Composition and kit for early screening of intestinal cancer and method for detecting methylation of KCNQ5 gene in early blood of intestinal cancer

Abstract

The invention provides a composition, a kit and a detection method for early screening of intestinal cancer. The composition comprises a specific primer and a probe of the KCNQ5 gene, and a reference gene primer and a probe. The detection method is based on sulfite treatment and fluorescent quantitative PCR amplification of cfDNA in plasma, the methylation level of the KCNQ5 gene is judged by calculating the Ct value difference (delta Ct) between the specific primer and the internal reference primer, and methylation is positive when the delta Ct value is smaller than 8.09. The method is easy to operate and non-invasive, healthy people and intestinal cancer patients can be efficiently distinguished, and convenience is provided for early screening of intestinal cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro diagnosis, and in particular to a composition and a kit for early screening of rectal cancer, and a method for detecting blood KCNQ5 gene methylation in early rectal cancer. Background Art

[0002] Colorectal cancer (CRC) is a cancerous lesion originating from the colorectal mucosal epithelium. According to the latest US cancer data released as of January 2024, colorectal cancer ranks third in the incidence of cancer in the United States and is also the second leading cause of cancer death in cancer patients. The prognosis of CRC is closely related to early diagnosis. Most early CRCs can be cured, with a 5-year survival rate of up to 90%, while the late stage is less than 10%. At present, the gold standard for the diagnosis of colorectal cancer is still colonoscopy and biopsy, but due to the invasiveness of this examination, the long preoperative intestinal preparation and the high price, the acceptance and participation rate of this examination are low. How to screen out high-risk groups for colonoscopy is the key to improving the early diagnosis rate of CRC. Due to the strong concealment of CRC, the current early detection rate is only 5% to 10%, which greatly affects the diagnosis and treatment of the disease. Therefore, there is an urgent need for an efficient and convenient early screening method for CRC in clinical practice. Summary of the invention

[0003] In order to solve the above technical problems, the purpose of the present invention is to provide a method and a kit for detecting blood KCNQ5 gene methylation, wherein the kit for detecting blood KCNQ5 gene methylation has accurate detection results, and in order to achieve the above technical effects, the present invention provides a composition for early screening of colorectal cancer, comprising a KCNQ5 target-specific primer pair, a KCNQ5-specific primer probe, an internal reference primer pair, and an internal reference primer probe, wherein:

[0004] The cytosine at the second CG site of the KCNQ5-specific primer probe sequence is a methylated cytosine, and the second CG site is sites 72623235 and 72623236 on chromosome 6.

[0005] It should be noted that the second CG site of the KCNQ5 gene (72623235, 72623236 on chromosome 6) is an important epigenetic marker site in the occurrence of colorectal cancer. The cytosine methylation status of this site directly affects the expression regulation of the KCNQ5 gene, and the abnormal expression of the KCNQ5 gene may be closely related to the early occurrence of colorectal cancer. By specifically targeting this methylation-sensitive area, it is possible to capture trace methylated DNA in blood samples of colorectal cancer patients, thereby providing an efficient molecular diagnostic tool for early screening. The introduction of methylated cytosine significantly enhances the thermodynamic stability of the binding of the probe to the target sequence. The presence of methylated cytosine reduces the occurrence of nonspecific binding by changing the hydrophobicity and spatial conformation of the DNA molecule, thereby improving the specificity and signal strength of the detection system. This enhancement effect enables the detection system to more sensitively capture trace methylated DNA signals while avoiding false positive or false negative results caused by competitive binding of unmethylated sequences.

[0006] As a preferred technical solution of a composition for early screening of colorectal cancer, the sequence of the KCNQ5 target-specific primer pair is as follows:

[0007] Forward primer: 5′-GGAGAAGGTGGTTAAGGTTTAGAG-3′;

[0008] Reverse primer: 5′-TACGCTTCTACTATCGCACCT-3′;

[0009] The sequences of the KCNQ5-specific primer probes are as follows:

[0010] 5'-ACGTTACTTCGCCTAACCTACTAACCTC-3';

[0011] The sequences of the internal reference primer pairs are as follows:

[0012] Forward primer: 5′-GTGATGGAGGAGGTTTAGTAA-3′;

[0013] Reverse primer: 5′-CCAATAAAACCTACTCCTCCCT-3′;

[0014] The sequences of the internal reference primer probe are as follows:

[0015] 5'-CACCCAACACACAATAAAAACACA-3'.

[0016] It should be noted that this technical solution achieves efficient detection of the methylation status of the KCNQ5 gene in early screening for colorectal cancer through precisely designed primer pairs and probes. The forward primer and reverse primer of the KCNQ5 target-specific primer pair can specifically bind to the target region of the target gene, guiding qPCR amplification to produce sufficient target fragments; the KCNQ5-specific primer probe targets the key methylation-sensitive sites in the amplified fragments, and its design ensures that the probe can accurately distinguish between methylated and unmethylated sequences, and reflects the amplification of the target fragments through fluorescent signals, thereby improving the sensitivity and specificity of the detection. At the same time, the introduction of internal reference primer pairs and internal reference probes provides a correction benchmark for the detection system by amplifying and monitoring the internal reference gene signals, effectively eliminating the influence of sample concentration differences or amplification efficiency fluctuations. This synergistic effect of primers and probes not only achieves highly sensitive detection of methylated DNA of the target gene, but also ensures the reliability of the detection system, making this method show significant technical advantages in early screening for colorectal cancer.

[0017] At the same time, the present invention also provides a kit for early screening of colorectal cancer, comprising the above-mentioned composition for early screening of colorectal cancer.

[0018] In a second aspect, the present invention also provides a method for detecting KCNQ5 gene methylation in blood in the early stage of colorectal cancer, comprising the following technical steps:

[0019] The KCNQ5 gene methylation was tested using a kit for early colorectal cancer screening;

[0020] Total cfDNA was extracted from plasma;

[0021] Add sulfite and incubate with cfDNA to convert methylated cytosine into uracil;

[0022] qPCR primers were added for amplification, and the FAM signal of the specific primer group and the CY-5 signal of the internal reference primer were collected. The ΔCt was obtained by calculating the difference between the ct values ​​of the specific primer and the internal reference primer to determine the methylation level of the blood KCNQ5 gene.

[0023] It should be noted that this method achieves efficient detection of KCNQ5 gene methylation in early screening of colorectal cancer through a series of precise technical steps. First, a specially designed kit was used to detect KCNQ5 gene methylation in blood samples. Then, total cfDNA (circulating free DNA) was extracted from plasma, which usually originates from cell apoptosis or necrosis and carries tumor-related epigenetic information. In the extracted cfDNA, unmethylated cytosine will be converted to uracil by sulfite treatment, while methylated cytosine remains unchanged. This step ensures the distinguishability of methylated and unmethylated states. Next, specially designed qPCR primers were added for amplification, in which the specific primer set and the internal reference primer targeted the KCNQ5 gene region and the internal reference gene region, respectively, and the FAM signal (from the specific primer) and the CY-5 signal (from the internal reference primer) were collected by real-time monitoring of qPCR. Finally, the methylation level of the KCNQ5 gene was further analyzed by calculating the difference (ΔCt) between the Ct values ​​of the specific primer and the internal reference primer. The size of the ΔCt value directly reflects the methylation status of the KCNQ5 gene. A smaller ΔCt value indicates a higher methylation level, providing accurate quantitative information for early screening of colorectal cancer risk. This technical process combines highly sensitive and specific qPCR technology, making the detection of KCNQ5 gene methylation in blood samples more accurate and reliable, and has great potential for clinical application.

[0024] As a preferred technical solution for the method of detecting blood KCNQ5 gene methylation in early colorectal cancer, the difference between the ct value of the specific primer and the internal reference primer is calculated to obtain ΔCt, thereby judging the blood KCNQ5 gene methylation level, including:

[0025] If the difference between the ct values ​​of the specific primer and the internal reference primer yields a ΔCT value < 8.09, it can be determined that the KCNQ5 gene is positively methylated;

[0026] If the difference between the ct values ​​of the specific primer and the internal reference primer yields a ΔCT value ≥ 8.09 or no fluorescent signal is detected, it is judged as negative for KCNQ5 gene methylation.

[0027] It should be noted that the preferred technical solution quantitatively determines the methylation level of the KCNQ5 gene in the blood by calculating the difference (ΔCt) between the Ct value of the specific primer and the internal reference primer, thereby achieving accurate early screening. When the ΔCt value is <8.09, it indicates that the methylation level of the KCNQ5 gene is high, so it can be judged as positive KCNQ5 gene methylation, which usually means that epigenetic changes related to colorectal cancer have occurred, and there may be an early risk of colorectal cancer; and when the ΔCt value is ≥8.09 or no fluorescent signal is detected, it indicates that the KCNQ5 gene does not show obvious methylation changes, so it is judged as negative KCNQ5 gene methylation, which means that colorectal cancer-related methylation markers have not been detected in the blood sample. In this way, the ΔCt value as a key indicator, combined with the monitoring of real-time fluorescent signals, can effectively distinguish between positive and negative methylation states, and provide high sensitivity and high specificity technical support for early screening of colorectal cancer. This quantitative analysis method is of great significance in clinical applications and can provide a reliable basis for early diagnosis and risk assessment.

[0028] The present invention also provides a kit for early screening of colorectal cancer for use in screening, diagnosing and treating cancer diseases.

[0029] Furthermore, the intestinal cancer includes rectal cancer and colon cancer.

[0030] Compared with the prior art, the beneficial effects of the present invention are: first, the use of blood samples for detection is more convenient and non-invasive than the traditional colonoscopy method, which greatly reduces the pain and psychological burden of patients during the screening process, and at the same time improves the acceptance of the test. Secondly, by detecting the methylation level of the KCNQ5 gene, the present invention can effectively identify the molecular markers of early colorectal cancer. Since abnormal methylation is an early event in tumorigenesis, this method realizes the early diagnosis and screening of colorectal cancer, helps to improve the early detection rate and significantly reduce the mortality rate. In addition, the present invention designs specific KCNQ5 gene primers and probes, and combines fluorescent quantitative PCR technology, and the detection results have high specificity and high sensitivity. β-actin is used as an internal reference gene to further ensure the accuracy and stability of the experiment and eliminate errors in samples and experimental operations. Finally, this method does not require electrophoresis or hybridization operations subsequent to PCR, reduces experimental steps and contamination risks, and greatly improves detection efficiency and convenience. These advantages make the detection method of the present invention have good clinical promotion value and can be widely used in the field of early screening and diagnosis of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the fluorescence quantitative amplification curve for KCNQ5 gene methylation detection;

[0032] Figure 2is the fluorescence quantitative amplification curve of the β-actin internal reference gene;

[0033] Figure 3 The test results of colorectal cancer tissue and adjacent normal tissue;

[0034] Figure 4 These are the test results and ROC curves of plasma samples from healthy people and colorectal cancer patients. DETAILED DESCRIPTION

[0035] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0036] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0037] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0038] Example 1. Preparation and use of the kit

[0039] This embodiment provides a kit for detecting blood KCNQ5 gene methylation, including a KCNQ5 target specific primer pair, a KCNQ5 specific primer probe, an internal reference primer pair, and an internal reference primer probe, the sequences of which are as follows:

[0040] KCNQ5 target-specific primer pairs:

[0041] Forward: 5'-GGAGAAGGTGGTTAAGGTTTAGAG-3';

[0042] Reverse: 5'-TACGCTTCTACTATCGCACCT-3';

[0043] KCNQ5-specific primer probe:

[0044] 5'-ACGTTACTTCGCCTAACCTACTAACCTC-3';

[0045] Internal reference primer pair:

[0046] Forward: 5'-GTGATGGAGGAGGTTTAGTAA-3';

[0047] Reverse: 5'-CCAATAAAACCTACTCCTCCCT-3';

[0048] Internal reference primer probe:

[0049] 5'-CACCCAACACACAATAACAAACACA-3';

[0050] The cytosine at the second CG site of the KCNQ5-specific primer probe sequence is a methylated cytosine, and the second CG site is sites 72623235 and 72623236 on chromosome 6.

[0051] Experimental process:

[0052] Extraction of plasma free DNA

[0053] The specific extraction method may include the following steps: According to the operation process of the magnetic bead method plasma free DNA extraction kit of Fanzhi Medical:

[0054] (1) Preparation before the experiment

[0055] Check whether there are crystals in Lysis Buffer 1 and Lysis Buffer 2. If there are crystals, melt them in 60℃ water, then invert them 10 times to mix well and set aside.

[0056] (2) Experimental procedures

[0057] ① Lysis: In a 15 ml centrifuge tube, add 40 ml proteinase K, 2 ml sample, and 100 μl lysis buffer 1 in sequence, shake well to mix, and then lyse at 60°C for 20 minutes.

[0058] ② Binding: Add 1.6 ml of lysis solution 2, 0.9 ml of isopropanol and 40 μl of magnetic beads CD to the centrifuge tube, shake thoroughly to mix, and then bind at room temperature for 10 minutes. During this period, shake several times to ensure that the magnetic beads remain suspended. Then place on the magnetic rack, wait for the solution to clarify, and discard the supernatant.

[0059] ③1st wash: Inject 1ml of wash solution CD1 into the centrifuge tube and shake for 20 seconds. Transfer the liquid and magnetic beads to a new 1.5ml centrifuge tube, place it on the magnetic rack, wait for the solution to become clear, then aspirate and discard the supernatant.

[0060] ④ Second washing: Inject 1 ml of washing solution CD1 into the centrifuge tube and shake for 20 seconds. Centrifuge briefly, place the centrifuge tube on the magnetic rack, wait for the solution to become clear, then aspirate and discard the supernatant.

[0061] ⑤ The third wash: inject 1 ml of washing solution CD2 into the centrifuge tube and shake for 20 seconds. Centrifuge briefly, place the centrifuge tube on the magnetic rack, wait for the solution to become clear, then aspirate and discard the supernatant.

[0062] ⑥Wash 4: Repeat step 5.

[0063] ⑦ Discard the liquid: Perform a brief centrifugation to collect the residual liquid in the tube; place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, absorb and discard the liquid in the tube, and dry it at room temperature for 5-10 minutes.

[0064] ⑧Storage: Centrifuge briefly to centrifuge the evaporated liquid on the tube cap, place the centrifuge tube on a magnetic rack, let it stand, and after the magnetic beads are completely adsorbed, transfer the supernatant to a new enzyme-free centrifuge tube and store it at -20℃ or -80℃.

[0065] 2. Sulfite Conversion

[0066] (1) Preparation before the experiment

[0067] Prepare Bisulfite Mix according to experimental requirements. When preparing, add 850 μl of BM buffer to each tube of dry powder, shake and mix for 5 minutes to completely dissolve the dry powder (the unused Bisulfite Mix is ​​divided into brown centrifuge tubes, and 94 μl of unused Bisulfite Mix is ​​added to each brown centrifuge tube, and stored in a -20° refrigerator, which can be stored for one month, and avoid repeated thawing and freezing).

[0068] (2) Experimental procedures

[0069] ① Prepare the bisulfite reaction system in a 200μl centrifuge tube. The specific preparation system is shown in Table 1.1:

[0070] Table 1.1 Bisulfate reaction system

[0071]

[0072]

[0073] ② After the reaction system is prepared, use an instrument that can set a temperature change program (such as a PCR instrument) to perform bisulfite conversion. The whole process takes about 1 hour. The detailed procedure is shown in Reference Table 1.2:

[0074] Table 1.2 Bisulfite cycle conditions

[0075] temperature time 95℃ 10 minutes 64℃ 30-60 minutes 4℃ Keep

[0076] Note: If the initial amount of DNA to be processed is less than 500 ng, it only needs to be treated at 64°C for 30 minutes. If the initial amount of DNA to be processed is greater than 500 ng, it needs to be treated at 64°C for 60 minutes. In addition, during the treatment process, make sure to tightly cover the cap of the centrifuge tube and perform hot cover treatment.

[0077] ③ Column balancing step: Add 500 μl of balancing solution BL to the adsorption column CB1 (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm for 1 minute, pour out the waste liquid in the collection tube, and then put the adsorption column back into the collection tube.

[0078] ④ After the bisulfite treatment procedure, briefly centrifuge the 200μl centrifuge tube and transfer the reaction system to a new 1.5ml centrifuge tube.

[0079] ⑤After the transfer is completed, add 5 times the volume (600 μl) of binding solution PB and mix thoroughly.

[0080] ⑥ Inject the solution obtained in the previous step into the adsorption column CB1 (the adsorption column is placed in the collection tube), let it stand at room temperature for 2 minutes, centrifuge it at 12,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and then put the adsorption column CB1 back into the collection tube.

[0081] ⑦ Add 600 μl of rinse solution PW to the adsorption column CB1, centrifuge at 12,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and then put the adsorption column CB1 back into the collection tube.

[0082] ⑧ Add 600 μl of solution DB to the adsorption column CB1, place it at room temperature (15-25°C) for 15 minutes, centrifuge it at 12,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and then put the adsorption column CB1 back into the collection tube.

[0083] ⑨ Inject 600 μl of rinse solution PW into the adsorption column CB1, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid in the collection tube.

[0084] ⑩Repeat step 9.

[0085] The adsorption column CB1 was placed in a collection tube, centrifuged at 12,000 rpm for 2 minutes, and the rinse solution was removed.

[0086] Place the adsorption column at room temperature for 7 minutes to ensure that it is fully dried to avoid the residual rinsing solution from affecting the subsequent experiments.

[0087] Take out the adsorption column CB1, put it into a clean centrifuge tube, add 20 ml of elution buffer EB to the center of the adsorption membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12000 rpm for 2 minutes to collect the DNA solution.

[0088] Add the solution obtained by centrifugation back into the centrifugal adsorption column and centrifuge again. If the treated DNA is used within 24 hours, store it in a 4°C refrigerator; if it needs to be stored for a long time (no longer than 1 month), store it in a -20°C refrigerator.

[0089] 3. qMSP detection

[0090] The above-mentioned primers and probes were used to perform methylation quantitative PCR on the above-mentioned sulfite-converted DNA.

[0091] In this embodiment, the KCNQ5 gene and the internal reference gene of the same sample are detected separately, that is, multiple detection is not performed.

[0092] The concentrations of the above primers and probes were both 10 μM.

[0093] The configuration of qMSP reaction system is shown in Table 1.3.

[0094] Table 1.3 Quantitative methylation-specific PCR reaction system

[0095]

[0096] ② Invert the prepared reaction system several times, vortex and centrifuge briefly, add it to the eight-tube strip in sequence, centrifuge it briefly, put it into the fluorescence quantitative PCR instrument, and set the reaction program according to Table 1.4.

[0097] Table 1.4 Quantitative methylation-specific PCR reaction conditions

[0098]

[0099] ③After the reaction is completed, export the experimental results data.

[0100] The fluorescence quantitative amplification curves of KCNQ5 gene and internal reference gene are shown in Figure 1 and Figure 2 As shown, the amplification effect is good and can be used for subsequent experiments.

[0101] Figure 1The fluorescence quantitative amplification curve of KCNQ5 gene methylation detection is shown, reflecting the real-time fluorescence signal changes of specific primers and probes during PCR amplification. The amplification curve is a typical S-shaped curve, showing good amplification efficiency and no non-specific amplification phenomenon, which proves the detection specificity and reliability of the kit. At the same time, the lower Ct value shows that this method has high sensitivity to low-concentration methylated DNA and can accurately identify the methylation status of the KCNQ5 gene.

[0102] Figure 2 The fluorescence quantitative amplification curve of the β-actin reference gene is shown to correct the difference in extraction efficiency between samples. Figure 1 Similarly, it shows that β-actin has a stable expression level in each sample and is suitable as an internal reference gene. Its smooth curve and absence of abnormal signals further ensure the accuracy of the detection method and provide a reliable basis for the correction of the test results.

[0103] By analyzing Figure 1 and Figure 2 It can be seen that the present invention adopts a dual protection mechanism of specific primers and internal reference genes in the detection process, which not only improves the sensitivity and specificity of the detection, but also enhances the reliability and consistency of the results, providing strong technical support for early screening of colorectal cancer.

[0104] Example 2. Kit for detecting tissue samples from patients with colorectal cancer

[0105] In this example, a method and a kit for detecting blood KCNQ5 gene methylation provided by the present invention are used to detect two pairs of colorectal cancer tissues and adjacent cancer samples. The method and the kit for detecting blood SEPTIN9 gene methylation can well distinguish the two sample sources.

[0106] Test results such as Figure 3 As shown, the test results showed that the fluorescence signal intensity of cancer tissue samples was significantly higher than that of adjacent normal tissues, clearly reflecting the significant increase in the methylation level of the KCNQ5 gene in cancer tissues. This significant signal difference verifies the diagnostic value of abnormal KCNQ5 gene methylation as a molecular marker for colorectal cancer. At the same time, the fluorescence signal intensity of adjacent normal tissues was low and highly consistent, indicating that the test has good specificity and can effectively eliminate the interference of non-methylated DNA. The test results showed no abnormalities or stray signals, further demonstrating the stability and reliability of the kit and detection method in practical applications. Overall, Figure 3 The results intuitively illustrate the high efficiency and accuracy of the detection method of the present invention in distinguishing cancer tissue from normal tissue, and provide reliable experimental support and theoretical basis for early screening of colorectal cancer.

[0107] Example 3. Kit for detecting plasma samples from patients with colorectal cancer

[0108] In this example, 31 healthy subjects and 34 colorectal cancer patients' plasma were used as samples to be tested, and the kit and the detection method provided by the present invention were used for detection. The specific detection method is as follows:

[0109] 1. Extraction of plasma free DNA

[0110] The specific extraction method may include the following steps: According to the operation process of the magnetic bead method plasma free DNA extraction kit of Fanzhi Medical:

[0111] (1) Preparation before the experiment

[0112] Check whether there are crystals in Lysis Buffer 1 and Lysis Buffer 2. If there are crystals, melt them in 60℃ water, then invert them 10 times to mix well and set aside.

[0113] (2) Experimental procedures

[0114] ① Lysis: In a 15 ml centrifuge tube, add 40 ml proteinase K, 2 ml sample, and 100 μl lysis buffer 1 in sequence, shake well to mix, and then lyse at 60°C for 20 minutes.

[0115] ② Binding: Add 1.6 ml of lysis solution 2, 0.9 ml of isopropanol and 40 μl of magnetic beads CD to the centrifuge tube, shake thoroughly to mix, and then bind at room temperature for 10 minutes. During this period, shake several times to ensure that the magnetic beads remain suspended. Then place on the magnetic rack, wait for the solution to clarify, and discard the supernatant.

[0116] ③1st wash: Inject 1ml of wash solution CD1 into the centrifuge tube and shake for 20 seconds. Transfer the liquid and magnetic beads to a new 1.5ml centrifuge tube, place it on the magnetic rack, wait for the solution to become clear, then aspirate and discard the supernatant.

[0117] ④ Second washing: Inject 1 ml of washing solution CD1 into the centrifuge tube and shake for 20 seconds. Centrifuge briefly, place the centrifuge tube on the magnetic rack, wait for the solution to become clear, then aspirate and discard the supernatant.

[0118] ⑤ The third wash: inject 1 ml of washing solution CD2 into the centrifuge tube and shake for 20 seconds. Centrifuge briefly, place the centrifuge tube on the magnetic rack, wait for the solution to become clear, then aspirate and discard the supernatant.

[0119] ⑥Wash 4: Repeat step 5.

[0120] ⑦ Discard the liquid: Perform a brief centrifugation to collect the residual liquid in the tube; place the centrifuge tube on a magnetic rack and let it stand until the magnetic beads are completely adsorbed, absorb and discard the liquid in the tube, and dry it at room temperature for 5-10 minutes.

[0121] ⑧Storage: Centrifuge briefly to centrifuge the evaporated liquid on the tube cap, place the centrifuge tube on a magnetic rack, let it stand, and after the magnetic beads are completely adsorbed, transfer the supernatant to a new enzyme-free centrifuge tube and store it at -20℃ or -80℃.

[0122] 2. Sulfite Conversion

[0123] (1) Preparation before the experiment

[0124] Prepare Bisulfite Mix according to experimental requirements. When preparing, add 850 μl of BM buffer to each tube of dry powder, shake and mix for 5 minutes to completely dissolve the dry powder (the unused Bisulfite Mix is ​​divided into brown centrifuge tubes, and 94 μl of unused Bisulfite Mix is ​​added to each brown centrifuge tube, and stored in a -20° refrigerator, which can be stored for one month, and avoid repeated thawing and freezing).

[0125] (2) Experimental procedures

[0126] ① Prepare the bisulfite reaction system in a 200μl centrifuge tube. The specific preparation system is shown in Table 3.1:

[0127] Table 3.1 Bisulfate reaction system

[0128]

[0129]

[0130] ② After the reaction system is prepared, use an instrument that can set a temperature change program (such as a PCR instrument) to perform bisulfite conversion. The whole process takes about 1 hour. The detailed procedure is shown in Reference Table 3.2;

[0131] Table 3.2 Bisulfite cycle conditions

[0132] temperature time 95℃ 10 minutes 64℃ 30-60 minutes 4℃ Keep

[0133] Note: If the initial amount of DNA to be processed is less than 500 ng, it only needs to be treated at 64°C for 30 minutes. If the initial amount of DNA to be processed is greater than 500 ng, it needs to be treated at 64°C for 60 minutes. In addition, during the treatment process, make sure to tightly cover the cap of the centrifuge tube and perform hot cover treatment.

[0134] ③ Column balancing step: Add 500 μl of balancing solution BL to the adsorption column CB1 (the adsorption column is placed in the collection tube), centrifuge at 12000 rpm for 1 minute, pour out the waste liquid in the collection tube, and then put the adsorption column back into the collection tube.

[0135] ④ After the bisulfite treatment procedure, briefly centrifuge the 200μl centrifuge tube and transfer the reaction system to a new 1.5ml centrifuge tube.

[0136] ⑤After the transfer is completed, add 5 times the volume (600 μl) of binding solution PB and mix thoroughly.

[0137] ⑥ Inject the solution obtained in the previous step into the adsorption column CB1 (the adsorption column is placed in the collection tube), let it stand at room temperature for 2 minutes, centrifuge it at 12,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and then put the adsorption column CB1 back into the collection tube.

[0138] ⑦ Add 600 μl of rinse solution PW to the adsorption column CB1, centrifuge at 12,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and then put the adsorption column CB1 back into the collection tube.

[0139] ⑧ Add 600 μl of solution DB to the adsorption column CB1, place it at room temperature (15-25°C) for 15 minutes, centrifuge it at 12,000 rpm for 1 minute, pour out the waste liquid in the collection tube, and then put the adsorption column CB1 back into the collection tube.

[0140] ⑨ Inject 600 μl of rinse solution PW into the adsorption column CB1, centrifuge at 12,000 rpm for 1 minute, and discard the waste liquid in the collection tube.

[0141] ⑩Repeat step 9.

[0142] The adsorption column CB1 was placed in a collection tube, centrifuged at 12,000 rpm for 2 minutes, and the rinse solution was removed.

[0143] Place the adsorption column at room temperature for 7 minutes to ensure that it is fully dried to avoid the residual rinsing solution from affecting the subsequent experiments.

[0144] Take out the adsorption column CB1, put it into a clean centrifuge tube, add 20 ml of elution buffer EB to the center of the adsorption membrane, let it stand at room temperature for 2 minutes, and centrifuge at 12000 rpm for 2 minutes to collect the DNA solution.

[0145] Add the solution obtained by centrifugation back into the centrifugal adsorption column and centrifuge again. If the treated DNA is used within 24 hours, store it in a 4°C refrigerator; if it needs to be stored for a long time (no longer than 1 month), store it in a -20°C refrigerator.

[0146] 3. qMSP detection

[0147] The kit in Example 1 was used to perform methylation quantitative PCR on the above-mentioned sulfite-converted DNA.

[0148] The test result criteria are:

[0149] ① After completing the PCR cycle program, collect fluorescence data for 45 cycles, and the instrument's accompanying software automatically calculates the KCNQ5 and ACTB CT values.

[0150] ②Confirm that all samples CY5 have signals, the amplification curve of the CY5 channel is a smooth S-shaped, and the CT value is less than 33, then continue the analysis, otherwise the experimental results are considered unreliable.

[0151] ③ Calculate the ΔCT value, FAMΔCT value = FAM CT value - CY5 CT value

[0152] ④ The ROC curve was drawn based on the ΔCT value of plasma cfDNA of all patients, with a cutoff value of 8.09 (see Figure 4 ).

[0153] ⑤KCNQ5 gene methylation negative and positive judgment

[0154] a. If the FAMΔCT value is less than 8.09, it can be judged that the KCNQ5 gene is methylated positive.

[0155] b. If the FAMΔCT value is ≥8.09 or no fluorescent signal is detected, it is judged as negative KCNQ5 gene methylation.

[0156] Figure 4 The test results of plasma samples from healthy people and colorectal cancer patients based on the detection method of the present invention and the ROC curve of risk assessment are shown. The test results show that the fluorescence signal of cancer patients is significantly higher than that of healthy people, further verifying the effectiveness of KCNQ5 gene methylation level as a cancer marker. The ROC curve evaluates the accuracy of the detection method by calculating the relationship between sensitivity and specificity. The AUC value under the curve is 0.683, indicating that the method of the present invention has good discrimination ability. When the cutoff value ΔCt is 8.09, the sensitivity of the detection is 64.7% and the specificity is 67.7%, which achieves effective distinction between healthy people and cancer patients. In addition, this method is implemented by fluorescent quantitative PCR, without the need for cumbersome follow-up operations, further improving the detection efficiency and applicability. Figure 4 The analysis results fully demonstrate that the detection method of the present invention has clinical potential in colorectal cancer screening, can provide a reliable basis for early diagnosis, improve the sensitivity and specificity of colorectal cancer screening, and reduce the misdiagnosis and missed diagnosis rates.

[0157] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A composition for early screening of colorectal cancer, characterized in that: It includes a KCNQ5 target-specific primer pair, a KCNQ5-specific primer probe, an internal reference primer pair and an internal reference primer probe, wherein the cytosine at the second CG site of the KCNQ5-specific primer probe sequence is a methylated cytosine, and the second CG site is sites 72623235 and 72623236 on chromosome 6.

2. The composition for early screening of colorectal cancer according to claim 1, characterized in that: The sequences of the KCNQ5 target-specific primer pairs are as follows: Forward primer: 5′-GGAGAAGGTGGTTAAGGTTTAGAG-3′; Reverse primer: 5′-TACGCTTCTACTATCGCACCT-3′; The sequences of the KCNQ5-specific primer probes are as follows: 5'-ACGTTACTTCGCCTAACCTACTAACCTC-3'; The sequences of the internal reference primer pairs are as follows: Forward primer: 5′-GTGATGGAGGAGGTTTAGTAA-3′; Reverse primer: 5′-CCAATAAAACCTACTCCTCCCT-3′; The sequences of the internal reference primer probe are as follows: 5'-CACCCAACACACAATAAAAACACA-3'.

3. A kit for early screening of colorectal cancer, characterized in that: Comprising the composition for early screening of colorectal cancer as described in claim 1 or 2.

4. A method for detecting KCNQ5 gene methylation in blood in the early stage of colorectal cancer, characterized in that: The technical steps include: Using the kit for early screening of colorectal cancer as claimed in claim 3 to detect KCNQ5 gene methylation; Total cfDNA was extracted from plasma; Add sulfite and incubate with cfDNA to convert methylated cytosine into uracil; qPCR primers were added for amplification, and the FAM signal of the specific primer group and the CY-5 signal of the internal reference primer were collected. The ΔCt was obtained by calculating the difference between the ct values ​​of the specific primer and the internal reference primer to determine the methylation level of the blood KCNQ5 gene.

5. The method for detecting KCNQ5 gene methylation in blood in early stage of colorectal cancer according to claim 4, characterized in that: The difference between the ct value of the specific primer and the internal reference primer is used to obtain ΔCt, which is used to determine the methylation level of the blood KCNQ5 gene, including: If the difference between the ct values ​​of the specific primer and the internal reference primer yields a ΔCT value < 8.09, it can be determined that the KCNQ5 gene is positively methylated; If the difference between the ct values ​​of the specific primer and the internal reference primer yields a ΔCT value ≥ 8.09 or no fluorescent signal is detected, it is judged as negative for KCNQ5 gene methylation.

6. Use of the kit for early screening of colorectal cancer as claimed in claim 3 in screening, diagnosis and treatment of cancer diseases.

7. The use according to claim 6, characterized in that: The intestinal cancer includes rectal cancer and colon cancer.

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