Kit, system and method for prognosis evaluation of kidney cancer

Abstract

The invention provides a kidney cancer prognosis evaluation kit, system and method, relates to the field of in vitro detection, and designs and develops a Methylight detection primer and probe based on a DNA sample kidney methylation related marker and application of the Methylight detection primer and probe in prediction of kidney cancer prognosis. The kit has the advantages of high sensitivity and specificity, stable and reliable detection result and low cost, and has important significance for prognosis evaluation of kidney cancer.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro detection, and in particular relates to a renal cancer prognosis assessment kit, system and method. Background Art

[0002] In recent years, more and more studies have shown that abnormal DNA methylation is involved in the occurrence and development of various diseases and tumors. It is reported that the methylation level in tumor cells plays a vital role in anti-tumor immunity and immunotherapy effects. Currently, more and more studies are focusing on the effect of DNA methylation on the immunotherapy effect of patients with renal cell carcinoma.

[0003] Renal cell carcinoma (RCC) is one of the most common malignant tumors of the urinary system. RCC is a rare solid tumor that is insensitive to both radiotherapy and chemotherapy. Surgical resection is currently the most effective treatment, but up to 40% of patients still relapse or metastasize after resection of the primary lesion.

[0004] The clinical progression of RCC is highly variable, ranging from slowly growing localized tumors to highly aggressive metastatic phenotypes, and its progression and prognosis are associated with diverse molecular events.

[0005] Therefore, exploring biomarkers that can be used to indicate RCC progression will help determine its invasive potential, thereby guiding the clinical management of patients and providing a theoretical reference and basis for predicting prognosis. Summary of the invention

[0006] In order to solve the above problems, the present invention provides a renal cancer prognosis assessment kit, system and method.

[0007] In one aspect, the present invention provides nucleotide sequences for detecting the methylation level of renal cancer-related methylation sites.

[0008] Specifically, the nucleotide sequence includes at least one of the following primer-probe combinations: The first primer-probe combination includes primers with sequences as shown in SEQ ID NO.1-2 and a probe with a sequence as shown in SEQ ID NO.3; The second primer-probe combination includes primers with sequences as shown in SEQ ID NO.4-5 and a probe with a sequence as shown in SEQ ID NO.6; The third primer-probe combination includes primers whose sequences are shown in SEQ ID NO.7-8 and a probe whose sequence is shown in SEQ ID NO.9.

[0009] More specifically, the probe carries a fluorescent label and a quenching label, the fluorescent group includes at least one of FAM, VIC and CY5 fluorescent groups, and the quenching group includes at least one of MGB, BHQ and TAMRA.

[0010] Specifically, the primer probe set also includes a fifth primer probe set for detecting an internal reference gene.

[0011] More specifically, the fifth primer-probe set for detecting the internal reference gene includes primers with sequences as shown in SEQ ID NO.10-SEQ ID NO.11 and a probe with a sequence as shown in SEQ ID NO.12, and the internal reference gene is ACTB.

[0012] On the other hand, the present invention provides the use of the above nucleotide sequence in preparing a product for evaluating the prognosis of renal cancer.

[0013] Specifically, the product includes, but is not limited to, at least one of a chip, a test kit, a test strip, a test card or a microfluidic device.

[0014] Further specifically, the product also includes other reagents required for detecting methylation levels.

[0015] More specifically, the steps of using the product include: (1) Extract DNA from the sample to be tested; (2) Bisulfite conversion and purification of the extracted DNA; (3) using the above nucleotide sequence to perform PCR amplification; (4) Prognosis assessment of renal cancer based on the methylation level of methylation sites.

[0016] Specifically, in step (3), the method for detecting the methylation level includes: any one or more of BSP, first-generation sequencing, second-generation sequencing, pyrophosphate sequencing, bisulfite conversion sequencing, methylation chip method, MSP methylation-specific PCR, digital PCR method, third-generation sequencing method, whole genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, methylation-specific PCR, and methylation fluorescent PCR.

[0017] Preferably, the method for detecting the methylation level is: methylation fluorescence PCR.

[0018] In yet another aspect, the present invention provides a kit for evaluating the prognosis of renal cancer.

[0019] Specifically, the kit includes the above-mentioned nucleotide sequence.

[0020] In yet another aspect, the present invention provides a system for prognosis assessment of renal cancer.

[0021] Specifically, the system includes a PCR instrument, a computing device and an output device.

[0022] In yet another aspect, the present invention provides a method for evaluating the prognosis of renal cancer using the above system.

[0023] Specifically, the method comprises the following steps: (1) Synthesize primer-probe combinations; (2) Extracting DNA from samples; (3) Configure the reaction system; (4) The reaction system is placed in the above system for PCR amplification, and the calculation device determines the result and outputs the result through the output device.

[0024] Compared with the prior art, the present invention has the following advantages: The present invention is simple and easy to operate, and has low cost. The present invention uses methylation fluorescence PCR to detect the methylation level of the patient's renal cancer-related methylation sites, providing a rapidly implementable, non-invasive marker that is more sensitive than traditional methods in evaluating the prognosis of renal cancer. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0026] Example 1 Primer Probe Synthesis Based on the four methylated cytosine-rich regions (CG regions) screened out, detection primers and probes targeting renal cancer-related methylation sites were designed. The sequences of the detection primers, probes and blocker primers are shown in Table 1: Table 1

[0027] Example 2 PCR The experimental process is as follows: Blood samples were collected and DNA was extracted using a blood genomic DNA extraction kit (TIANGEN, catalog number DP348) according to the instructions.

[0028] (2) Use the DNA Bisulfite Conversion Kit (TIANGEN, Cat. No. DP215) to perform bisulfite conversion and purification on the extracted DNA according to the instructions.

[0029] (3) The purified bisulfite-converted DNA was used with detection primers and probes designed for the three CG regions to prepare the PCR reaction system as shown in Table 2.

[0030] Table 2

[0031] (4) After the PCR reaction system is prepared, vortex gently to mix, then place it into the ABI7500 according to the following procedure and run according to Table 3.

[0032] Table 3

[0033] A total of 50 plasma samples were selected from 20 patients with renal cancer, 10 patients with kidney disease, 10 patients with other cancers and 10 healthy subjects. The selection criteria for the renal cancer samples were: primary renal cancer diagnosed by pathology, no previous history of malignant tumors and serious medical diseases, and no preoperative anti-tumor treatment. The control samples included kidney disease, other cancers and healthy samples. The selection criteria for the control samples were kidney disease including compensated chronic renal failure and acute renal failure, other cancers including gastric cancer, lung cancer and liver cancer, and the diagnostic criteria were based on ICD-10. The healthy samples were physical examination samples without abnormalities.

[0034] IBM SPSS Statistics 24 software package was used to perform statistical analysis on methylation markers. The independent sample t-test of non-parametric test was used to analyze the statistical differences of the test results, and P value <0.05 was considered to be statistically significant. The diagnostic performance of a single methylation marker for renal cancer is shown by the ROC curve (receiver operating characteristic curve). The risk prediction performance and statistical parameters of a single methylation marker are shown in Table 4: Table 4

[0035] The risk prediction performance and statistical parameters of multiple methylation markers are shown in Table 5: One group: cg26006951, cg11304234 and cg04100971 Group 2: cg26006951 and cg11304234 Group 3: cg11304234 and cg04100971 Group 4: cg26006951 and cg04100971 Table 5

[0036] Example 3 Regression analysis of methylation markers for survival prognosis After confirming the association between the above methylation markers and renal cancer, we selected 212 renal cancer patients for follow-up. The selection criteria for renal cancer patients were: primary renal cancer diagnosed by pathology, no previous history of malignant tumors and serious medical diseases, and no preoperative anti-tumor treatment. COX multivariate regression analysis was used to evaluate the predictive value of methylation markers and clinical data age, gender, and degree of surgical resection for the survival prognosis of renal cancer patients in the 212 patients. The results are shown in Table 6.

[0037] Table 6

[0038] And according to the follow-up results, the result interpretation criteria of the kit of the present invention are given, and the specific criteria are shown in Table 7: Table 7

[0039] If the result is negative, the risk of renal cancer recurrence is low and regular follow-up is recommended; if the result is positive, the risk of renal cancer recurrence is high and microscopic examination or tissue biopsy is recommended for confirmation.

[0040] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A nucleotide sequence for detecting the methylation level of a methylation site associated with renal cancer, characterized in that: Include at least one of the following primer-probe combinations: The first primer-probe combination includes primers with sequences as shown in SEQ ID NO.1-2 and probes with sequences as shown in SEQ ID NO.3; The second primer-probe combination includes primers with sequences as shown in SEQ ID NO.4-5 and a probe with a sequence as shown in SEQ ID NO.6; The third primer-probe combination includes primers whose sequences are shown in SEQ ID NO.7-8 and a probe whose sequence is shown in SEQ ID NO.

9.

2. The nucleotide sequence according to claim 1, characterized in that The probe carries a fluorescent label and a quenching label, wherein the fluorescent label comprises at least one of FAM, VIC and CY5 fluorescent groups, and the quenching label comprises at least one of MGB, BHQ and TAMRA.

3. The nucleotide sequence according to claim 2, characterized in that The primer probe set also includes a fifth primer probe set for detecting an internal reference gene, the fifth primer probe set for detecting an internal reference gene includes primers with sequences shown in SEQ ID NO.10-SEQ ID NO.11 and a probe with a sequence shown in SEQ ID NO.12, and the internal reference gene is ACTB.

4. Use of the nucleotide sequence according to any one of claims 1 to 3 in the preparation of a product for renal cancer prognosis assessment.

5. The use according to claim 4, characterized in that: The renal cancer prognosis assessment product also includes other reagents required for detecting methylation levels.

6. The use according to claim 5, characterized in that: The steps for using the product include: (1) Extract DNA from the sample to be tested; (2) Bisulfite conversion and purification of the extracted DNA; (3) performing PCR amplification using the nucleotide sequence; (4) Prognosis assessment of renal cancer based on the methylation level of methylation sites.

7. The use according to claim 6, characterized in that: In step (3), the method for detecting the methylation level includes: any one or more of BSP, first-generation sequencing, second-generation sequencing, pyrophosphate sequencing, bisulfite conversion sequencing, methylation chip method, MSP methylation-specific PCR, digital PCR method, third-generation sequencing method, whole genome methylation sequencing method, DNA enrichment detection method, simplified bisulfite sequencing technology, HPLC method, methylation-specific PCR, and methylation fluorescent PCR.

8. A kit for evaluating the prognosis of renal cancer, characterized in that: The kit comprises the nucleotide sequence according to any one of claims 1 to 3.

9. A system for evaluating the prognosis of renal cancer, characterized in that: The system includes a PCR instrument, a computing device and an output device.

10. A method for evaluating the prognosis of renal cancer using the system of claim 9, characterized in that: The following steps are involved: (1) Synthesize primer-probe combinations; (2) Extracting DNA from samples; (3) Configure the reaction system; (4) The reaction system is placed in the system for PCR amplification, the computing device determines the result, and the result is output through the output device.

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