product specifications. If the manufacturer of the reagents or equipment used is not specified, they are conventional products that can be purchased commercially.

[0050] Example 1 The inventors have determined target nucleic acids for diagnosing colorectal cancer according to the following method and steps. First, the inventors screened and determined candidate genes for methylation testing: CDH4, ZNF132, PPP2R5C, LONRF2, ADHFE1, SDC2, NDRG4, and BMP3, by comparing the significant discrimination shown by different genes in the Cancer Genome Atlas (TCGA) database between tumor and non-tumor samples in terms of methylation levels. Next, specific primers and probes were designed for the candidate genes. During the primer and probe design process, the inventors discovered that, for testing the methylation of target nucleic acids, after the target genome sequence is transformed by bisulfite, most Cs are transformed to Us, resulting in low genome complexity. Therefore, when designing primers and probes, many consecutive single bases are encountered, and the annealing temperature is generally low, resulting in a relatively small region for primer design.

[0051] Subsequently, using the primers and probes designed above, candidate genes were initially screened using small amounts of tumor and non-tumor samples. The sensitivity / specificity results for each gene were CDH4 (61.5% / 61.5%), ZNF132 (50% / 100%), PPP2R5C (78.9% / 100%), LONRF2 (77.8% / 72%), ADHFE1 (100% / 91.3%), SDC2 (88.9% / 100%), NDRG4 (70% / 95%), and BMP3 (65% / 90%). BMP3, NDRG4, ADHFE1, PPP2R5C, and SDC2 showed good sensitivity / specificity.

[0052] Furthermore, when we compared the methylation data in the TCGA database with the five candidate genes with good sensitivity / specificity, we found that the ADHFE1, PPP2R5C, and SDC2 genes all had lower background levels in cancer-adjacent tissues than the BMP3 and NDRG4 genes, as shown in Figure 1, and showed more significant differences between cancer tissues and cancer-adjacent tissues.

[0053] Therefore, the inventors finally determined that the ADHFE1, PPP2R5C, and SDC2 genes were the target nucleic acids for diagnosing colorectal cancer, and further designed and optimized specific primer and probe sequences for the target genes, as shown in Table 1.

[0054] Example 2 The inventors will use the primers and probes for the target nucleic acids (ADHFE1, PPP2R5C, and SDC2 genes) for colorectal cancer diagnosis obtained in Example 1 to perform fluorescent quantitative PCR amplification of positive and negative cell lines to verify the effectiveness of the primers and probes.

[0055] Step 1. Sample collection: Positive standard DNA (CpGenome Human Methylated DNA Standard Set, Sigma-Aldrich, S8001M), negative standard DNA (CpGenome Human Non-Methylated DNA Standard Set, Sigma-Aldrich, S8001U) Step 2. DNA modification: Take 10 ng of each of the above standard DNAs and perform bisulfite transformation using the EZ DNA Methylation-Gold Kit (ZYMO RESEARCH). After column purification, redissolve in 33 μl of NF water for preliminary use.

[0056] Step 3. PCR test: Using the primers and probes in Table 1 as examples, fluorescent quantitative PCR testing is performed according to the following PCR system and PCR reaction procedure, where the primers and probes for ADHFE1 are SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:127; the primers and probes for 127;PPP2R5C are SEQ ID NO:53, SEQ ID NO:54 and SEQ ID NO:139; the primers and probes for SDC2 are SEQ ID NO:89, SEQ ID NO:90 and SEQ ID NO:101; and the primers and probes for the internal reference GAPDH gene are SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:99.

[0057] wherein the 5' end of probe SEQ ID NO: 127 is FAM and the 3' end is BHQ1; The 5' end of probe SEQ ID NO:139 is CY5 and the 3' end is BHQ3; The 5' end of probe SEQ ID NO:101 is VIC and the 3' end is BHQ1; The 5' end of probe SEQ ID NO:99 is ROX and the 3' end is MGB.

[0058] (1) The PCR system is shown in Table 2.

[0059] [Table 6]

[0060] (2) The PCR reaction procedure is shown in Table 3.

[0061] [Table 7]

[0062] Step 4, result analysis: The difference (ΔCt value) between the amplification Ct values ​​of the ADHFE1, PPP2R5C, and SDC2 genes and the amplification Ct value of the internal reference GAPDH gene is calculated, and the sample gene methylation level is determined to be normal or not based on the decision threshold set by analyzing a large number of clinical samples with known pathological information.

[0063] The results of amplification using the above primers and probes are shown in FIGS.

[0064] Example 3 The inventors have designed and developed a reagent kit for diagnosing colorectal cancer, which comprises specific primers and probes for specifically testing the methylation of the ADHFE1 gene, the PPP2R5C gene, and the SDC2 gene, wherein the specific primers contained in different reagent kits are selected from the primers shown in Table 1, and the probes are selected from the probes shown in Table 1. Each reagent kit comprises at least one forward / reverse primer for specifically testing the ADHFE1 gene, at least one forward / reverse primer for specifically testing the PPP2R5C gene, and at least one forward / reverse primer for specifically testing the SDC2 gene, and optionally at least one probe for testing the ADHFE1 gene, at least one probe for testing the PPP2R5C gene, and at least one probe for testing the SDC2 gene.

[0065] The developed reagent kit may optionally include forward and reverse primers and probes for testing an internal reference gene, for example, GAPDH.

[0066] A fluorescent group such as FAM, VIC, ROX or CY5 may be attached to the 5' end of the probe, and a quencher group such as MGB may be attached to the 3' end.

[0067] The primer and probe sequences in the reagent kit can be provided in powder form.

[0068] In addition, the provided reagent kit may optionally contain a PCR reaction buffer, polymerase, DNA methylation transformation reagent, etc., thereby enabling those skilled in the art to easily diagnose colorectal cancer according to the method of the present invention.

[0069] Example 4 The inventors used the reagent kit for diagnosing colorectal cancer shown in Example 3 to perform methylation testing on samples by the following method, the steps of which are shown in Figure 4 and include the following steps:

[0070] 1. Sample collection: A total of 201 colorectal cancer samples, 184 healthy controls, 4 adenoma samples and 7 polyp samples were collected.

[0071] 2. Sample DNA Extraction: Extract DNA from the collected samples using a commercially available fecal sample DNA extraction reagent kit.

[0072] 3.DNA modification: The DNA was subjected to bisulfite transformation, column purification, and resolubilization using the EZ DNA Methylation-Gold Kit (ZYMO RESEARCH) for preliminary use.

[0073] 4. PCR testing: In this example, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:127 were selected as primers and probes for ADHFE1, SEQ ID NO:53, SEQ ID NO:54, and SEQ ID NO:139 were selected as primers and probes for PPP2R5C, SEQ ID NO:89, SEQ ID NO:90, and SEQ ID NO:101 were selected as primers and probes for SDC2, and SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:99 were selected as primers and probes for the internal reference GAPDH gene. Fluorescent quantitative PCR tests were performed on the samples collected according to the PCR system and reaction conditions described in Example 2 above.

[0074] 5.Result analysis: The test was performed using the SLAN96S qPCR instrument, with the baseline and threshold set to the default values, and the threshold Ct value for each gene was 37.

[0075] Here, if the Ct value of GAPDH is greater than 37, it is determined that the quality control has failed, If the ADHFE1, PPP2R5C, and SDC2 genes are greater than 37, the result is negative. When the Ct values ​​of the ADHFE1, PPP2R5C, and SDC2 genes were less than or equal to 37, the difference (ΔCt) between the amplification Ct values ​​of the ADHFE1, PPP2R5C, and SDC2 genes and the amplification Ct value of the internal reference GAPDH gene was calculated. ROC curves for the ΔCt values ​​of the three tested genes were generated using SPSS software. The ROC curves are shown in Figure 5, and the performance and area under the curve (AUC) of each gene are listed in Table 4.

[0076] [Table 8]

[0077] To further improve the performance of the reagent kit, the △Ct values ​​of the three genes were analyzed together, and the AUC reached 0.945. Finally, if △Ct(ADHFE1)≦8, △Ct(PPP2R5C)≦5, and △Ct(SDC2)≦12, the gene test was determined to be positive; if any gene was positive, the sample test was determined to be positive. Under these conditions, the specificity was 94.02%, and the sensitivity was 87.56%.

[0078] Here, sensitivity is used to indicate the proportion of positive samples or patients diagnosed with colorectal cancer on final clinical pathology by the test.

[0079] Specificity refers to the proportion of samples or patients diagnosed as normal by the test in the final clinical pathology.

[0080] From the above results, it can be seen that the present invention can achieve better testing performance than single gene testing by testing multiple genes in combination.

[0081] In the description herein, the reference terms such as "one embodiment," "some embodiments," "examples," "specific examples," or "some examples" mean that the specific features, structures, materials, or characteristics described in combination with the embodiment or example are included in at least one embodiment or example of the present invention. In the description herein, exemplary descriptions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in an appropriate manner in one or more embodiments or examples. Furthermore, if there is no conflict between them, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described herein.

[0082] Although embodiments of the present invention have been presented and described, the above embodiments are illustrative and should not be construed as limiting the present invention, and those skilled in the art will appreciate that various changes, modifications, substitutions and variations can be made to the above embodiments within the scope of the present invention.

Claims

1. A composition for examining colorectal cancer, comprising reagents for simultaneously examining the methylation status of the ADHFE1 gene, the PPP2R5C gene, and the SDC2 gene, A reagent for testing the ADHFE1 gene comprises a forward primer consisting of the sequence shown in SEQ ID NO: 7, a reverse primer consisting of the sequence shown in SEQ ID NO: 8, and a probe consisting of the sequence shown in SEQ ID NO: 127; A reagent for detecting the PPP2R5C gene comprises a forward primer consisting of the sequence shown in SEQ ID NO: 53, a reverse primer consisting of the sequence shown in SEQ ID NO: 54, and a probe consisting of the sequence shown in SEQ ID NO: 139; The reagent for testing the SDC2 gene includes a forward primer consisting of the sequence shown in SEQ ID NO: 89, a reverse primer consisting of the sequence shown in SEQ ID NO: 90, and a probe consisting of the sequence shown in SEQ ID NO:

101. A composition for detecting colorectal cancer, comprising:

2. The composition of claim 1, further comprising a reagent for testing the GAPDH gene, wherein the reagent for testing the GAPDH gene comprises a forward primer consisting of the sequence shown in SEQ ID NO: 1, a reverse primer consisting of the sequence shown in SEQ ID NO: 2, and a probe consisting of the sequence shown in SEQ ID NO:

99.

3. The composition comprises:

2. The composition according to claim 1, comprising at least one selected from a PCR buffer solution, salt ions, dNTPs, DNA polymerase, and a target nucleic acid.

4. A reagent kit for diagnosing colorectal cancer, comprising the composition according to any one of claims 1 to 3.

5. The reagent kit according to claim 4, further comprising at least one of a DNA extraction reagent, a DNA methylation transformation reagent, and a nucleic acid purification reagent.

6. A method for determining the methylation status of genes in a sample, wherein the genes are ADHFE1 gene, PPP2R5C gene, and SDC2 gene; The method comprises: (1) obtaining genomic DNA from the sample; (2) methylation conversion of genomic DNA, using a sulfite, bisulfite, or bisulfite-containing chemical reagent or a commercially available kit for bisulfite conversion of genomic DNA, in which unmethylated cytosine is converted to uracil and methylated cytosine is not subjected to such conversion, to obtain converted genomic DNA; (3) using the reagent to perform a fluorescent quantitative PCR test on the methylation conversion product in the same environment to determine the methylation status of the gene in the sample; A reagent for testing the ADHFE1 gene comprises a forward primer consisting of the sequence shown in SEQ ID NO: 7, a reverse primer consisting of the sequence shown in SEQ ID NO: 8, and a probe consisting of the sequence shown in SEQ ID NO: 127; A reagent for detecting the PPP2R5C gene comprises a forward primer consisting of the sequence shown in SEQ ID NO: 53, a reverse primer consisting of the sequence shown in SEQ ID NO: 54, and a probe consisting of the sequence shown in SEQ ID NO: 139; The reagent for testing the SDC2 gene includes a forward primer consisting of the sequence shown in SEQ ID NO: 89, a reverse primer consisting of the sequence shown in SEQ ID NO: 90, and a probe consisting of the sequence shown in SEQ ID NO:

101. A method for determining the methylation status of a gene in a sample, comprising:

7. 7. The method of claim 6, wherein the sample comprises at least one selected from the group consisting of a tissue sample, a blood sample, a secretion, and an excretion.

8. The method of claim 7, wherein the excrement comprises a fecal sample.

9. The method according to claim 6, wherein the fluorescent quantitative PCR test conditions include a total of 45 cycles of 95°C for 10 minutes, 95°C for 15 seconds, 55°C for 30 seconds, and 72°C for 30 seconds.

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