Kit for early detection of kidney cancer and application

Abstract

The invention discloses a kidney cancer early detection kit and application, and relates to the technical field of biology. The invention provides application of a reagent for detecting the methylation level of a methylation site marker of kidney cancer in preparation of a kidney cancer detection product. The reagent comprises a methylation detection primer pair, the methylation detection primer pair comprises primer pairs used for detecting sites cg01362243, cg07351192, cg06829686 and cg26595643, the kidney cancer can be effectively diagnosed in the early stage, the survival rate of patients is increased, and the methylation detection primer pair is easy and convenient to operate, accurate in result and high in specificity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a kit for early detection of renal cancer and its application. Background Art

[0002] Renal cancer originates from renal tubular epithelial cells. Risk factors for renal cancer include smoking, obesity, hypertension, and a history of chronic kidney disease. Renal cancer is also associated with gene mutations and other environmental exposures. The cause of renal cancer is hidden, and early diagnosis has the following three main problems: (1) No valuable molecular markers for early screening or diagnosis of renal cancer have been reported; (2) Abdominal ultrasound is the most commonly used method for renal cancer screening in clinical practice, but its efficacy in early diagnosis of renal cancer is low, especially for renal cancer with a small lesion; (3) Some special benign renal lesions (such as fat-poor angiomyolipoma and complex cysts) are difficult to effectively identify with abdominal imaging examinations, which may lead to overtreatment of these patients.

[0003] The diagnosis of renal cancer mainly relies on imaging examinations, and the final diagnosis depends on pathological biopsy. However, histopathological biopsy has certain limitations, including sampling bias, heterogeneity, hysteresis, inability to repeat sampling, and operational risks.

[0004] Chinese patent CN111411156A discloses a kit and a method for early detection of cancer, wherein the kit includes a primer-probe combination for detecting the DNA methylation level of PRKY, HOXD3b, HOXA7 or GSTP1 gene loci, a DNA polymerase and a reaction system. The invention can rapidly and highly specifically detect the DNA methylation level of the target loci of PRKY, HOXD3b, HOXA7 or GSTP1 genes, and has the advantages of high sensitivity, strong specificity, simple and fast operation, etc., and can provide an important reference for clinicians in the early diagnosis and differential diagnosis of prostate cancer, kidney cancer or bladder cancer. However, it does not improve the accuracy of kidney cancer detection.

[0005] Chinese patent CN110004231A discloses a renal cancer exosome marker group and its application, and the application of the marker group detection reagent in the preparation of renal cancer diagnostic reagents and / or kits, characterized in that the marker group includes: exosome-derived VHL gene mRNA, exosome-derived PBRM1 gene mRNA, exosome-derived ATM gene mRNA, exosome-derived PTEN gene mRNA and exosome-derived SETD2 gene mRNA. The marker group described in the invention can distinguish renal cancer patients from non-renal cancer patients by detecting the mRNA of exosome-derived VHL gene, PBRM1 gene, ATM gene, PTEN gene and SETD2 gene and the corresponding diagnostic model, especially the distinction between early renal cancer samples (such as clinical T1 renal cancer) and non-renal cancer samples (such as normal people and simple renal cysts, benign renal space-occupying lesions such as hamartomas), solving the defect that early renal cancer is difficult to diagnose in a timely and accurate manner in clinical practice. However, its detection process is relatively complicated.

[0006] Therefore, there is an urgent need to provide a simple, easy and highly accurate detection method to achieve early and accurate diagnosis of renal cancer. Summary of the invention

[0007] The purpose of the present invention is to provide a kit for early detection of renal cancer and its application.

[0008] To achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0009] In one aspect, the present invention provides the use of a reagent for detecting the methylation level of a methylation site marker of renal cancer in the preparation of a renal cancer detection product, wherein the reagent comprises a methylation detection primer pair; the methylation detection primer pair comprises the following primer pair:

[0010] The primer pair used to detect site cg01362243, the sequence is shown in SEQ ID NO: 1-2;

[0011] The primer pair used to detect site cg07351192, the sequence is shown in SEQ ID NO:3-4;

[0012] The primer pair used to detect site cg06829686, the sequence is shown in SEQ ID NO:5-6;

[0013] The primer pair used to detect site cg26595643 has a sequence as shown in SEQ ID NO: 7-8.

[0014] Specifically, the reagent further includes a methylation detection probe, and the methylation detection probe includes the following probes:

[0015] A probe for detecting site cg01362243, the sequence of which is shown in SEQ ID NO:9;

[0016] The probe for detecting site cg07351192 has a sequence as shown in SEQ ID NO: 10;

[0017] A probe for detecting site cg06829686, the sequence of which is shown in SEQ ID NO: 11;

[0018] The probe used to detect site cg26595643 has a sequence as shown in SEQ ID NO:12.

[0019] Specifically, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group.

[0020] Further, the fluorescent reporter group is selected from any one of FAM, ROX, HEX, CY5, VIC, TET, JOE, Cy3, Cy7, RED610, Texas Red, RED670, NED, AMCA, Pacific Blue, Atto 425, BODIPY FL, Atto 590, and Cy5.5;

[0021] Furthermore, the fluorescent reporter group is selected from any one of FAM, ROX, HEX, and CY5.

[0022] According to some embodiments of the present invention, the fluorescent reporter group is selected from FAM.

[0023] Furthermore, the fluorescence quenching group is selected from any one of 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, Dabcyl, Eclipse, MGB, and QYS-7;

[0024] Furthermore, the fluorescence quenching group is selected from any one of BHQ-1 and BHQ-2.

[0025] According to some embodiments of the present invention, the fluorescence quenching group is BHQ-1.

[0026] Specifically, the reagents also include a primer pair and a probe for detecting an internal reference gene.

[0027] Furthermore, the internal reference gene is selected from any one of GAPDH, β-actin, ACTB, B2M, ARBP, and HPRT1.

[0028] Furthermore, the internal reference gene is ACTB.

[0029] Furthermore, the sequence of the primer pair of the internal reference gene is shown in SEQ ID NO:13-14, and the sequence of the probe is shown in SEQ ID NO:15.

[0030] Specifically, the renal cancer includes but is not limited to renal clear cell carcinoma, papillary renal carcinoma, renal chromophobe cell carcinoma, renal medullary carcinoma, multilocular cystic renal cell carcinoma, mucinous tubular and spindle cell carcinoma.

[0031] Specifically, the products include any physical products such as test kits, chips, devices, systems, or virtual products based on computer programs.

[0032] In another aspect, the present invention provides a kit for detecting renal cancer, wherein the kit comprises a methylation detection primer pair; the methylation detection primer pair comprises the following primer pair:

[0033] The primer pair used to detect site cg01362243, the sequence is shown in SEQ ID NO: 1-2;

[0034] The primer pair used to detect site cg07351192, the sequence is shown in SEQ ID NO:3-4;

[0035] The primer pair used to detect site cg06829686, the sequence is shown in SEQ ID NO:5-6;

[0036] The primer pair used to detect site cg26595643 has a sequence as shown in SEQ ID NO: 7-8.

[0037] Specifically, the kit also includes a methylation detection probe, and the methylation detection probe includes the following probes:

[0038] A probe for detecting site cg01362243, the sequence of which is shown in SEQ ID NO:9;

[0039] The probe for detecting site cg07351192 has a sequence as shown in SEQ ID NO: 10;

[0040] A probe for detecting site cg06829686, the sequence of which is shown in SEQ ID NO: 11;

[0041] The probe used to detect site cg26595643 has a sequence as shown in SEQ ID NO:12.

[0042] Specifically, the test sample of the kit is selected from one or more of blood, serum, plasma, urine, saliva, sweat, mucus, tears, lymph, feces, interstitial fluid, and tissue samples.

[0043] Specifically, the kit also includes a primer pair and a probe for detecting an internal reference gene.

[0044] Furthermore, the internal reference gene is selected from any one of GAPDH, β-actin, ACTB, B2M, ARBP, and HPRT1.

[0045] Furthermore, the internal reference gene is ACTB.

[0046] Furthermore, the sequence of the primer pair of the internal reference gene is shown in SEQ ID NO:13-14, and the sequence of the probe is shown in SEQ ID NO:15.

[0047] Specifically, the final concentration of the primers in the kit is 90-100 mM;

[0048] Furthermore, the final concentration of the primers in the kit is 100 mM.

[0049] Specifically, the final concentration of the primers in the kit is 90-100 mM;

[0050] Furthermore, the final concentration of the primers in the kit is 100 mM.

[0051] Specifically, the kit also includes a positive quality control product and a negative quality control product.

[0052] Furthermore, the positive quality control product is human methylated standard DNA, and the negative quality control product is human unmethylated standard DNA.

[0053] Specifically, the kit can be used for the following detection platforms: fluorescent quantitative PCR method, PCR amplification method, digital PCR method, second-generation sequencing method, third-generation sequencing method, and methylation chip method.

[0054] Specifically, the kit also includes a PCR reaction system, and the PCR reaction system includes an upstream primer, a downstream primer, a probe, magnesium ions, dNTPs, Taq polymerase, a buffer and a template.

[0055] According to some embodiments of the present invention, the kit includes 0.125 μL upstream primer, 0.125 μL downstream primer, 0.05 μL probe, 4 μL magnesium ions, 1 μL dNTPs, 0.5 μL Taq polymerase, 5 μL buffer, and 1 μL template.

[0056] Specifically, the PCR reaction program of the kit is: 95°C, 5 min; 95°C, 20 s, 63°C, 30 s, 45 cycles; 40°C, 5 min.

[0057] In yet another aspect, the present invention provides use of the above-mentioned kit in preparing a product for detecting renal cancer.

[0058] The beneficial effects of the present invention are:

[0059] The present invention provides a reagent for detecting the methylation level of a methylation site marker of renal cancer, wherein the reagent comprises a methylation detection primer pair; the methylation detection primer pair comprises a primer pair for detecting sites cg01362243, cg07351192, cg06829686, and cg26595643, and can effectively diagnose renal cancer at an early stage, improve the survival rate of patients, and is simple to operate, accurate in results, and highly specific. DETAILED DESCRIPTION

[0060] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further explained in conjunction with specific embodiments below, but the following embodiments are only preferred embodiments of the present invention, not all. Based on the embodiments in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following embodiments, unless otherwise specified, the operating methods used are conventional operating methods, the equipment used are conventional equipment, and the equipment materials used in each embodiment are the same.

[0061] Example 1 Renal cancer detection composition and primers thereof

[0062] The specific sequences of primers and probes of the renal cancer detection composition are shown in Table 1 below.

[0063] Table 1 Primer and probe sequences

[0064]

[0065]

[0066] Example 2 Preparation of a Kidney Cancer Detection Kit

[0067] The kidney cancer detection kit of the present invention includes the primers and probes in Table 1 above, PCR reaction solution, positive quality control and negative quality control, the positive quality control is human methylated standard DNA, and the negative quality control is human non-methylated standard DNA. The kidney cancer detection kit prepared by the present invention is used to detect the sample, and the specific steps of the detection are:

[0068] Step 1: DNA extraction

[0069] The QIAamp DNAMini Kit was used to extract the whole genome DNA of the tissue, and the DNA concentration was detected by NanoDrop2000 ultra-micro spectrophotometer.

[0070] Step 2: Methylation modification

[0071] Using EZ DNAMethylation Gold TM Kit methylation conversion kit (purchased from Zymo Research) converts unmethylated cytosine in DNA sequences into uracil.

[0072] Step 3: qPCR amplification

[0073] (1) Preparation of primer and probe combination

[0074] Prepare according to Table 2 below:

[0075] Table 2 Primer probe composition

[0076]

[0077]

[0078] (2) Preparation of qPCR reaction solution

[0079] Prepare the PCR reaction solution according to Table 3 below, mix and centrifuge the prepared PCR reaction solution, and then put it into the PCR reaction tank.

[0080] Table 3 qPCR reaction system

[0081] Components Dosage (μL) Upstream primer 0.125 Downstream primer 0.125 Probe 0.05 <![CDATA[Mg 2+ ,25mM)]]> 4 dNTPs(10mM) 1 Taq polymerase (5unit / ul) 0.5 5× Buffer 5 <![CDATA[ddH2O]]> 13.2 template 1 total 25

[0082] (3) Sample addition: Add the DNA sample to the PCR reaction tube. The sample volume for both the positive quality control and the negative quality control is 5 μL. After sample addition, cover the tube and centrifuge to mix.

[0083] (4) PCR reaction procedure:

[0084] PCR amplification was performed according to Table 4 below, with 3 replicate wells for each sample and water as a blank control.

[0085] Table 4 PCR amplification program

[0086]

[0087] (5) Interpretation of test results

[0088] The internal reference gene ACTB channel of the sample to be tested has an obvious amplification curve and the Ct value is less than 35, which means that the test sample is valid. On the premise that the sample to be tested is valid, the detection channel has an obvious amplification curve, and the Ct value is less than 35, which can be judged as positive, and the Ct value is greater than 35, which can be judged as negative.

[0089] Comparative Example 1

[0090] The difference between Comparative Example 1 and Example 2 is that the preparation of the primer-probe combination is different, and the site cg01362243 is replaced by cg11304234. The preparation of the primer-probe in Comparative Example 1 is shown in Table 5 below:

[0091] Table 5 Primer probe composition

[0092]

[0093]

[0094] Comparative Example 2

[0095] The difference between Comparative Example 2 and Example 2 is that the preparation of the primer-probe combination is different. The preparation of the primer-probe in Comparative Example 2 is shown in Table 6 below.

[0096] Table 6 Primer probe composition

[0097]

[0098] Experimental Example 1

[0099] The samples were collected from 65 patients diagnosed with renal cancer by histopathology (30 patients with renal clear cell carcinoma and 35 patients with papillary renal carcinoma) and 20 healthy controls, and the samples were tested using the kits of Example 2 and Comparative Examples 1-2, respectively.

[0100] (1) DNA extraction

[0101] The QIAamp DNAMini Kit was used to extract the whole genome DNA of the tissue, and the DNA concentration was detected by NanoDrop2000 ultra-micro spectrophotometer.

[0102] (2) Methylation modification

[0103] Using EZ DNA Methylation Gold TM Kit methylation conversion kit (purchased from Zymo Research) converts unmethylated cytosine in DNA sequences into uracil.

[0104] Step 3: qPCR amplification

[0105] (1) Preparation of primer and probe combination

[0106] Prepare according to Table 7 below:

[0107] Table 7 Primer probe composition

[0108]

[0109] (2) Preparation of qPCR reaction solution

[0110] Prepare the PCR reaction solution according to Table 8 below, mix and centrifuge the prepared PCR reaction solution, and then put it into the PCR reaction tank.

[0111] Table 8 qPCR reaction system

[0112] Components Dosage (μL) Upstream primer 0.125 Downstream primer 0.125 Probe 0.05 Magnesium ion (25mM) 4 dNTPs(10mM) 1 Taq polymerase (5unit / ul) 0.5 5× Buffer 5 <![CDATA[ddH2O]]> 13.2 template 1 total 25

[0113] (3) Sample addition: Add the DNA sample to the PCR reaction tube. The sample volume for both the positive quality control and the negative quality control is 5 μL. After sample addition, cover the tube and centrifuge to mix.

[0114] (4) PCR reaction procedure:

[0115] PCR amplification was performed according to Table 9 below, with 3 replicate wells for each sample and water as a blank control.

[0116] Table 9 PCR amplification program

[0117]

[0118] The test results are shown in the following table:

[0119] Table 10 Test results

[0120]

[0121] As can be seen from the above table, the sensitivity of Example 2 is 96.9% and the specificity is 95%; the sensitivity of Comparative Example 1 is 89.2% and the specificity is 85%; the sensitivity of Comparative Example 2 is 84.6% and the specificity is 80%. The sensitivity of the early detection kit for renal cancer of the present invention is greater than 96% and the specificity is greater than 90%, which has good sensitivity and specificity.

[0122] Experimental Example 2

[0123] Another 20 healthy controls and 10 gastric cancer patients were selected, and the detection method was the same as that of Experimental Example 1. The detection results are shown in Table 11 below.

[0124] Table 11

[0125]

[0126]

[0127] It can be seen from the above table that the method for detecting renal cancer of the present application has good specificity.

[0128] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a reagent for detecting the methylation level of a methylation site marker of renal cancer in the preparation of a renal cancer detection product, characterized in that: The reagents include a methylation detection primer pair; the methylation detection primer pair includes the following primer pairs: The primer pair used to detect site cg01362243, the sequence is shown in SEQ ID NO: 1-2; The primer pair used to detect site cg07351192, the sequence is shown in SEQ ID NO:3-4; The primer pair for detecting site cg06829686, the sequence of which is shown in SEQ ID NO:5-6; The primer pair used to detect site cg26595643 has a sequence as shown in SEQ ID NO: 7-8.

2. The use according to claim 1, characterized in that: The reagent also includes a methylation detection probe, and the methylation detection probe includes the following probes: A probe for detecting site cg01362243, the sequence of which is shown in SEQ ID NO:9; The probe for detecting site cg07351192 has a sequence as shown in SEQ ID NO: 10; A probe for detecting site cg06829686, the sequence of which is shown in SEQ ID NO: 11; The probe used to detect site cg26595643 has a sequence as shown in SEQ ID NO:

12.

3. The use according to claim 2, characterized in that: The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a fluorescent quencher group.

4. The use according to claim 2, characterized in that: The fluorescent reporter group is selected from any one of FAM, ROX, HEX, CY5, VIC, TET, JOE, Cy3, Cy7, RED610, Texas Red, RED670, NED, AMCA, Pacific Blue, Atto425, BODIPY FL, Atto 590, and Cy5.5; the fluorescent quencher group is selected from any one of 6-TAMRA, BHQ-1, BHQ-2, BHQ-3, Dabcyl, Eclipse, MGB, and QYS-7.

5. The use according to claim 1, characterized in that: The reagent also includes a primer pair and a probe for detecting an internal reference gene; the sequence of the primer pair of the internal reference gene is shown in SEQ ID NO:13-14, and the sequence of the probe is shown in SEQ ID NO:

15.

6. A kit for detecting renal cancer, characterized in that: The kit comprises a methylation detection primer pair; the methylation detection primer pair comprises the following primer pairs: The primer pair used to detect site cg01362243, the sequence is shown in SEQ ID NO: 1-2; The primer pair used to detect site cg07351192, the sequence is shown in SEQ ID NO:3-4; The primer pair used to detect site cg06829686, the sequence is shown in SEQ ID NO:5-6; The primer pair used to detect site cg26595643, the sequence is shown in SEQ ID NO:7-8; The kit also includes a methylation detection probe, which includes the following probes: A probe for detecting site cg01362243, the sequence of which is shown in SEQ ID NO:9; The probe for detecting site cg07351192 has a sequence as shown in SEQ ID NO: 10; A probe for detecting site cg06829686, the sequence of which is shown in SEQ ID NO: 11; The probe used to detect site cg26595643 has a sequence as shown in SEQ ID NO:

12.

7. The kit according to claim 6, characterized in that The test sample of the kit is selected from one or more of blood, serum, plasma, urine, saliva, sweat, mucus, tears, lymph, feces, interstitial fluid, and tissue samples.

8. The kit according to claim 6, characterized in that The final concentration of the primers in the kit is 90-100 mM.

9. The kit according to claim 6, characterized in that The kit also includes a positive quality control product and a negative quality control product; the positive quality control product is a human methylated standard DNA, and the negative quality control product is a human non-methylated standard DNA.

10. Use of the kit according to claims 6-9 in preparing a product for detecting renal cancer.

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