Colorectal cancer diagnosis system and application thereof

Abstract

The invention discloses a colorectal cancer diagnosis system and application thereof, and the colorectal cancer diagnosis system comprises human SDC2, SFRP2 and TFPI2 gene methylation PCR detection reagents, 20 ng / L of unmethylated human genome standard substances, 50 excrement sample nucleic acids with the concentration of 5-40 ng / L and clinically and clearly diagnosed as normal people, and a kit for detecting the colorectal cancer. The kit is characterized in that the kit comprises 20 ng / L of 100% methylated human genome standard substance and 30 cases of excrement sample nucleic acid of patients with colorectal cancer stage I clinically and clearly diagnosed, wherein the concentration of the excrement sample nucleic acid is 5-40 ng / L. A multi-layer composite interpretation system formed by a multi-component system or a calculation method is used for effectively interpreting whether methylation of the gene in the sample to be detected is positive or negative, the positive rate of colorectal cancer diagnosis can be effectively increased, and therefore the value of early screening of the colorectal cancer is greatly increased.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of disease detection, in particular to a colorectal cancer diagnosis system and application thereof. BACKGROUND

[0002] Colorectal cancer (CRC) is one of the common malignant tumors worldwide. According to the Global Cancer Burden Report in 2020, the prevalence of CRC ranks third in the world, and the mortality rate ranks second. The incidence of CRC is higher in developed countries, but since the 1980s, due to the nationwide screening program and the popularity of colonoscopy, the mortality rate of CRC is declining in some developed countries. However, the early diagnosis rate of CRC in China is low, and the prevalence and mortality rates are on the rise. In 2020, more than 550,000 people in China were newly diagnosed with CRC, with an incidence rate ranking second among all cancers in China, and more than 280,000 people died of CRC. The estimated age-standardized incidence rate was 23.9 / 100,000, and the mortality rate was 12.0 / 100,000. The prognosis of CRC is closely related to the disease stage at the time of diagnosis. Early CRC lacks typical clinical symptoms, and most CRC patients are diagnosed at an advanced stage. Therefore, finding an effective early CRC screening and diagnosis method can reduce the prevalence and mortality rates of CRC, and has great significance for early detection, early treatment, improving the quality of life of patients, and improving the survival rate of patients.

[0003] The Chinese Expert Consensus on Early Diagnosis and Screening Strategy for Colorectal Cancer (2018) and the Chinese Guideline for Colorectal Cancer Screening and Early Diagnosis and Treatment (2020, Beijing) recommend the following methods for colorectal cancer screening and early diagnosis: colonoscopy, fecal occult blood test, fecal DNA methylation detection, and multi-target fecal FIT-DNA detection. Colonoscopy is an invasive test, and people's compliance is poor, making it difficult to be used as a general screening method. The specificity of fecal occult blood test and fecal FIT detection is not high, making it difficult to accurately diagnose early. Only fecal DNA methylation detection has the three characteristics of "high compliance", "high specificity", and "early screening of precancerous lesions". In the past five years, the use of fecal DNA methylation detection for early or auxiliary diagnosis of colorectal cancer has gradually increased, and a large number of colorectal cancer-related genes have been discovered, and various combinations have been reported.

[0004] However, the process of cancer caused by methylation is a gradual quantitative process, and there is usually no intuitive difference between yes and no, so the interpretation of methylation positivity in methylation detection reagents is usually a difficult problem in accurately diagnosing reagents. If a conventional qPCR reagent only uses one CutOff value to define the methylation positivity of a gene, the early diagnosis of cancer is usually inaccurate, and the false negative and false positive rates are high. SUMMARY

[0005] To solve the above problems, the present application provides a colorectal cancer diagnostic system, which comprises the following components: component 1, which is a human SDC2, SFRP2 and TFPI2 gene methylation PCR detection reagent, and a reagent for detecting an internal reference gene, which is B2M; component 2, which is an unmethylated human genome standard of 20 ng / µL; component 3, which is 50 cases of fecal sample nucleic acid of normal persons clinically diagnosed as 5-40 ng / µL; component 4, which is a 100% methylated human genome standard of 20 ng / µL; component 5, which is 30 cases of fecal sample nucleic acid of patients clinically diagnosed as colorectal cancer stage I of 5-40 ng / µL; component 2 is diluted 10 times, 100 times, 1000 times and 10000 times with pure water, i.e. the final concentration of the unmethylated human genome standard is 2 ng / µL, 0.2 ng / µL, 0.02 ng / µL and 0.002 ng / µL; based on the PCR amplification Ct value of the internal reference B2M gene of the 1000 times diluted component 2, N(B2M) is obtained according to the "mean-2SD"; according to the PCR amplification SDC2 gene amplification Ct value of the 10 times, 100 times, 1000 times and 10000 times gradient diluted component 2 and component 3 of 50 cases of samples, N(SDC2) is obtained according to the "mean-2SD"; in turn, N(SFRP2) and N(TFPI2) are obtained; using the above 0.02 ng / µL component 2 as a dilution matrix, the above component 4 is diluted 2 times, 4 times, 10 times, 20 times and 100 times, respectively, i.e. the final methylation proportion and concentration of the methylated human genome standard are (50%, 10 ng / µL), (25%, 5 ng / µL), (10%, 2 ng / µL), (5%, 1 ng / µL) and (1%, 0.2 ng / µL); then using pure water as a dilution matrix, the (1%, 0.2 ng / µL) diluent is diluted 10 times to obtain (1%, 0.02 ng / µL) methylated human genome standard; using the above (1% methylation, 0.P1(SDC2) is obtained according to "mean-2SD" from the Ct value of the SDC2 gene amplification of component 4 at a concentration of 02 ng / µL; P1(SFRP2) and P1(TFPI2) are obtained by analogy; the Ct values of the three genes SDC2, SFRP2 and TFPI2 of the samples selected from the samples in component 5 are located between P1 and N, and the Ct values of the three genes SDC2, SFRP2 and TFPI2 of these samples are calculated with the Ct values of B2M to obtain ΔCt(SDC2), ΔCt(SFRP2) and ΔCt(TFPI2), wherein ΔCt(SDC2) = Ct(SDC2)-Ct(B2M), ΔCt(SFRP2) = Ct(SFRP2)-Ct(B2M) and ΔCt(TFPI2) = Ct(TFPI2)-Ct(B2M); the median of ΔCt(SDC2) of the above samples is selected to obtain P2(SDC2); P2(SFRP2) and P2(TFPI2) are obtained by analogy; based on the Ct value results of the samples to be tested and the three groups of parameters N(SDC2), N(SFRP2) and N(TFPI2); P1(SDC2), P1(SFRP2) and P1(TFPI2); and P2(SDC2), P2(SFRP2) and P2(TFPI2), the determination of whether the sample to be tested is a colorectal cancer sample is realized.

[0006] In an embodiment, the PCR amplification primers and probes for detecting the methylation of the SDC2 gene in component 1 are SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively; the PCR amplification primers and probes for detecting the methylation of the SFRP2 gene are SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively; and the PCR amplification primers and probes for detecting the methylation of the TFPI2 gene are SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

[0007] In an embodiment, the internal reference gene detected in component 1 is B2M, and the PCR amplification primers and probes for detecting the methylation of the B2M gene are SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, respectively.

[0008] In an embodiment, the application provides the use of the above-mentioned colorectal cancer diagnosis system in the preparation of reagents for detecting whether a sample is a colorectal cancer sample.

[0009] In one embodiment, the application includes the following judgment method: For the internal reference gene B2M, when the Ct of the internal reference gene B2M during the detection of the test sample is ≤ N(B2M), it is judged as B2M positive; when Ct > N(B2M), it is judged as B2M negative; for SDC2, as a methylation indicator target, the first-layer positive judgment: when Ct ≤ P1(SDC2), it is judged as SDC2 positive; the second-layer positive judgment: when the Ct value is in the range of P1(SDC2) < Ct ≤ N and at the same time △Ct(SDC2) ≤ P2(SDC2), it is judged as SDC2 positive; the first-layer negative judgment: when Ct > N(SDC2), it is judged as SDC2 negative; the second-layer negative judgment: when the Ct value is in the range of P1(SDC2) < Ct ≤ N and at the same time △Ct(SDC2) > P2(SDC2), it is judged as SDC2 negative; for SFRP2, as a methylation indicator target, the first-layer positive judgment: when Ct ≤ P1(SFRP2), it is judged as SFRP2 positive; the second-layer positive judgment: when the Ct value is in the range of P1(SFRP2) < Ct ≤ N and at the same time △Ct(SFRP2) ≤ P2(SFRP2), it is judged as SFRP2 positive; the first-layer negative judgment: when Ct > N(SFRP2), it is judged as SFRP2 negative; the second-layer negative judgment: when the Ct value is in the range of P1(SFRP2) < Ct ≤ N and at the same time △Ct(SFRP2) > P2(SFRP2), it is judged as SFRP2 negative; for TFPI2, as a methylation indicator target, the first-layer positive judgment: when Ct ≤ P1(TFPI2), it is judged as TFPI2 positive; the second-layer positive judgment: when the Ct value is in the range of P1(TFPI2) < Ct ≤ N and at the same time △Ct(TFPI2) ≤ P2(TFPI2), it is judged as TFPI2 positive; the first-layer negative judgment: when Ct > N(TFPI2), it is judged as TFPI2 negative; the second-layer negative judgment: when the Ct value is in the range of P1(TFPI2) < Ct ≤ N and at the same time △Ct(TFPI2) > P2(TFPI2), it is judged as TFPI2 negative; when B2M is judged as positive: When any one of the three genes SDC2, SFRP2, and TFPI2 is positive, it is diagnosed as positive for colorectal cancer; when all three genes are negative, it is diagnosed as negative for colorectal cancer.

[0010] This invention utilizes a multi-component system to form a multi-layered composite interpretation system or calculation method, effectively interpreting the methylation positivity or negativity of genes in the test sample. This can significantly improve the positive rate of colorectal cancer diagnosis, thereby greatly enhancing the value of early colorectal cancer screening. Expanded validation using nucleic acid analysis of 100 fecal samples with clear clinical diagnoses demonstrated that, with a negative concordance rate exceeding 90%, the "two-layer composite interpretation principle" significantly improved the overall positive concordance rate for advanced adenoma (precancerous lesions), stage I cancer, and all stages of cancer compared to the "single Ct threshold interpretation method," and met the clinical trial requirement of a concordance rate >90%. Detailed Implementation

[0011] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the embodiments are merely examples and cannot limit the present invention. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application. The experimental methods used in this invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0012] Example 1: Establishment of the Colorectal Cancer Diagnostic System of the Present Invention

[0013] The colorectal cancer diagnostic system comprises the following components: Component 1: Human SDC2, SFRP2, and TFPI2 gene methylation detection kit, the primer sequence information of which is shown in Table 1 below, and the detection reaction solution formulation and amplification program are shown in Table 2. This component is used to detect the methylation status of the three colorectal cancer-highly related genes SDC2, SFRP2, and TFPI2, as well as the internal reference gene B2M, in the sample to be tested; Component 2: 20 ng / µL of unmethylated human genomic standard; Component 3: Nucleic acid from 50 fecal samples of clinically diagnosed normal individuals at a concentration of 5-40 ng / µL; Component 4: 20 ng / µL of 100% methylated human genomic standard; Component 5: Nucleic acid from 30 fecal samples of patients clinically diagnosed with stage I colorectal cancer at a concentration of 5-40 ng / µL. Table 1 shows the primer sequence information for the human SDC2, SFRP2, and TFPI2 gene methylation detection kits, and Table 2 shows the reaction solution formulation and amplification procedure for the human SDC2, SFRP2, and TFPI2 gene methylation detection kits.

[0014] Table 1

[0015]

[0016] The diagnostic system was established using the above five components.

[0017] I. Pretreatment of Components 2 and 4 before detection

[0018] 1. Using pure water as the dilution matrix, component 2 was diluted 10-fold, 100-fold, 1000-fold, and 10000-fold, respectively, to obtain unmethylated human genome standards with final concentrations of 2 ng / µL, 0.2 ng / µL, 0.02 ng / µL, and 0.002 ng / µL. 2. Using a 1000-fold dilution of component 2 (0.02 ng / µL) as the dilution matrix, component 4 was diluted 2, 4, 10, 20, and 100 times, respectively, to obtain methylated human genome standards with methylation ratios and concentrations of (50%, 10 ng / µL), (25%, 5 ng / µL), (10%, 2 ng / µL), (5%, 1 ng / µL), and (1%, 0.2 ng / µL), respectively. Then, the (1%, 0.2 ng / µL) dilution was diluted 10 times with pure water as the dilution matrix to obtain methylated human genome standards of (1%, 0.02 ng / µL).

[0019] II. Using component 1, perform qPCR detection on the gradient dilutions of component 2, component 3, component 4, and component 5 according to Table 2 above. 1. Detection Ct values ​​of each analyte or its dilution Statistical analysis of N values ​​(see Table 3 for details): The mean - 2SD of Ct for each replicate well of the internal control B2M gene in component 2 at a 1000-fold dilution was 38, denoted as N(B2M); the mean - 2SD of Ct for each replicate well of component 2 at serial dilutions (10-fold, 100-fold, 1000-fold, 10000-fold) and for each sample in component 3 for the SDC2 gene was 39.5, denoted as N(SDC2); the mean - 2SD of Ct for the SFRP2 gene was 39.5, denoted as N(SFRP2); and the mean - 2SD of Ct for the TFPI2 gene was 39, denoted as N(TFPI2). Table 3 shows the Ct values ​​of each target in the nucleic acid detection results of the tested components used for statistical analysis of N values.

[0020] Statistical analysis of P1 values ​​(see Table 4 for details): The mean - 2SD of Ct for SDC2 gene amplification in component 4 (1% methylation, 0.02 ng / µL concentration) was 35, denoted as P1(SDC2); the mean - 2SD of Ct for SFRP2 gene amplification was 35, denoted as P1(SFRP2); and the mean - 2SD of Ct for TFPI2 gene amplification was 33, denoted as P1(TFPI2). Table 4 shows the Ct values ​​of each target in the nucleic acid detection results of the tested components used for P1 value statistical analysis. Statistical analysis of P2 values (see Table 5 for details): Among the 11 samples in Component 5 (Component 5 - P6, P8, P9, P13, P15, P19, P23, P24, P26, P28, P29), for the three genes SDC2, SFRP2, and TFPI2, the detected Ct values were in the range of P1 < Ct < N. Calculate the Ct difference (△Ct) between the detected Ct values of these three genes SDC2, SFRP2, and TFPI2 and the Ct value detected by B2M for each of these samples: Obtain △Ct(SDC2), △Ct(SFRP2), and △Ct(TFPI2), where △Ct(SDC2) = Ct(SDC2) - Ct(B2M), △Ct(SFRP2) = Ct(SFRP2) - Ct(B2M), and △Ct(TFPI2) = Ct(TFPI2) - Ct(B2M). The "median" of △Ct(SDC2) is 10.3, denoted as P2(SDC2); the "median" of △Ct(SFRP2) is 6.7, denoted as P2(SFRP2); the "median" of △Ct(TFPI2) is 6.4, denoted as P2(TFPI2). Table 5 shows the Ct values of each target in the nucleic acid detection results of the tested components for P2 value statistical analysis.

[0021] Table 3

[0022] (Note: The corresponding Ct for no amplification is recorded as 40; the nucleic acids of 50 fecal samples from clinically diagnosed normal individuals in Component 3 are recorded as "Component 3 - N1~N50").

[0023] Table 4

[0024] (Note: The corresponding Ct for no amplification is recorded as 40).

[0025] Table 5

[0026] (Note: The corresponding Ct for no amplification is recorded as 40; the nucleic acids of 30 fecal samples from patients with stage I colorectal cancer clinically diagnosed in Component 5 are recorded as "Component 5 - P1~P30", and the data re - marked for P2 statistics is shown).

[0027] III. Nucleic acids of 100 fecal samples with clear clinical diagnosis results For the nucleic acids of 100 fecal samples with clear clinical diagnosis results, Component 1 was used for detection. According to the statistically obtained N, P1, and P2 values above, the single Ct - threshold interpretation method and the two - layer composite interpretation principle were used for interpretation respectively, and the results of the two interpretation methods were statistically analyzed.

[0028] 1. Nucleic acid sample information of 100 fecal samples with clear clinical diagnoses: 30 normal colonoscopy subjects, 10 patients with colonoscopy-confirmed advanced adenomas (precancerous stage), 35 cases of stage I colorectal cancer, 10 cases of stage II colorectal cancer, 10 cases of stage III colorectal cancer, and 5 cases of stage IV colorectal cancer.

[0029] 2. The Ct results of the above samples after testing with component 1 are shown in Table 6. Table 6 shows the Ct values ​​of each target in the nucleic acid test results of 100 fecal samples with clear clinical diagnoses.

[0030] Table 6 Sample Name B2M SDC2 SFRP2 TFPI2 KN1 29.02 40 40 40 KN2 29.57 40 40 40 KN3 29.44 40 40 40 KN4 35.92 40 40 40 KN5 31.75 40 40 40 KN6 33.21 40 40 40 KN7 29.82 40 40 40 KN8 28.74 36.53 36.62 40 KN9 29.16 40 40 40 KN10 29.39 40 40 40 KN11 29.59 40 40 40 KN12 32.81 40 40 40 KN13 34.63 40 40 40 KN14 31.47 40 40 40 KN15 32.98 40 40 37.14 KN16 34.62 40 40 40 KN17 30.57 40 40 40 KN18 30.64 40 40 40 KN19 30.53 40 40 40 KN20 34.47 40 40 40 KN21 31.06 40 40 40 KN22 34.11 40 40 40 KN23 30.83 40 40 40 KN24 35.32 40 40 40 KN25 31.24 40 40 40 KN26 34.61 40 40 40 KN27 34.29 40 40 40 KN28 29.9 40 40 40 KN29 30.85 40 40 40 KN30 29.02 40 40 40 AA1 29.78 40 40 40 AA2 36.17 40 40 40 AA3 31.26 40 40 40 AA4 31.82 40 40 40 AA5 30.21 35.66 38.14 40 AA6 32.48 40 40 40 AA7 29.04 36.33 35.78 33.96 AA8 34.54 40 40 40 AA9 26.49 35.71 36.27 36.26 AA10 31.44 39.32 37.33 40 K1P1 30.54 35.11 32.54 32.25 K1P2 31.26 40 40 40 K1P3 25.25 30.45 29.71 29.33 K1P4 29.39 36.12 35.69 40 K1P5 25.87 30.59 31.4 40 K1P6 23.31 29.76 30.05 31.08 K1P7 23.85 30.21 30.67 31.17 K1P8 29.11 33.76 33.22 40 K1P9 35.7 37.57 40 40 K1P10 33.67 40 40 40 K1P11 24.79 35.25 33.89 35.58 K1P12 22.66 28.82 29.09 40 K1P13 23.86 27.98 28.3 26.12 K1P14 27.52 31.55 32.35 33.64 K1P15 26.16 33.11 33.8 40 K1P16 27.91 32.88 34.19 34.49 K1P17 28.82 40 33.01 40 K1P18 27.17 29.24 28.12 26.5 K1P19 30.72 38.42 36.84 34.16 K1P20 31.04 40 40 40 K1P21 30.82 40 32.93 40 K1P22 31.05 37.55 37.52 40 K1P23 28.73 30.48 30.87 30.21 K1P24 30.37 35.83 35.33 40 K1P25 30.22 40 36.96 30.56 K1P26 23.27 40 35.68 31.42 K1P27 27.69 30.77 31.61 30.68 K1P28 28.69 31.55 30.95 30.85 K1P29 28.71 31.65 30.68 31.04 K1P30 29.87 40 40 40 K1P31 28.83 37.65 34.81 40 K1P32 29.59 34.68 36.1 40 K1P33 30.13 35.45 36.1 40 K1P34 24.33 31.62 35.76 40 K1P35 29.89 40 34.58 40 K2P1 28.34 40 40 40 K2P2 27 32.53 31.55 31.37 K2P3 23.2 28.64 28.42 25.86 K2P4 27.08 35.64 35.6 36.83 K2P5 24.7 29.96 30.3 27.57 K2P6 25.16 31.41 30.35 30.11 K2P7 28 32.57 32.54 31.09 K2P8 22.07 25.64 25.16 23.15 K2P9 26.97 35.77 34.77 40 K2P10 27.14 33.63 38.94 40 K3P1 24.8 29.85 30.22 28.17 K3P2 25.57 32.23 34.04 31.68 K3P3 29.07 30.52 30.49 28.96 K3P4 31.49 40 40 40 K3P5 22.83 25.59 25.29 24.07 K3P6 27.02 28.75 28.82 27.85 K3P7 25.72 29.03 28.86 29.22 K3P8 24.56 26.91 28.66 25.43 K3P9 26.63 35.09 36.68 34.61 K3P10 29.68 36.38 34.69 40 K4P1 24.49 31.7 33.7 36.26 K4P2 28.53 32.41 33.86 31.22 K4P3 27.03 37.73 33.15 40 K4P4 28.41 37.69 34.99 40 K4P5 28.22 37.21 35.16 40 .

[0031] (Note: The corresponding Ct for no amplification is denoted as 40; 30 clinically diagnosed normal individuals are labeled KN1~KN30; 10 patients with colonoscopy-confirmed adenomas are labeled AA1~AA10; 35 patients clinically diagnosed with stage I colorectal cancer are labeled K1P1~K1P35; 10 patients clinically diagnosed with stage II colorectal cancer are labeled K2P1~K2P10; 10 patients clinically diagnosed with stage III colorectal cancer are labeled K3P1~K3P10; and 5 patients clinically diagnosed with stage IV colorectal cancer are labeled K4P1~K4P5) 3. Statistical analysis of the interpretation results of the two methods

[0032] 3.1 Single Ct Threshold Interpretation Method

[0033] Based on the N and P1 values ​​obtained above, for the internal reference gene, a value ≤ N is interpreted as positive for internal reference amplification, and a value > N is interpreted as negative for internal reference amplification. For the target gene, a value ≤ P1 for each target is interpreted as positive for target amplification, and a value > P1 is interpreted as negative for target amplification.

[0034] For B2M, it is used as an internal reference. When Ct≤N(B2M), that is, when Ct≤38, B2M is interpreted as positive; when Ct>N(B2M), that is, when Ct>38, B2M is interpreted as negative. For SDC2, as a methylation indicator target, when Ct≤P1(SDC2), i.e. when Ct≤35, SDC2 is interpreted as positive; when Ct>P1(SDC2), i.e. when Ct>35, SDC2 is interpreted as negative. For SFRP2, as a methylation indicator target, when Ct≤P1(SFRP2), i.e., when Ct≤35, SFRP2 is interpreted as positive; when Ct>P1(SFRP2), i.e., when Ct>35, SFRP2 is interpreted as negative. For TFPI2, as a methylation indicator target, when Ct≤P1(TFPI2), i.e., when Ct≤33, TFPI2 is interpreted as positive; when Ct>P1(TFPI2), i.e., when Ct>33, TFPI2 is interpreted as negative. When B2M is positive: if any one of the three genes SDC2, SFRP2 and TFPI2 is positive, the diagnosis is positive for colorectal cancer; if all three genes are negative, the diagnosis is negative for colorectal cancer.

[0035] If the B2M reading is negative, the test is invalid and needs to be repeated.

[0036] 3.2 Statistical Analysis of Single Ct Threshold Interpretation Results

[0037] According to principle 3.1 above, in 100 clinical samples, the positive concordance rate for colorectal cancer was 76.7%, and the negative concordance rate for colorectal cancer (normal individuals) was 100% (Table 7). Among them, the positive concordance rate for stage I colorectal cancer was "(26 / 35)×100%=74.3%"; advanced adenomas are a benign lesion before cancer, and if they can be diagnosed in advance at this stage, it will greatly ensure the safety of patients' lives. This type of sample is used as an extreme test of the testing reagent, and the positive concordance rate for interpreting the single Ct threshold is calculated separately as "(0 / 10)×100%=0%". From this, we can see that the positive concordance rate of interpreting the single Ct threshold is less than 90%, which is difficult to meet the minimum 90% requirement of clinical trials. Table 7 shows the consistency analysis results of the results of negative and positive samples of single Ct threshold interpretation and clinical diagnosis.

[0038] Table 7

[0039] 3.3 Principles of Two-Layer Composite Interpretation Based on the N, P1, and P2 values ​​obtained from the above statistics, the internal reference gene was interpreted as positive for amplification if the value was ≤N, and negative if the value was >N. For the three genes SDC2, SFRP2, and TFPI2, a two-tiered interpretation was used.

[0040] For B2M, it is used as an internal reference. When Ct≤N(B2M), that is, when Ct≤38, B2M is interpreted as positive; when Ct>N(B2M), that is, when Ct>38, B2M is interpreted as negative. For SDC2, as a methylation indicator target, the first - layer positive interpretation: When Ct ≤ P1(SDC2), that is, when Ct ≤ 35, SDC2 is interpreted as positive; the second - layer positive interpretation: When the Ct value is in the range of P1(SDC2) < Ct ≤ N and at the same time △Ct(SDC2) (△Ct(SDC2)=Ct(SDC2)-Ct(B2M)), that is, 35 < Ct ≤ 39.5 and at the same time △Ct(SDC2) ≤ 10.3, SDC2 is interpreted as positive; the first - layer negative interpretation: When Ct > N(SDC2), that is, when Ct > 39.5, SDC2 is interpreted as negative; the second - layer negative interpretation: When the Ct value is in the range of P1(SDC2) < Ct ≤ N and at the same time △Ct(SDC2)>P2(SDC2), that is, 35 < Ct ≤ 39.5 and at the same time △Ct(SDC2)>10.3, SDC2 is interpreted as negative; For SFRP2, as a methylation indicator target, the first - layer positive interpretation: When Ct ≤ P1(SFRP2), that is, when Ct ≤ 35, SFRP2 is interpreted as positive; the second - layer positive interpretation: When the Ct value is in the range of P1(SFRP2) < Ct ≤ N and at the same time △Ct(SFRP2) (△Ct(SFRP2)=Ct(SFRP2)-Ct(B2M)) ≤ P2(SFRP2), that is, 35 < Ct ≤ 39.5 and at the same time △Ct(SFRP2) ≤ 6.7, SFRP2 is interpreted as positive; the first - layer negative interpretation: When Ct > N(SFRP2), that is, when Ct > 39.5, SFRP2 is interpreted as negative; the second - layer negative interpretation: When the Ct value is in the range of P1(SFRP2) < Ct ≤ N and at the same time △Ct(SFRP2)>P2(SFRP2), that is, 35 < Ct ≤ 39.5 and at the same time △Ct(SFRP2)>6.7, SFRP was interpreted as negative; For TFPI2, as a methylation indicator target, the first - layer positive interpretation: When Ct ≤ P1(TFPI2), that is, when Ct ≤ 33, TFPI2 is interpreted as positive; the second - layer positive interpretation: When the Ct value is in the range of P1(TFPI2) < Ct ≤ N and at the same time △Ct(TFPI2) (△Ct(TFPI2)=Ct(TFPI2)-Ct(B2M)) ≤ P2(TFPI2), that is, 37 < Ct ≤ 39 and at the same time △Ct(TFPI2) ≤ 6.4, TFPI2 is interpreted as positive; the first - layer negative interpretation: When Ct > N(TFPI2), that is, when Ct > 39, TFPI2 is interpreted as negative; the second - layer negative interpretation: When the Ct value is in the range of P1(TFPI2) < Ct ≤ N and at the same time △Ct(TFPI2)>P2(TFPI2), that is, 37 < Ct ≤ 39 and at the same time △Ct(TFPI2)>6.4, TFPI2 is interpreted as negative; When B2M is positive: if any one of the three genes SDC2, SFRP2 and TFPI2 is positive, the diagnosis is positive for colorectal cancer; if all three genes are negative, the diagnosis is negative for colorectal cancer.

[0041] If the B2M reading is negative, the test is invalid and needs to be repeated.

[0042] Based on the above principles, in 100 clinical samples, the positive concordance rate for colorectal cancer was 91.7%, and the negative concordance rate was 93.3% (see Table 8 for details). Among them, the positive concordance rate for stage I colorectal cancer was "(32 / 35)×100%=91.4%". For advanced adenoma samples, the positive concordance rate using the two-layer composite interpretation principle was "(4 / 10)×100%=40%". Table 8 shows the consistency analysis results between the two-layer composite interpretation principle and the clinical diagnosis of negative and positive samples.

[0043] Table 8

[0044] Therefore, after expanding the verification of nucleic acid from 100 fecal samples with clear clinical diagnoses, it was demonstrated that when the negative concordance rate was higher than 90%, the overall positive concordance rate of advanced adenoma (precancerous lesion), stage I cancer, and all stages of cancer was significantly higher than that of the "single Ct threshold interpretation method" when using the "two-layer composite interpretation principle" for interpretation, and it met the concordance rate requirement of >90% in clinical trials.

[0045] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0046] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A colorectal cancer diagnostic system characterized by comprising: The diagnostic system comprises the following components: Component 1, which is a human SDC2, SFRP2 and TFPI2 gene methylation PCR detection reagent, and a reagent for detecting a reference gene, which is B2M; Component 2, which is an unmethylated human genome standard at 20 ng / µL; Component 3, which is 50 cases of fecal sample nucleic acid of clinically diagnosed normal persons at a concentration of 5-40 ng / µL; Component 4, which is a 100% methylated human genome standard at 20 ng / µL; Component 5, which is 30 cases of fecal sample nucleic acid of clinically diagnosed colorectal cancer stage I patients at a concentration of 5-40 ng / µL; Component 2 is diluted 10 times, 100 times, 1000 times and 10000 times with pure water, i.e. finally obtaining an unmethylated human genome standard at a final concentration of 2 ng / µL, 0.2 ng / µL, 0.02 ng / µL and 0.002 ng / µL; based on the PCR amplification Ct value of the reference B2M gene of the 1000-fold diluted component 2, N(B2M) is obtained according to "mean-2SD"; according to the PCR amplification Ct value of the SDC2 gene of the 10-fold, 100-fold, 1000-fold and 10000-fold gradient diluted component 2 and the 50 cases of samples in component 3, N(SDC2) is obtained according to "mean-2SD"; and in sequence, N(SFRP2) and N(TFPI2) are obtained; The above-mentioned 0.02 ng / µL component 2 is used as a dilution matrix, and the above-mentioned component 4 is diluted 2 times, 4 times, 10 times, 20 times and 100 times, respectively, i.e. finally obtaining a methylated human genome standard at a methylation ratio and concentration of (50%, 10 ng / µL), (25%, 5 ng / µL), (10%, 2 ng / µL), (5%, 1 ng / µL) and (1%, 0.2 ng / µL); then the diluent (1%, 0.2 ng / µL) is diluted 10 times with pure water as a dilution matrix, to obtain a methylated human genome standard at (1%, 0.02 ng / µL); the SDC2 gene amplification Ct value of the above-mentioned (1% methylation, 0.02 ng / µL concentration) component 4 is used to obtain P1(SDC2) according to "mean-2SD"; and in sequence, P1(SFRP2) and P1(TFPI2) are obtained. The Ct values of the three genes SDC2, SFRP2 and TFPI2 in the selected samples from the component 5 samples are located between P1 and N, and the Ct values of the three genes SDC2, SFRP2 and TFPI2 in the selected samples are calculated with the Ct values of B2M to obtain Ct difference values ΔCt, ΔCt(SDC2), ΔCt(SFRP2) and ΔCt(TFPI2), wherein ΔCt(SDC2) = Ct(SDC2) - Ct(B2M), ΔCt(SFRP2) = Ct(SFRP2) - Ct(B2M) and ΔCt(TFPI2) = Ct(TFPI2) - Ct(B2M); the median of ΔCt(SDC2) of the selected samples is selected to obtain P2(SDC2); and P2(SFRP2) and P2(TFPI2) are obtained in the same way. Based on the Ct values of the to-be-tested sample and the three groups of parameters N(SDC2), N(SFRP2) and N(TFPI2), P1(SDC2), P1(SFRP2) and P1(TFPI2), and P2(SDC2), P2(SFRP2) and P2(TFPI2), it is determined whether the to-be-tested sample is a colorectal cancer sample.

2. The colorectal cancer diagnostic system according to claim 1, characterized by, The PCR amplification primers and probes for detecting the methylation of the SDC2 gene in component 1 are SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, respectively; the PCR amplification primers and probes for detecting the methylation of the SFRP2 gene are SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, respectively; and the PCR amplification primers and probes for detecting the methylation of the TFPI2 gene are SEQ ID NO. 7, SEQ ID NO. 8 and SEQ ID NO. 9, respectively.

3. The colorectal cancer diagnostic system according to claim 1, characterized by, The internal reference gene detected in component 1 is B2M, and the PCR amplification primers and probes for detecting the methylation of the B2M gene are SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12, respectively.

4. The colorectal cancer diagnosis system according to any one of claims 1-3 is used for preparing a reagent for detecting whether a sample is a colorectal cancer sample.

5. Use according to claim 4, characterized in that, The application includes the following determination method: For the internal reference gene B2M, when the Ct of the internal reference gene B2M is ≤ N(B2M) when the to-be-tested sample is detected, the B2M is determined to be positive; and when the Ct > N(B2M), the B2M is determined to be negative. For SDC2 as a methylation indicator target, the positive first layer interpretation: when Ct≤P1(SDC2), SDC2 is positive; the positive second layer interpretation: when the Ct value is in P1(SDC2)<Ct≤N and at the same time, △Ct(SDC2)≤P2(SDC2), SDC2 is positive; the negative first layer interpretation: when Ct>N(SDC2), SDC2 is negative; the negative second layer interpretation: when the Ct value is in P1(SDC2)<Ct≤N and at the same time, △Ct(SDC2)>P2(SDC2), SDC2 is negative; For SFRP2 as a methylation indicator target, the positive first layer interpretation: when Ct≤P1(SFRP2), SFRP2 is positive; the positive second layer interpretation: when the Ct value is in P1(SFRP2)<Ct≤N and at the same time, △Ct(SFRP2)≤P2(SFRP2), SFRP2 is positive; the negative first layer interpretation: when Ct>N(SFRP2), SFRP2 is negative; the negative second layer interpretation: when the Ct value is in P1(SFRP2)<Ct≤N and at the same time, △Ct(SFRP2)>P2(SFRP2), SFRP2 is negative; For TFPI2 as a methylation indicator target, the positive first layer interpretation: when Ct≤P1(TFPI2), TFPI2 is positive; the positive second layer interpretation: when the Ct value is in P1(TFPI2)<Ct≤N and at the same time, △Ct(TFPI2)≤P2(TFPI2), TFPI2 is positive; the negative first layer interpretation: when Ct>N(TFPI2), TFPI2 is negative; the negative second layer interpretation: when the Ct value is in P1(TFPI2)<Ct≤N and at the same time, △Ct(TFPI2)>P2(TFPI2), TFPI2 is negative; When B2M is positive: any one of the above three genes of SDC2, SFRP2 and TFPI2 is positive, which is diagnosed as positive for colorectal cancer; when all three genes are negative, it is diagnosed as negative for colorectal cancer.

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