Nucleotide composition for early diagnosis of prostatic cancer, kit and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a nucleotide composition for early diagnosis of prostatic cancer, a kit and application of the nucleotide composition. The detection kit for early diagnosis of prostatic cancer comprises a urinary sediment DNA bisulfite modification reagent, a PCR reaction premix solution and a quality control product, abnormal methylation of cast-off cells PAQR3, DACT-2 and RASSF1A gene promoter regions is detected, the detection method is high-sensitivity methylation specific PCR, and a detection sample is forepart urine after prostate massage. The method has the advantages of high sensitivity, good specificity, short detection time, high popularization degree and stable detection result.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer set and a kit for early diagnosis of prostate cancer and applications thereof. Background Art

[0002] Prostate cancer refers to an epithelial malignant tumor that occurs in the prostate. The early symptoms of prostate cancer are not obvious. As the disease progresses, some symptoms may gradually appear. The symptoms can be divided into two categories: one is compression symptoms. The gradually enlarged prostate gland compresses the urethra and can cause progressive dysuria, which manifests as dysuria, slow urine flow, easy interruption of urine flow, incomplete urination, frequent urination, urgency, and increased nocturia. The diseased part compresses the rectum and can cause difficulty in defecation or intestinal obstruction, compresses the vas deferens and causes lack of ejaculation, and compresses the nerves and causes local pain. The other is the metastasis symptom. Prostate cancer may invade the seminal vesicles and vascular nerve bundles, causing hematuria and hematospermia.

[0004] Prostate cancer is diagnosed by: 1. Rectal examination: It is an important and commonly used method for diagnosing prostate cancer. It is also the main method for diagnosing early prostate cancer. Because the prostate is very close to the rectum, rectal examination can be used to understand the shape, size, hardness, etc. of the prostate. This method is simple and easy to operate, but it is more difficult to detect early tumors.

[0005] 2. Transrectal ultrasound: Transrectal ultrasound has good clinical efficacy and can detect abnormal prostate nodules. It has the advantages of high sensitivity, easy operation, and cost-effectiveness. However, its detection rate for early tumors is low.

[0006] 3. Prostate specific antigen (PSA) test: Serum prostate specific antigen refers to a substance secreted by prostate tissue, which is closely related to the occurrence of prostate cancer. The determination of PSA is currently an effective method for diagnosing prostate cancer. Studies have reported that the sensitivity of PSA detection is related to the stage of prostate cancer: I and II are lower than 25%, and III and IV are higher than 50%. It can be seen that the PSA test has a low detection rate for early prostate cancer.

[0007] DNA methylation is one of the earliest DNA modification pathways discovered by scientists. Under the catalysis of methyltransferase, methyl groups are selectively added to the cytosine of the two nucleotides CG of DNA to form 5-methylcytosine. DNA methylation can cause changes in chromatin structure, DNA stability, etc., thereby controlling the expression of genes in the body. In a broad sense, DNA methylation refers to the chemical modification process in which a specific base on the DNA sequence obtains a methyl group through covalent bond binding under the catalysis of DNA methyltransferase (DNMT) with S-adenosyl methionine (SAM) as a methyl donor.

[0008] The methylation level of the promoter region of cancer-related genes in tumor cells is significantly different from that in normal cells. These differences are used as biomarkers to detect the occurrence and recurrence of tumors and predict the prognosis of tumor patients. DNA methylation is involved in specific regulatory mechanisms, which can determine the gene expression pattern of cells and can be inherited to daughter cells without changes in DNA sequence. DNA methylation testing can be used to detect early tumor occurrence and early recurrence of tumors.

[0009] Currently, there is an urgent need to provide a kit for early diagnosis of prostate cancer with good specificity, high sensitivity and low cost. Summary of the invention

[0010] The purpose of the present invention is to provide a detection kit for early diagnosis of prostate cancer, which has the advantages of high sensitivity, good specificity, short detection time, high popularity and stable detection results; the present invention also provides the application of the kit for early diagnosis of prostate cancer, which can simultaneously analyze the methylation abnormalities of three prostate cancer markers through high-sensitivity detection of abnormal DNA methylation in urine sediment, and has a detection sensitivity of as low as 0.1% for abnormal DNA in urine sediment, thereby achieving early detection of prostate cancer.

[0011] The detection kit for early diagnosis of prostate of the present invention comprises a urine sediment DNA bisulfite modification reagent, a PCR reaction premix and a quality control product, wherein the PCR reaction premix comprises a PCR reaction enzyme, dNTP, a buffer and a nucleotide composition. The nucleotide composition comprises 6 primers, 3 probes and 3 nucleotide repressors.

[0012] The primers for the PAQR3 gene are PAQR3-F and PAQR3-R, and the sequences are shown in SEQ ID NOs. 01 and 02; The probe of the PAQR3 gene is PAQR3-Pro, which is the sequence shown in SEQ ID NO.03; The repressor sequence of the PAQR3 gene is PAQR3-Blocker, which is the sequence shown in SEQ ID NO.04; The primers for the DACT-2 gene are DACT-2-F and DACT-2-R, and the sequences are shown in SEQ ID NOs. 05 and 06; The probe of DACT-2 gene is DACT-2-Pro, which is the sequence shown in SEQ ID NO.07; The repressor sequence of the DACT-2 gene is DACT-2-Blocker, which is the sequence shown in SEQ ID NO.08; The primers for the RASSF1A gene are RASSF1A-F and RASSF1A-R, and the sequences are shown in SEQ ID NOs. 09 and 10; The probe of the RASSF1A gene is RASSF1A-Pro, which is the sequence shown in SEQ ID NO.11; The repressor sequence of the RASSF1A gene is RASSF1A-Blocker, which is the sequence shown in SEQ ID NO.12; The 18th adenine deoxynucleotide from the 5' end to the 3' end of PAQR3-F is modified with a locked nucleic acid, the 17th uracil deoxynucleotide from the 5' end to the 3' end of PAQR3-R is modified with a locked nucleic acid, the 5' end of PAQR3-Pro is modified with either Oregon Green or FAM, the 3' end is modified with either Dabcyl or BHQ1, and the 3' end of PAQR3-Blocker is modified with either spacer 3 or phosphorylation.

[0013] The 16th deoxynucleotide from the 5' end to the 3' end of DACT-2-F is modified with a locked nucleic acid, the 16th deoxynucleotide from the 5' end to the 3' end of DACT-2-R is modified with a locked nucleic acid, the 5' end of DACT-2-Pro is modified with one of JOE, HEX or CY3, the 3' end is modified with BHQ1, and the 3' end of DACT-Blocker is modified with one of spacer 3 or phosphorylation.

[0014] The 17th deoxynucleotide from the 5' end to the 3' end of RASSF1A-R is modified with a locked nucleic acid, the 15th deoxynucleotide from the 5' end to the 3' end of RASSF1A-F is modified with a locked nucleic acid, the 5' end of RASSF1A-2-Pro is modified with either CY5 or CY5.5 and the 3' end with BHQ2, and the 3' end of RASSF1A-Blocker is modified with either spacer 3 or phosphorylation.

[0015] The urine sediment sample is obtained by collecting urine after prostate massage and centrifuging at 4000 rpm for 10 minutes.

[0016] The bisulfite modification reagent comprises a bisulfite modification reagent, a DNA protection solution, a binding solution, a desulfonation solution, a rinsing solution, an elution solution and a silica magnetic bead suspension solution.

[0017] The bisulfite modification reagent is a mixture of sodium bisulfite and sodium sulfate, wherein the mass ratio of sodium bisulfite to sodium sulfate is 5:1, or 4:1, or 3:1, or 2:1, and preferably, the mass ratio of sodium bisulfite to sodium sulfate is 4:1.

[0018] The DNA protection solution is a dimethyl ether solution of water-soluble VE, with a concentration of 3-4.5M, preferably, a concentration of 3.8M.

[0019] The binding solution is a guanidine bisulfate solution with a concentration of 6-9M, preferably 8M.

[0020] The desulfurization liquid is a sodium hydroxide solution with a concentration of 1.1-2.2M, preferably, a concentration of 1.8M.

[0021] The rinsing solution is a mixture of 75% ethanol and Tris-HCl, the concentration of Tris-HCl is 8.2-12.1Mm, preferably, the concentration is 10.1M.

[0022] The eluent is a Tris-HCl solution with a concentration of 10 mmol / L.

[0023] The silica magnetic bead suspension is prepared by suspending silica magnetic beads in ultrapure water containing 1% ethanol, wherein the concentration of the silica magnetic beads is 50-90 mg / ml, preferably, the concentration is 78 mg / ml.

[0024] The PCR reaction enzyme is hot start Taq enzyme, and the buffer is polymerase buffer.

[0025] The quality control products are positive quality control product (PC), negative quality control product (NC) and blank quality control product (NTC), the positive quality control product is transmethylated DNA, the negative quality control product is demethylated DNA, and the blank quality control product is deionized water.

[0026] The kit for early diagnosis of prostate cancer of the present invention comprises the following steps: (1) Extract sample DNA; (2) Bisulfite modification of sample DNA and quality control DNA using urine sediment bisulfite modification reagent; (3) Perform PCR detection on sample DNA and quality control DNA using PCR reaction premix.

[0027] The sample described in step (1) is urine, and the volume of the sample is 10-50 ml.

[0028] The PCR detection system described in step (3) is as follows: PCR reaction premix 18 μl PCR grade deionized water 10 μl Sample DNA or control DNA 8μl; The PCR detection procedure described in step (3) is as follows: ① React at 37℃ for 35min; ② React at 95℃ for 2min; ③ 50s at 58℃, 15s at 95℃, cycle 5 times: ④ React at 63℃ for 2 seconds, 56℃ for 35 seconds, and 94℃ for 15 seconds, cycle 35 times, and collect the fluorescence signal; ⑤React at 40℃ for 1s.

[0029] In step ③, no fluorescence signal was collected for 5 cycles, and in step ④, fluorescence signal was collected for 35 cycles.

[0030] There are three channels for collecting fluorescence signals in the PCR detection described in step ④, and the excitation-emission wavelengths of the three channels are 470-510nm, 530-565nm and 630-665nm respectively.

[0031] The sequences of SEQID NO.01~12 are shown in Table 1.

[0032] Table 1 SEQ ID NO. 01~12 sequences

[0033] The kit of the present invention is a highly sensitive and specific urine sediment DNA methylation multi-target detection kit for early diagnosis of prostate cancer. The kit includes a urine sediment DNA bisulfite modification reagent, a PCR reaction premix, and a quality control reagent. The PCR reaction premix includes a PCR reaction enzyme, dNTP, a buffer, and a nucleotide composition. Among them, the nucleotide composition includes 6 primers, 3 probes, and 3 nucleotide repressors. The primer carries a locked nucleic acid modification group, the probe carries a fluorescent group and a fluorescent quenching group, and the repressor sequence carries an arm modification group or a phosphorylation modification group.

[0034] Locked nucleic acid (LNA) is a modified RNA, in which the 2' and 4' carbons on a portion of the ribose in LNA are linked together. Locked nucleic acid modification can increase the melting temperature (Tm value) of primers or probes, and enhance the stability of primer or probe binding. LNA can increase primer specific binding and increase the primer Tm value. In the present invention, by performing LNA modification on the portion of the primer near the 3' end, the binding stability of the primer to the positive template is increased, and the binding stability of the primer to the negative template is reduced, thereby effectively improving the specificity of the primer and achieving the purpose of improving the detection sensitivity of the PCR system.

[0035] The PAQR3-Blocker, DACT-2-Blocker and RASSF1A-Blocker sequences in the present invention are designed for negative template sequences, and can effectively block negative templates, preventing positive primers or probes from binding to negative templates, thereby affecting the detection rate.

[0036] The kit has the characteristics of good specificity, high sensitivity, low cost and easy popularization. The present invention provides a method for diagnosing prostate cancer by detecting DNA methylation in urine sediment The beneficial effects of the present invention are as follows: (1) Good specificity: Conventional tumor markers may also increase under non-tumor pathological or physiological conditions, and there is a certain proportion of false positives in clinical testing. However, the methylation markers of exfoliated cells only occur in abnormal cells and therefore have good specificity.

[0037] (2) Can track the course of the disease: DNA methylation testing of exfoliated cells after prostate cancer massage can reflect whether there are prostate tumor cells in the patient's urine on the day of sampling. After surgical resection of the tumor tissue or other treatment methods, if the treatment is thorough, the urine obtained through prostate massage after surgery and other treatments can be tested as negative for DNA methylation of exfoliated cells, thus being able to reflect the dynamic progression of the tumor in real time.

[0038] (3) Higher sensitivity: Relying on high-sensitivity detection technology of urine sediment, the detection kit of the present invention can perform qualitative analysis on DNA methylation of single-copy tumor exfoliated cells, thereby achieving high-sensitivity detection of prostate cancer.

[0039] (4) Recurrence risk assessment: After surgical resection of tumor tissue, the kit of the present invention can be used to detect methylation of urine exfoliated cells to determine whether the surgery was complete and whether there are any tiny lesions remaining, thereby assessing the risk of recurrence after treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Attached Figure 1 , Test results of 8 healthy people in implementation case 1.

[0041] Attached Figure 2 , Figure 2 shows the test results of 8 prostate cancer samples in implementation case 1.

[0042] Attached Figure 3 , Detection lower limit verification result diagram in implementation case 3.

[0043] The present invention is further described below with reference to the embodiments. Example 1

[0044] The composition of the kit for early diagnosis of prostate cancer is shown in Table 2.

[0045] Table 2 Composition of the kit for early diagnosis of prostate cancer

[0046] The kit component bisulfite modification reagent comprises bisulfite modification reagent, DNA protection solution, binding solution, desulfonation solution, rinsing solution, elution solution and silica magnetic bead suspension.

[0047] The bisulfite modification reagent is a mixture of sodium bisulfite and sodium sulfate, wherein the mass ratio of sodium bisulfite to sodium sulfate is 5:1, or 4:1, or 3:1, or 2:1, and preferably, the mass ratio of sodium bisulfite to sodium sulfate is 4:1.

[0048] The DNA protection solution is a dimethyl ether solution of water-soluble VE, with a concentration of 3-4.5M, preferably, a concentration of 3.8M.

[0049] The binding solution is a guanidine bisulfate solution with a concentration of 6-9M, preferably 8M.

[0050] The desulfurization liquid is a sodium hydroxide solution with a concentration of 1.1-2.2M, preferably, a concentration of 1.8M.

[0051] The rinsing solution is a mixture of 75% ethanol and Tris-HCl, the concentration of Tris-HCl is 8.2-12.1Mm, preferably, the concentration is 10.1M.

[0052] The eluent is a Tris-HCl solution with a concentration of 10 mmol / L.

[0053] The silica magnetic bead suspension is prepared by suspending silica magnetic beads in ultrapure water containing 1% ethanol, wherein the concentration of the silica magnetic beads is 50-90 mg / ml, preferably, the concentration is 78 mg / ml.

[0054] The PCR reaction premix of the kit components contains hot start Taq enzyme, dNTP, PCR Buffer and nucleotide composition.

[0055] 2× polymerase buffer; Hot start Taq enzyme, concentration 0.28U; dNTPs, concentration 4.0 mM; Nucleotide composition The nucleotide composition includes 6 primers, 3 probes, and 3 nucleotide repressors.

[0056] The 18th adenine deoxynucleotide from the 5' end to the 3' end of PAQR3-F is modified with a locked nucleic acid, the 17th uracil deoxynucleotide from the 5' end to the 3' end of PAQR3-R is modified with a locked nucleic acid, the 5' end of PAQR3-Pro is modified with FAM and the 3' end with BHQ1, and the 3' end of PAQR3-Blocker is modified with spacer 3.

[0057] The 16th deoxynucleotide from the 5' end to the 3' end of DACT-2-F is modified with a locked nucleic acid, the 16th deoxynucleotide from the 5' end to the 3' end of DACT-2-R is modified with a locked nucleic acid, the 5' end of DACT-2-Pro is modified with HEX and the 3' end with BHQ1, and the 3' end of DACT-Blocker is modified with spacer 3.

[0058] The 17th deoxynucleotide from the 5' end to the 3' end of RASSF1A-R is modified with a locked nucleic acid, the 15th deoxynucleotide from the 5' end to the 3' end of RASSF1A-F is modified with a locked nucleic acid, the 5' end of RASSF1A-2-Pro is modified with CY5 and the 3' end with BHQ2, and the 3' end of RASSF1A-Blocker is modified with spacer 3.

[0059] The urine sediment sample is obtained by collecting urine after prostate massage and centrifuging at 4000 rpm for 10 minutes.

[0060] The PCR reaction enzyme is hot start Taq enzyme, and the buffer is polymerase buffer.

[0061] The quality control products are positive quality control product (PC), negative quality control product (NC) and blank quality control product (NTC), the positive quality control product is transmethylated DNA, the negative quality control product is demethylated DNA, and the blank quality control product is deionized water.

[0062] The application of the detection kit for early diagnosis of prostate cancer comprises the following steps: 8 prostate cancer samples, 8 healthy samples, 1 positive quality control, 1 negative quality control, and 1 blank control sample were tested. The healthy subjects were volunteers.

[0063] The information of prostate cancer samples is shown in Table 3, and the information of healthy human samples is shown in Table 4.

[0064] Table 3 Prostate cancer sample information

[0065] Table 4 Sample information of healthy people

[0066] Step 1: Enrich the nucleic acid of the sample and quality control product, perform bisulfite modification after elution with the eluent, and obtain the test template of the kit after purification; use the "MagMAX™ Nucleic Acid Extraction Kit (Large Volume)" (Thermo Scientific, Catalog No.: AM1939) reagent to extract urine sediment DNA. The operation steps are as follows: (1) Take 10 ml of urine, which is the front urine collected after prostate massage; (2) Pour the urine into a centrifuge tube, centrifuge at 6000 rpm / min for 10 min, and pour out the supernatant; (3) Add 6 ml of PBS buffer to the urine sediment, vortex to resuspend, centrifuge at 6000 rpm / min for 10 min, and pour out the supernatant; (4) Add 200 μl of protein digestion solution and 40 μl of proteinase K (20 mg / ml) solution, cover the tube tightly, vortex to resuspend, and incubate in a metal bath at 65°C for 1 h (mix several times during this period); (5) Incubate in a metal bath at 92°C for 1 h to obtain a digestion mixture; (6) After the metal bath is completed, add 300 μl of lysis buffer and mix well, then transfer the entire mixture into the adsorption column; (7) Centrifuge at 12,000 rpm for 1 min and discard the waste liquid in the collection tube; (8) Add 800 μl of washing solution A to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube; (9) Add 800 μl of washing solution B to the adsorption column, centrifuge at 12,000 rpm for 1 min, and discard the waste liquid in the collection tube; (10) Return the sample to the collection tube and centrifuge the empty tube at 12,000 rpm for 1 min. (11) Place the adsorption column in a new 1.5 ml centrifuge tube, open the tube cap, and leave it at room temperature for 5 min to completely dry the adsorption column; (12) Add 80 μl of elution buffer to the adsorption column, let it stand at room temperature for 5 min, and centrifuge at 12,000 rpm for 1 min to collect the DNA product.

[0067] Step 2: Bisulfite modification of the obtained DNA.

[0068] Add 900 ul of RNase-free deionized water to the tube containing the bisulfite modification reagent and shake and mix for 5 minutes to obtain a bisulfite modification reagent solution.

[0069] Take 30ul of the obtained DNA template and add it to a 200ul EP tube, then add 15 μl of DNA protection solution and 85 μl of bisulfite modification reagent solution, and use deionized water without RNase to make up to 150 μl. Centrifuge it for a moment and put it into the PCR instrument. Set the program to 98℃ for 10 min and 64℃ for 100 min. After the operation, get the reaction mixture. Finally, perform the following steps: (1) In a 2 ml EP tube, add the above reaction mixture, then add 1.6 ml binding solution, vortex to mix, centrifuge briefly, and then add 20 ul of silica magnetic bead suspension; (2) Aspirate the liquid into a centrifuge tube, centrifuge at 13,000 rpm / min for 40 s, and discard the effluent; (3) Repeat the previous step; (4) Add 250 μl of rinse buffer, centrifuge at 13,000 rpm / min for 10 s, and discard the flow-through; (5) Add 250 μl of desulfurization solution, place at room temperature for 15 min, centrifuge at 13,000 rpm / min for 10 s, and discard the effluent; (6) Add 250 μl of rinse buffer, centrifuge at 13,000 rpm / min for 15 s, and discard the flow-through; (7) Add 250 μl of rinse buffer, centrifuge at 13,000 rpm / min for 2 min, and discard the flow-through; (8) Add 20 μl of elution buffer to the center of the centrifuge column, incubate at room temperature for 5 min, and centrifuge at 13,000 rpm / min for 3 min. The effluent is the purified DNA.

[0070] Step 3: PCR testing.

[0071] In a 200 μl EP tube, add 18 μl of PCR reaction premix, 10 μl of PCR-grade deionized water, and 8 μl of sample DNA or control DNA.

[0072] Place the EP tube with the reaction solution in the real-time quantitative fluorescence PCR instrument. The detection procedure is as follows: Step 1: react at 37°C for 35 min; Step 2: 95°C for 2 min; Step 3: 58℃ for 50s, 95℃ for 15s, cycle 5 times: Step 4: react at 63°C for 2 seconds, 56°C for 35 seconds, and 94°C for 15 seconds, cycle 35 times, and collect the fluorescence signal; Step 5: react at 40℃ for 1s.

[0073] PCR detection requires three channels for collecting fluorescence signals, and the excitation-emission wavelengths of the three channels are 470-510 nm, 530-565 nm, and 630-665 nm, respectively.

[0074] Step 4: Analyze the test data.

[0075] Record the test data and interpret the results according to the following standards.

[0076] (1) Positive test interpretation: On the premise that there is no invalid test interpretation, the amplification Ct value of the PAQR3 gene is ≤38, or the amplification Ct value of the DACT-2 gene is ≤38, or the amplification Ct value of the RASSF1A gene is ≤38.

[0077] (2) Negative test interpretation: The amplification Ct value of the PAQR3 gene is greater than 38, the amplification Ct value of the DACT-2 gene is greater than 38, and the amplification Ct value of the RASSF1A gene is greater than 38.

[0078] Step 5: Conclusion.

[0079] The test results are shown in Table 5. A total of 8 prostate cancer patient samples were tested, 7 of which were positive, with a positive rate of 87.5%; a total of 8 healthy people were tested, all of which were negative, with a negative rate of 100%; the test results are shown in the attached figure. Figure 1 , Attachment Figure 2 The test results show that the kit of the present invention has good clinical performance.

[0080] Table 5 Test results Example 2

[0081] Verification of the recovery rate of urine sediment nucleic acid enrichment system Detection method: The test sample is the reference product, which is transmethylated DNA (positive DNA). After NanoDrop quantification, it is put into deionized water to prepare the reference product. The reference product volume is 1 ml, and the concentration of positive DNA is 5 ng / ml, 20 ng / ml, and 80 ng / ml. The reference product is extracted using the "MagMAX™ Nucleic Acid Extraction Kit (Large Volume)" (ThermoScientific, Catalog No.: AM1939) reagent.

[0082] Testing instruments and equipment include: High-speed centrifuge, fluorescence quantitative PCR instrument (ABI 7500), vortex shaker, instant centrifuge, refrigerator, magnetic rack, clean bench, NanoDrop 2000c (Thermo Scientific), etc.

[0083] The extraction method is the same as that in Example 1.

[0084] Test results and discussion: The purified and recovered DNA was quantified using ddPCR, and ddPCR primers were designed. The primers can only amplify DNA. The recovery rates obtained are shown in Table 6. From the results, it can be seen that the reagent of the present invention has a higher recovery rate. Example 3

[0085] PCR detection system detection off-line analysis (1) Testing instruments and equipment High-speed centrifuge, fluorescence quantitative PCR instrument (ABI), vortex shaker, instant centrifuge, refrigerator, magnetic rack, clean bench, NanoDrop 2000c (Thermo Scientific), etc.

[0086] (2) Configuration reference: The reference material was prepared using transmethylated DNA (positive DNA) and deionized water. The DNA was quantified using ddPCR. The proportion of positive DNA was 0.2%, and the test was repeated 8 times for each sample.

[0087] In a 200 μl EP tube, add 18 μl of PCR reaction premix, 10 μl of PCR-grade deionized water, and 8 μl of sample DNA or control DNA.

[0088] Place the EP tube with the reaction solution in the real-time quantitative fluorescence PCR instrument. The detection procedure is as follows: Step 1: react at 37°C for 35 min; Step 2: 95°C for 2 min; Step 3: 58℃ for 50s, 95℃ for 15s, cycle 5 times: Step 4: react at 63°C for 2 seconds, 56°C for 35 seconds, and 94°C for 15 seconds, cycle 35 times, and collect the fluorescence signal; Step 5: react at 40℃ for 1s.

[0089] PCR detection requires three channels for collecting fluorescence signals, and the excitation-emission wavelengths of the three channels are 470-510 nm, 530-565 nm, and 630-665 nm, respectively.

[0090] (3) Test results and discussion In the 8 tests of the reference products, the PAQR3 gene, DACT-2 gene, and RASSF1A gene were all detected, with a detection rate of 100%, indicating that this kit has good detection performance. The test results are attached. Figure 3 .

Claims

1. A primer set and a kit for early diagnosis of prostate cancer, characterized in that: The kit components include: urine sediment DNA bisulfite modification reagent, PCR reaction premix and quality control products.

2. The kit according to claim 1, characterized in that The PCR reaction premix includes PCR reaction enzyme, dNTP, buffer and nucleotide composition.

3. The nucleotide composition according to claim 2, characterized in that Includes 6 primers, 3 probes, 3 nucleotide repressors, The primers for the PAQR3 gene are PAQR3-F and PAQR3-R, and the sequences are shown in SEQ ID NOs. 01 and 02; The probe of the PAQR3 gene is PAQR3-Pro, which is the sequence shown in SEQ ID NO.03; The repressor sequence of the PAQR3 gene is PAQR3-Blocker, which is the sequence shown in SEQ ID NO.04; The primers for the DACT-2 gene are DACT-2-F and DACT-2-R, and the sequences are shown in SEQ ID NOs. 05 and 06; The probe of DACT-2 gene is DACT-2-Pro, which is the sequence shown in SEQ ID NO.07; The repressor sequence of the DACT-2 gene is DACT-2-Blocker, which is the sequence shown in SEQ ID NO.08; The primers for the RASSF1A gene are RASSF1A-F and RASSF1A-R, and the sequences are shown in SEQ ID NOs. 09 and 10; The probe of the RASSF1A gene is RASSF1A-Pro, which is the sequence shown in SEQ ID NO.11; The repressor sequence of the RASSF1A gene is RASSF1A-Blocker, which is the sequence shown in SEQ ID NO.

12.

4. The detection kit according to claim 1, characterized in that The 18th adenine deoxynucleotide from the 5' end to the 3' end of the PAQR3-F is modified with a locked nucleic acid; The 17th uracil deoxynucleotide from the 5′ to 3′ end of PAQR3-R carries a locked nucleic acid modification; PAQR3-Pro has one with Oregon Green modification or FAM modification at the 5′ end and one with Dabcyl modification or BHQ1 modification at the 3′ end; The 3' end of PAQR3-Blocker has either spacer 3 modification or phosphorylation modification.

5. The kit for early diagnosis of prostate cancer according to claim 1, characterized in that The 16th deoxynucleotide from the 5' end to the 3' end of the DACT-2-F is modified with a locked nucleic acid; The 16th deoxynucleotide from the 5′ to 3′ end of DACT-2-R carries a locked nucleic acid modification; DACT-2-Pro has one of the JOE, HEX, or CY3 modifications at its 5′ end and a BHQ1 modification at its 3′ end; The 3' end of DACT-Blocker has either spacer 3 or phosphorylation modification.

6. The kit for early diagnosis of prostate cancer according to claim 1, characterized in that The 17th deoxynucleotide from the 5' end to the 3' end of the RASSF1A-R is modified with a locked nucleic acid; The 15th deoxynucleotide from the 5′ end to the 3′ end of RASSF1A-F carries a locked nucleic acid modification; RASSF1A-2-Pro has one of the CY5 or CY5.5 modifications at its 5′ end and a BHQ2 modification at its 3′ end; The 3' end of RASSF1A-Blocker has either spacer 3 or phosphorylation modification.

7. The kit for early diagnosis of prostate cancer according to claim 1, characterized in that The urine sediment DNA bisulfite modification reagent comprises a bisulfite modification reagent, a DNA protection solution, a binding solution, a desulfonation solution, a rinsing solution, an elution solution and a silica magnetic bead suspension, wherein the bisulfite modification reagent is a mixture of sodium bisulfite and sodium sulfate, the DNA protection solution is a dimethyl ether solution of water-soluble VE, the binding solution is a guanidine isosulfate solution, the desulfonation solution is a sodium hydroxide solution, the rinsing solution is a mixture of 75% ethanol and Tris-HCl, the elution solution is a Tris-HCl solution, and the silica magnetic bead suspension is prepared by suspending silica magnetic beads in ultrapure water containing 1% ethanol.

8. The detection kit for early diagnosis of prostate cancer according to claim 1, characterized in that The quality control products include positive quality control product, negative quality control product and blank quality control product. The positive quality control product is transmethylated DNA, the negative quality control product is demethylated DNA, and the blank quality control product is deionized water.

9. Use of a kit for early diagnosis of prostate cancer according to any one of claims 1 to 8, characterized in that The sample in step (1) is urine after prostate massage, and the volume of the sample is 10-50 ml; it is characterized by comprising the following steps: (1) Extract sample DNA; (2) Bisulfite modification of sample DNA and quality control DNA using urine sediment bisulfite modification reagent; (3) Perform PCR detection on sample DNA and quality control DNA using PCR reaction premix.

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