Primer combinations, kits and uses thereof for detecting intestinal polyps or colorectal cancer

Abstract

The present application provides primer combinations, kits and uses thereof for detecting intestinal polyps and / or colorectal cancer. The related products for detecting intestinal polyps or colorectal cancer provided by the present application include primer combinations, reagents and kits, which are based on detecting the RNA level of at least one of EPCAM, CCND1, S100A4 and SHNG7 genes in a fecal sample, to achieve non-invasive, accurate and simple detection of intestinal polyps or colorectal cancer with extremely high sensitivity and specificity, especially for detecting advanced adenoma (>1cm adenomatous intestinal polyps) and / or early colorectal cancer.

Description

Technical Field

[0001] This invention relates to the field of medical technology, specifically to primer combinations, kits, and their uses for detecting intestinal polyps and / or colorectal cancer. Background Technology

[0002] Colorectal cancer is one of the most common malignant tumors of the digestive tract, and it is highly prevalent worldwide. Most colorectal cancers develop from intestinal polyps, and the disease process is relatively slow, with a benign tumor development stage lasting 3-17 years before becoming cancerous. Therefore, colorectal cancer is one of the cancers most likely to be controlled through early screening. Adenomatous polyps are a common type of intestinal polyp, often referred to as adenomas, and are more prone to becoming cancerous. When an adenoma is larger than 1 cm, its risk of developing into colorectal cancer is far greater than that of an adenoma smaller than 1 cm. Therefore, adenomas larger than 1 cm are considered advanced adenomas, precancerous lesions, and have an extremely high risk of becoming cancerous. If advanced adenomas are detected and surgically removed in time, colorectal cancer can be avoided. Meanwhile, for patients with early-stage colorectal cancer, if they are diagnosed and treated in time, the 5-year survival rate can exceed 90% (Pamela Buchwald, et al, ANZ J Surg. 2018 Mar; 88(3): E114-E117. PMID: 27618786).

[0003] Currently, colorectal cancer screening in my country generally involves initial screening using questionnaires and fecal occult blood tests, followed by colonoscopy and pathological confirmation for high-risk individuals identified in the initial screening. The fecal occult blood test detects hemoglobin in stool; a positive result indicates gastrointestinal bleeding. However, due to the numerous causes of gastrointestinal bleeding, the fecal occult blood test has extremely low sensitivity for detecting colorectal cancer and colorectal polyps. Colonoscopy is the "gold standard" for colorectal cancer detection, enabling a basic definitive diagnosis. However, colonoscopy requires complex preoperative preparation procedures such as bowel preparation and anesthesia, which can easily lead to complications such as bleeding, intestinal perforation, and infection. Furthermore, many people are unwilling to undergo colonoscopy due to psychological factors such as embarrassment and fear. These factors significantly reduce adherence to colonoscopy.

[0004] Therefore, it is necessary to develop a highly accurate and compliant early screening method for colorectal cancer to achieve "early detection, early diagnosis, and early treatment" of colorectal cancer.

[0005] Fecal-based nucleic acid molecular testing can significantly improve adherence to colorectal cancer screening. In 2014, the US FDA approved the Cologuard fecal-based genetic screening kit. Individuals undergoing screening simply send their stool samples to the testing company. The kit detects multiple gene DNA methylation and mutation levels in the stool, combined with a fecal occult blood test, to diagnose colorectal cancer. However, Cologuard's multi-target DNA testing based on stool has high sample quality requirements, requires different technologies for different indicators, involves complex sample processing, and is time-consuming, resulting in high testing costs. Following the success of Cologuard's fecal multi-target DNA methylation colorectal cancer detection product, similar products based on fecal DNA methylation for colorectal cancer detection have emerged in the Chinese market. Examples include the fecal DNA methylation detection kit from Guangzhou Kangliming Biotechnology Co., Ltd. (patent number CN110317871B, detecting SDC2, COL4A1 / COL4A2, and ITGA4 methylation), the kit from Wuhan Aimeisen Life Science Co., Ltd. (patent number CN106893777B, detecting NDRG4, SDC2, TFPI2, and BMP3 methylation), and the kit from Shanghai Ruiyi Biotechnology Co., Ltd. (patent number CN108977543B, detecting SDC2 and SFRP2 gene methylation). However, these fecal DNA methylation-based products exhibit low sensitivity for detecting advanced adenomas (>1cm adenomatous polyps, also known as precancerous lesions). For instance, the sensitivity of Cologuard's product for advanced adenomas is only 42%.

[0006] In May 2024, the U.S. FDA approved ColoSense, a fecal multi-target RNA colorectal cancer screening kit developed by Geneoscopy. Individuals can send their stool samples to the testing company for enrichment of human cells in the stool, detection of the mRNA expression levels of eight genes, and integration with fecal occult blood testing to diagnose colorectal cancer. ColoSense's fecal multi-target RNA detection product achieves 100% sensitivity for early-stage colorectal cancer (Stage I) and 45% sensitivity for advanced adenomas, both significantly superior to all currently available fecal DNA methylation colorectal cancer diagnostic products. However, the ColoSense kit involves complex sample processing, requiring multiple steps to enrich human cells from stool and simultaneous detection of the mRNA expression of eight genes (GAPDH, ACY1, AREG, EGLN2, TNFRSF10B, KRAS, SMAD4, CDH1) using digital PCR, which is time-consuming and costly. In addition, ColoSense's fecal multiRNA detection kit is not sensitive enough for advanced adenomas. Summary of the Invention

[0007] To address the aforementioned issues, and through repeated experimentation, the inventors of this application have developed a method and related products for the non-invasive, accurate, and simple detection of intestinal polyps and / or colorectal cancer with extremely high sensitivity and specificity.

[0008] Specifically, this application provides the following technical solution:

[0009] On the one hand, this application provides primer combinations selected from one or more pairs of primers for the specific identification of one or more of the following genes: EPCAM, CCND1, S100A4 and SNHG7.

[0010] On the other hand, this application provides the use of the provided primer combination in the preparation of products for the diagnosis or auxiliary diagnosis of intestinal polyps and / or colorectal cancer.

[0011] In another aspect, this application provides reagents or kits for detecting intestinal polyps and / or colorectal cancer, which include the provided primer combination.

[0012] In addition, this application also provides the use of reagents for specifically identifying one or more of the EPCAM, CCND1, S100A4 and SNHG7 genes in the preparation of products (e.g., kits) for screening intestinal polyps and / or colorectal cancer via fecal samples.

[0013] The products (including primer combinations, reagents, and kits) and related uses provided in this application for detecting intestinal polyps or colorectal cancer use fecal RNA as the detection sample. The samples are easy to obtain, the sampling process is convenient, the sample processing process is simple, the detection time from sample to final result is short, and the products are non-invasive and have high compliance.

[0014] Furthermore, this application uses one or more of the EPCAM, CCND1, S100A4 and SNHG7 genes as detection markers, which not only achieves satisfactory sensitivity and specificity for the detection of colorectal cancer, especially early colorectal cancer, but also improves the sensitivity and specificity for the detection of intestinal polyps, especially advanced adenomas (>1cm adenomatous polyps, also known as precancerous lesions). Attached Figure Description

[0015] Figure 1 The amplification curve (A) and melting curve (B) of the cDNA corresponding to the RNA of EPCAM detected by the optimal primer pair are shown respectively.

[0016] Figure 2 The amplification curve (A) and melting curve (B) of the sample detection of cDNA corresponding to the RNA of CCND1 using the optimal primer pair are shown respectively.

[0017] Figure 3The amplification curve (A) and melting curve (B) of the sample detection of cDNA corresponding to the RNA of S100A4 with the optimal primer pair are shown respectively.

[0018] Figure 4 The amplification curve (A) and melting curve (B) of the sample detection of cDNA corresponding to SNHG7 RNA using the optimal primer pair are shown respectively.

[0019] Figure 5 The sample detection amplification curve (A) and melting curve (B) of the cDNA corresponding to the RNA used to detect ACTB are shown respectively.

[0020] Figure 6 The ROC curves of EPCAM RNA detection for colorectal cancer (normal vs. colorectal cancer, A) and advanced adenoma (normal vs. advanced adenoma, B) are shown respectively.

[0021] Figure 7 The ROC curves of CCND1 for detecting colorectal cancer (normal vs. colorectal cancer, A) and advanced adenoma (normal vs. advanced adenoma, B) are shown respectively.

[0022] Figure 8 The ROC curves of S100A4 RNA detection for colorectal cancer (normal vs. colorectal cancer, A) and advanced adenoma (normal vs. advanced adenoma, B) are shown respectively.

[0023] Figure 9 The ROC curves of SNHG7 RNA detection in colorectal cancer (normal vs. colorectal cancer, A) and advanced adenoma (normal vs. advanced adenoma, B) are shown respectively.

[0024] Figure 10 The ROC curves of two genes combined as biomarkers (CCND1+S100A4(A), CCND1+EPCAM(B), CCND1+SNHG7(C), EPCAM+SNHG7(D), EPCAM+S100A4(E), SNHG7+S100A4(F)) are shown for detecting colorectal cancer (normal vs. colorectal cancer).

[0025] Figure 11 The ROC curves of three genes combined as biomarkers (CCND1+S100A4+EPCAM(A), CCND1+S100A4+SNHG7(B), EPCAM+S100A4+SNHG7(C), CCND1+EPCAM+SNHG7(D)) for detecting colorectal cancer (normal vs. colorectal cancer) are shown respectively.

[0026] Figure 12The ROC curves of four genes combined as biomarkers (CCND1+S100A4+EPCAM+SNHG7) for detecting colorectal cancer (normal vs. colorectal cancer) are shown.

[0027] Figure 13 The ROC curves of two genes combined as biomarkers (CCND1+S100A4(A), CCND1+SNHG7(B), CCND1+EPCAM(C), EPCAM+S100A4(D), EPCAM+SNHG7(E), SNHG7+S100A4(F)) are shown for detecting advanced adenomas (normal vs. advanced adenomas).

[0028] Figure 14 The ROC curves of three genes combined as biomarkers (CCND1+S100A4+SNHG7(A), CCND1+S100A4+EPCAM(B), EPCAM+S100A4+SNHG7(C), CCND1+EPCAM+SNHG7(D)) are shown for detecting advanced adenomas (normal vs. advanced adenomas).

[0029] Figure 15 The ROC curves of four genes combined as biomarkers (CCND1+S100A4+EPCAM+SNHG7) for detecting advanced adenoma (normal vs. advanced adenoma) are shown. Detailed Implementation

[0030] With the development of RNA sequencing technology, numerous studies in recent years have compared the transcriptomics of colorectal cancer tissues with adjacent normal tissues, thereby identifying a variety of potential diagnostic and therapeutic biomarkers and targets. It is noteworthy that many RNAs exhibit significant differences in expression at the tissue and cellular levels, but when used as screening media such as feces and blood, or when different sample processing methods are employed, their sensitivity and specificity for colorectal cancer decrease significantly. Some RNAs even show no significant difference between normal and colorectal cancer groups. This is mainly because feces have a complex composition, with bacteria comprising the majority of cells and human cells making up only a small portion, and containing a large number of inhibitors of downstream reactions. For example, the levels of mRNAs of genes such as GAPDH, ACY1, and EGLN2 detected by the ColoSense kit show significant differences between normal individuals and colorectal cancer patients' feces, but data analysis from internationally recognized databases such as TCGA (The Cancer Genome Atlas) shows no significant difference in their mRNA expression between colorectal cancer tissues and normal intestinal tissues. For example, some gene markers, such as the vimentin gene, have a sensitivity of 83% for the diagnosis of colorectal cancer in tissues, but this drops to 46% in stool samples (Chen et al., JNatl Cancer Inst., 2005, 97(15):1124-32, PMID:16077070).

[0031] Although multi-target molecular detection has become the consensus for fecal-based colorectal cancer screening, the functional overlap of many colorectal cancer biomarkers makes it extremely difficult to screen for complementary biomarker combinations. Therefore, when researching fecal-based molecular detection methods for colorectal cancer, it is not sufficient to simply select differentially expressed molecules at the tissue or cellular level. Instead, it is necessary to define proper fecal sample processing and purification methods, validate the results with a large number of clinical fecal samples, and optimize the combination of different biomarkers.

[0032] Through extensive research and exploration, the inventors of this application have obtained molecular markers for detecting intestinal polyps and colorectal cancer based on fecal samples. Based on this, they have developed a method and related products for detecting intestinal polyps and / or colorectal cancer by detecting the RNA levels of the EPCAM, CCND1, S100A4, and SHNG7 genes in fecal samples. This method enables non-invasive, accurate, and simple detection of intestinal polyps and / or colorectal cancer with extremely high sensitivity and specificity, particularly for detecting advanced adenomas (precancerous lesions) or early-stage colorectal cancer larger than 1 cm.

[0033] In this article, "advanced adenoma" refers to adenomatous intestinal polyps larger than 1 cm, also known as "advanced intestinal polyps". When an intestinal polyp (adenoma) is larger than 1 cm, its risk of developing into colorectal cancer is much greater than that of an intestinal polyp (adenoma) smaller than 1 cm. Therefore, advanced adenomas larger than 1 cm are considered precancerous lesions with an extremely high risk of becoming cancerous.

[0034] Specifically, this application provides the following technical solution:

[0035] In a first aspect, this application provides primer combinations selected from at least one pair (e.g., one pair, two pairs, three pairs, or four pairs) of primers for the specific identification of at least one (e.g., one, two, three, or four) of the following genes: EPCAM, CCND1, S100A4, and SNHG7.

[0036] In a specific implementation, this application provides primer combinations selected from at least two pairs (e.g., two, three, or four pairs) of primers used for the specific identification of at least two (e.g., two, three, or four) of the following genes: EPCAM, CCND1, S100A4, and SNHG7.

[0037] In a specific implementation, this application provides primer combinations selected from at least three pairs (e.g., three or four pairs) of primers for the specific identification of at least three (e.g., three or four) of the following genes: EPCAM, CCND1, S100A4, and SNHG7.

[0038] In a specific implementation, this application provides primer combinations selected from four pairs of primers used for the specific identification of four of the following genes: EPCAM, CCND1, S100A4, and SNHG7.

[0039] In specific implementation schemes, the primer pairs are selected from one or more of the following primer pairs: primer pairs for specific identification of the EPCAM gene: forward primer: 5'-TAAGGCCAAGCAGTGCAACG-3' (SEQ ID NO.3), and reverse primer: 5'-TTGTCTGTTCTTCTGACCCCAG-3' (SEQ ID NO.4); primer pairs for specific identification of the CCND1 gene: forward primer: 5'-GGCGGAGGAGAACAAACAGA-3' (SEQ ID NO.7), and reverse primer (SEQ ID NO.8): 5'-GGAGGGCGGATTGGAAATGA-3'; primer pairs for specific identification of the S100A4 gene: forward primer: 5'-GCCCAGCTTCTTGGGGAAAA-3' (SEQ ID NO.19), and reverse primer: 5'-ATGGCGATGCAGGACAGGAA-3' (SEQ ID NO.19). NO.20); Primers for specific identification of the SNHG7 gene: forward primer: 5'-TTGCTGGCGTCTCGGTTAAT-3' (SEQ ID NO.21), and reverse primer: 5'-TCCATCACAGGCGAAGTCAC-3' (SEQ ID NO.22).

[0040] In a specific embodiment, the primer set also includes primers for the specific identification of an internal reference / housekeeping gene. Any internal reference gene known in the art is suitable for this application.

[0041] In a specific implementation plan, the internal reference gene is selected from ACTB gene, GAPDH gene, 36B4 gene, RPL13A gene, etc.

[0042] In a specific implementation, the primers used for specific identification of the ACTB gene are: forward primer: 5'-TGGCATCCACGAAACTACC-3' (SEQ ID NO.27) and reverse primer: 5'-GTGTTGGCGTACAGGTCTT-3' (SEQ ID NO.28).

[0043] In the specific implementation plan, the primer combination is used to detect fecal samples.

[0044] Secondly, this application provides the use of the primer combination of the first aspect in the preparation of products for detecting intestinal polyps (especially advanced adenomas) and / or colorectal cancer (especially early colorectal cancer).

[0045] In a specific implementation plan, the product is a reagent or a kit.

[0046] Thirdly, this application provides reagents or kits for detecting intestinal polyps (especially advanced adenomas) or colorectal cancer (especially early colorectal cancer), which include the primer combination of the first aspect.

[0047] In a specific implementation, the reagent or kit further includes one or more of the following: a biological sample preservation reagent, an RNA extraction reagent, an RNA reverse transcription reagent, and a PCR reaction reagent. The biological sample preservation reagent is used to preserve collected fecal samples; the RNA extraction reagent is used to extract RNA from the fecal samples; the RNA reverse transcription reagent is used to reverse transcribe the extracted RNA to obtain cDNA; and the PCR reaction reagent is used to amplify the obtained cDNA.

[0048] In a specific implementation, the reagent or kit includes one or more of the following: primer combinations for specific identification of at least one (e.g., one, two, three or four) of the genes EPCAM, CCND1, S100A4, SNHG7 and ACTB, as well as biological sample preservation solutions, RNA extraction solutions, RNA reverse reaction solutions, PCR reaction solutions, positive controls, negative controls, process control samples, standards, etc.

[0049] Fourthly, this application provides a method for using the reagents or kits of the third aspect to detect fecal samples for screening of intestinal polyps (especially advanced adenomas) and / or colorectal cancer (especially early colorectal cancer).

[0050] In a specific implementation scheme, the method includes the following steps: extracting RNA from the fecal sample to be tested using an RNA extraction reagent; synthesizing cDNA by reverse transcription of the extracted RNA using an RNA reverse transcription reagent; and detecting the synthesized cDNA by quantitative PCR using a PCR reaction reagent and the primer combination of the first aspect.

[0051] In a specific implementation plan, the kit and application provided in this application for detecting colorectal cancer-related RNA in fecal samples, such as a method for the diagnosis or auxiliary diagnosis of intestinal polyps (especially advanced adenomas) or colorectal cancer (especially early-stage colorectal cancer), comprises the following steps:

[0052] (1) RNA extraction: Fecal samples were lysed with Trizol reagent and centrifuged to obtain supernatant. 1 / 5 volume of chloroform was added and mixed thoroughly. The supernatant was then centrifuged again. An equal volume of anhydrous ethanol was added to the supernatant and mixed thoroughly. The mixture was then added to a centrifuge column with an adsorption membrane. The column was repeatedly rinsed and centrifuged with RNA rinsing buffer. RNA elution buffer was then added to elute the RNA from the adsorption membrane. The RNA solution was collected by centrifugation.

[0053] (2) RNA reverse transcription: The purified RNA was used to synthesize cDNA using an RNA reverse transcription kit;

[0054] (3) PCR reaction: Quantitative PCR detection of cDNA targeting multiple RNAs was performed using cDNA-specific primer pairs for colorectal cancer-related RNAs; and optionally,

[0055] (4) Analyze the test results.

[0056] Fifthly, this application also provides the use of reagents for specifically identifying at least one (e.g., one, two, three, or four) of the EPCAM, CCND1, S100A4, and SNHG7 genes in the preparation of products for screening for intestinal polyps (especially advanced adenomas) or colorectal cancer (especially early colorectal cancer) via fecal samples.

[0057] In the specific implementation plan, the product is a reagent kit.

[0058] In a specific implementation, the reagent includes at least one pair (e.g., one pair, two pairs, three pairs, or four pairs) of primers for the specific identification of at least one (e.g., one, two, three, or four) of the EPCAM, CCND1, S100A4, and SNHG7 genes.

[0059] In a specific implementation scheme, the primers used for specific identification of the EPCAM gene are: forward primer: 5'-TAAGGCCAAGCAGTGCAACG-3' (SEQ ID NO.3), and reverse primer: 5'-TTGTCTGTTCTTCTGACCCCAG-3' (SEQ ID NO.4).

[0060] In the specific implementation scheme, the primers used for specific identification of the CCND1 gene are: forward primer: 5'-GGCGGAGGAGAACAAACAGA-3' (SEQ ID NO.7) and reverse primer: 5'-GGAGGGCGGATTGGAAATGA-3' (SEQ ID NO.8).

[0061] In a specific implementation scheme, the primers used for specific identification of the S100A4 gene are: forward primer: 5'-GCCCAGCTTCTTGGGGAAAA-3' (SEQ ID NO.19), and reverse primer: 5'-ATGGCGATGCAGGACAGGAA-3' (SEQ ID NO.20).

[0062] In the specific implementation scheme, the primers used for specific identification of the SNHG7 gene are: forward primer: 5'-TTGCTGGCGTCTCGGTTAAT-3' (SEQ ID NO.21) and reverse primer: 5'-TCCATCACAGGCGAAGTCAC-3' (SEQ ID NO.22).

[0063] In a specific implementation, the reagent also includes primers for the specific identification of the internal reference gene.

[0064] In a specific implementation plan, the internal reference genes are ACTB gene, GAPDH gene, 36B4 gene, RPL13A gene, etc.

[0065] In a specific implementation, the primers used for specific identification of the ACTB gene are: forward primer: 5'-TGGCATCCACGAAACTACC-3' (SEQ ID NO.27) and reverse primer: 5'-GTGTTGGCGTACAGGTCTT-3' (SEQ ID NO.28).

[0066] In specific embodiments, one or more of the EPCAM, CCND1, S100A4, and SNHG7 genes can also be identified by one or more methods selected from the following: gene sequencing, microarray, gene chip, or Northern blotting, etc. Any gene identification method known in the art is applicable to this application.

[0067] Example

[0068] The following embodiments are used to illustrate this application, but are not intended to limit the scope of this application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0069] Unless otherwise specified, all reagents used in the examples are commercially available and all technical means used in the examples are conventional means well known to those skilled in the art.

[0070] Example 1

[0071] In this embodiment, RNA was isolated and extracted from fecal samples using an RNA extraction kit (RNAiso Plus, Takara, 9109), and cDNA was synthesized using an RNA reverse transcription kit (HiScript Q RT SuperMix, Vazyme, R223-01). The steps are as follows:

[0072] 1) Feces from hospital patients or normal intestinal controls were collected using a custom-designed fecal collection box, containing fecal RNA preservation solution. The preservation solution used contained 1M guanidine thiocyanate and 250mM Tris buffer (pH=5.5), which was used to protect the RNA from degradation by nucleases in the feces.

[0073] 2) After thoroughly mixing the fecal sample with the sample preservation solution, take 1.5 mL of the mixture into a 15 mL centrifuge tube, add 3 times the volume of total RNA extraction reagent Trizol, mix thoroughly, let stand at room temperature for 5 min, centrifuge at 12000g for 5 min, and transfer the supernatant to a new centrifuge tube.

[0074] 4) Add 1 / 5 volume of chloroform, mix thoroughly, let stand on ice for 5 minutes, centrifuge at 12000g for 15 minutes, and then transfer the supernatant to a new centrifuge tube.

[0075] 5) Add an equal volume of anhydrous ethanol, mix thoroughly, and then perform adsorption column purification. Transfer 750 μL of the mixture to the corresponding numbered adsorption column, centrifuge at 12000g for 30 seconds, discard the waste liquid in the collection tube, and return the adsorption column to the collection tube. Repeat this step, loading no more than 750 μL each time, until all the liquid has passed through the adsorption column. Rinse twice with 75% ethanol, then transfer 750 μL each time to the adsorption column in the collection tube, centrifuge at 12000g for 30 seconds, and discard the waste liquid in the collection column. Place the adsorption column in a new collection tube, add 50 μL of DEPC water to the adsorption column, and let it stand for 3 minutes. Centrifuge at 12000g for 3 minutes and collect the RNA solution.

[0076] 6) Detect RNA concentration, of which 3 μg is used for cDNA reverse conversion, and the remainder is stored at -80℃.

[0077] 7) Reverse transcription was performed using an RNA reverse transcription kit. Add 800-1000 ng of RNA and 2 μL of genomic DNA-removing enzyme solution to a PCR tube, and incubate at 42°C for 2 minutes. Then add 2 μL of reverse transcriptase to the PCR tube for reverse transcription. The reaction conditions were: 25°C, 10 minutes → 50°C, 35 minutes → 85°C, 5 minutes → 4°C to finish the reaction. After the reaction, dilute the reaction solution with 100 μL of enzyme-free DEPC water for subsequent quantitative real-time PCR experiments.

[0078] Example 2

[0079] In this embodiment, 3-4 pairs of primer sequences were designed for each of the EPCAM, CCND1, S100A4, and SNHG7 genes. These primers were used to amplify the fecal sample cDNA synthesized in Example 1. The primers were optimized through amplification and melting curve analysis, and finally, one optimal primer pair was selected for each gene. ACTB was used as an internal reference gene.

[0080]

[0081]

[0082] The quantitative real-time PCR reaction system contained cDNA template equivalent to 50 ng RNA, with a primer pair final concentration of 200 nM. SYBR Green was used as the fluorescent dye for amplification. The PCR reaction system is as follows:

[0083] SYBR green Master mix 6μL 1x Primers (forward + reverse) 0.12μL 200nM DEPC water 1.88μL cDNA 4μL 50ng

[0084] The PCR reaction program included equilibration at 50℃ for 2 min; pre-denaturation at 95℃ for 10 min; denaturation at 95℃ for 15 s; annealing and extension at 60℃ for 1 min; collection of fluorescence signals; and 40 cycles. Melting curve analysis was performed using the following steps: 95℃, 15 s → 60℃, 1 min → 95℃, 15 s → 60℃, 15 s.

[0085] The results are as follows: Figures 1 to 5 As shown, cDNA synthesized from RNA of three normal intestinal samples is presented. Amplification and melting curves of EPCAM (SEQ ID NO. 3 and 4), CCND1 (SEQ ID NO. 7 and 8), S100A4 (SEQ ID NO. 19 and 20), SNHG7 (SEQ ID NO. 21 and 22), and ACTB (SEQ ID NO. 27 and 28) were detected using the designed optimal primer pairs, indicating that each primer pair can yield a single PCR product.

[0086] Example 3

[0087] In this embodiment, EPCAM, CCND1, S100A4, and SNHG7 were tested on 120 stool samples collected from the hospital. These included stool samples from patients diagnosed with colorectal cancer and advanced adenomas (>1cm adenomatous polyps, precancerous lesions) by colorectal endoscopy.

[0088] After removing some watery stool samples, the RNA expression Ct < 36 of the internal reference gene ACTB was used as the standard for sample quality. A total of 101 stool samples met the quality requirements, including 45 colorectal cancer samples (stage I and II colorectal cancer), 30 normal intestinal samples, and 26 advanced adenoma (>1cm adenomatous intestinal polyps) samples.

[0089] 1. Single-gene testing for colorectal cancer or advanced adenoma

[0090] The Ct values ​​of EPCAM, CCND1, S100A4, SNHG7 and ACTB were obtained respectively, and the Ct value of the sample for which no signal was detected (Ct>40) was set to 40.

[0091] Calculate the expression of a single gene: Subtract the Ct value of the internal reference gene ACTB from the Ct value of each gene to obtain ΔCt, and substitute it into 2. -ΔCt The value obtained from the formula is used as the relative expression value of the gene. For example, if the Ct value of the EPCAM gene in a sample is 35, and the Ct value of its internal reference gene ACTB is 32, then ΔCt = Ct(EPCAM) - Ct(ACTB) = 3. Substituting into 2... -ΔCt The formula yielded a relative expression value of EPCAM of 0.125 in this sample.

[0092] The relative expression values ​​of EPCAM, CCND1, S100A4, and SNHG7 were calculated. Statistical analysis was performed on the three groups of samples using GraphPad Prism software version 8.0 (GraphPad Software Inc., La Jolla, CA). Sensitivity and specificity were calculated using the following formulas:

[0093] Sensitivity (True Positive Rate) = (Number of True Positives / (Number of True Positives + Number of False Negatives)) * 100%

[0094] Specificity (true negative rate) = (number of true negatives / (number of true negatives + number of false positives)) * 100%. The optimal cutoff value is determined using the Youden index (sensitivity + specificity - 1), thus obtaining the sensitivity and specificity of each gene.

[0095] Figures 6 to 9 The receiver operating characteristic (ROC) curves for EPCAM, CCND1, S100A4, and SNHG7 as biomarkers for colorectal cancer (normal vs. tumor) and advanced adenoma (normal vs. polyp) are shown below:

[0096]

[0097]

[0098] It is evident that EPCAM, CCND1, S100A4, and SNHG7, when used alone as biomarkers for the detection of colorectal cancer and advanced adenomas (>1cm adenomatous intestinal polyps), all achieve high sensitivity and specificity.

[0099] 2. Multi-gene combined detection of colorectal cancer

[0100] To explore the effectiveness of multiple genes as combined biomarkers in detecting colorectal cancer, the relative expression of each gene was reassigned a score: based on the ROC curve of each gene in detecting colorectal cancer, the value at which the sensitivity reached 80% was set as the low cutoff value, the value at which the specificity reached 90% was set as the middle cutoff value, and the value at which the specificity reached 100% was set as the high cutoff value. The final score of the gene was 0 (< low cutoff value), 1 (between low cutoff value and middle cutoff value), 2 (between middle cutoff value and high cutoff value), or 3 (> high cutoff value).

[0101] When performing combined gene detection, the scores of the selected genes are summed to obtain the sample's value. Statistical analysis (GraphPad Prism software version 8.0) is then performed to obtain sensitivity and specificity. For example, if a sample has a score of 2 for S100A4 and a score of 1 for SNHG7, in studying the effect of S100A4 and SNHG7 as combined biomarkers for detecting colorectal cancer, the sample's value would be 2 + 1 = 3. Based on this algorithm, the detection performance of dual, triple, and quadruple detection methods for colorectal cancer was studied, and ROC curves were obtained. The results are shown in [Figure number missing]. Figures 10 to 12 The details are as follows:

[0102] 3. Multi-gene combined detection of advanced adenomas

[0103] Accordingly, the statistical analysis results of these genes combined as biomarkers for detecting advanced adenomas (>1cm adenomatous intestinal polyps) are shown in [the table below]. Figures 13 to 15 The details are as follows:

[0104]

[0105] It is evident that the combined use of multiple genes as biomarkers for detecting colorectal cancer or advanced adenoma (>1cm adenomatous polyps) is significantly superior to that of single-gene biomarkers. In particular, when the three genes EPCAM+S100A4+SNHG7 are combined as biomarkers, the sensitivity for colorectal cancer and advanced adenoma (>1cm adenomatous polyps) is 93.33% and 61.54%, respectively, and the specificity for both is 93.33%.

[0106] The application has been described in detail above with general descriptions and specific implementation schemes. Any modifications or improvements made based on this application without departing from its spirit shall fall within the scope of protection claimed in this application.

Claims

1. The use of primer combinations for detecting RNA expression levels of the genes EPCAM, CCND1, S100A4, and SNHG7 in fecal samples in the preparation of products for the diagnosis or auxiliary diagnosis of intestinal polyps or colorectal cancer; wherein the intestinal polyps are advanced adenomas, and the colorectal cancers are early-stage colorectal cancers.

2. The use as described in claim 1, wherein the product is a reagent or a kit.

3. The use as described in claim 1 or 2, wherein the primer combination comprises: Primer pair used to detect RNA expression levels of the EPCAM gene: 5'-TAAGGCCAAGCAGTGCAACG-3' (SEQ ID NO. 3), and 5'-TTGTTCTGTTCTTCTGACCCCAG-3' (SEQ ID NO.4); Primer pair used to detect the RNA expression level of the CCND1 gene: 5'-GGCGGAGGAGAACAAACAGA-3' (SEQ ID NO. 7), and 5'-GGAGGGCGGATTGGAAATGA-3' (SEQ ID NO. 8); Primer pair used to detect RNA expression levels of the S100A4 gene: 5'-GCCCAGCTTCTTGGGGAAAA-3' (SEQ ID NO. 19), and 5'-ATGGCGATGCAGGACAGGAA-3' (SEQ ID NO. 20); and Primer pair used to detect the RNA expression level of the SNHG7 gene: 5'-TTGCTGGCGTCTCGGTTAAT-3' (SEQ ID NO.21), and 5'-TCCATCACAGGCGAAGTCAC-3' (SEQ ID NO. 22).

4. The use as described in claim 2, wherein the reagent or kit further comprises primers for detecting the RNA expression level of the internal reference gene.

5. The use as described in claim 4, wherein the internal reference gene is GAPDH, 36B4, RPL13A, or ACTB.

6. The use as described in claim 5, wherein the primer for detecting the RNA expression level of the ACTB gene is: 5'-TGGCATCCACGAAACTACC-3' (SEQ ID NO. 27), and 5'-GTGTTGGCGTACAGGTCTT-3' (SEQ ID NO. 28).

7. The use as described in claim 2, wherein the reagent or kit further comprises one or more of the following: biological sample preservation reagents, RNA extraction reagents, RNA reverse transcription reagents, and PCR reaction reagents.

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