A primer-probe combination, kit and application for detecting renal cancer

Abstract

The present invention discloses a primer-probe combination, a kit and an application for detecting renal cancer. The present invention provides a primer-probe combination for detecting renal cancer, the primer-probe combination comprising a primer-probe combination for detecting at least one of SYTL3, GBX2 and FLRT2; the nucleotide sequence of the primer for detecting SYTL3 is shown in SEQ ID NO: 1 and 2, and the nucleotide sequence of the probe for detecting SYTL3 is shown in SEQ ID NO: 3; the nucleotide sequence of the primer for detecting GBX2 is shown in SEQ ID NO: 4 and 5, and the nucleotide sequence of the probe for detecting GBX2 is shown in SEQ ID NO: 6; the nucleotide sequence of the primer for detecting FLRT2 is shown in SEQ ID NO: 7 and 8, and the nucleotide sequence of the probe for detecting FLRT2 is shown in SEQ ID NO: 9. The specificity is high and the sensitivity is strong.

Description

Technical Field

[0001] The present invention relates to the technical field of in vitro detection, and in particular to a primer-probe combination, a kit and an application thereof for detecting renal cancer. Background Art

[0002] Kidney cancer is one of the most common malignant tumors in the world, and its incidence is on the rise. The incidence and mortality of kidney cancer are high worldwide, which indicates that there are challenges in prevention, early diagnosis and treatment.

[0003] Imaging examination of renal cancer mainly relies on different imaging techniques, which can diagnose and evaluate renal cancer by revealing the structural and functional abnormalities of the kidney. However, subjective visual assessment and qualitative description by radiologists are usually required, which may affect the accuracy and reliability of the diagnosis. Imaging examinations usually cannot provide molecular and functional information of the tumor, and the molecular characteristics of the tumor (such as gene mutations, protein expression, etc.) are crucial for developing individualized treatment plans.

[0004] Although the application of biochemical markers in the detection of renal cancer is of great value, they have limitations such as insufficient specificity, low sensitivity, susceptibility to interference from multiple factors, insufficient accuracy in evaluating treatment response, and lack of unified standards, which restrict their application in clinical diagnosis and treatment.

[0005] In immunohistochemistry testing, factors such as antibody quality and specificity, complex operating procedures, subjectivity in result interpretation, high cost, dependence on sample quality, and specificity limitations may affect the accuracy and reliability of the test.

[0006] In the second-generation sequencing technology, the costs of sequencing instruments, reagents and consumables are relatively expensive, coupled with the complex data analysis process, resulting in a high overall testing cost.

[0007] At present, there is still a need to develop detection products and methods that are fast, easy to operate, low cost, have good specificity and are simple to interpret. Summary of the invention

[0008] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and to provide a primer-probe combination, a kit and an application for detecting renal cancer.

[0009] The present invention designs a primer-probe combination for methylated renal cancer samples and performs qPCR amplification to detect renal cancer. It can accurately identify the target sequence, effectively avoid the interference of non-target sequences, and has high specificity and sensitivity.

[0010] The first object of the present invention is to provide a primer-probe combination for detecting renal cancer.

[0011] The second object of the present invention is to provide the use of the primer-probe combination in preparing a kit for detecting renal cancer.

[0012] The third object of the present invention is to provide a kit for detecting renal cancer.

[0013] In order to achieve the above object, the present invention is implemented by the following scheme:

[0014] A primer-probe combination for detecting renal cancer, the primer-probe combination comprising a primer-probe combination for detecting at least one of a SYTL3 gene, a GBX2 gene, and a FLRT2 gene;

[0015] The nucleotide sequences of the primers for detecting the SYTL3 gene are shown in SEQ ID NOs: 1 and 2, and the nucleotide sequence of the probe for detecting the SYTL3 gene is shown in SEQ ID NO: 3;

[0016] The nucleotide sequences of the primers for detecting the GBX2 gene are shown in SEQ ID NOs: 4 and 5, and the nucleotide sequence of the probe for detecting the GBX2 gene is shown in SEQ ID NO: 6;

[0017] The nucleotide sequences of the primers for detecting the FLRT2 gene are shown in SEQ ID NOs: 7 and 8, and the nucleotide sequence of the probe for detecting the FLRT2 gene is shown in SEQ ID NO: 9.

[0018] The Gene ID of the SYTL3 gene in NCBI is: 84958, the Gene ID of the GBX2 gene in NCBI is: 2637, and the Gene ID of the FLRT2 gene in NCBI is: 23768.

[0019] In a specific embodiment of the present invention, the 5' end of the probe for detecting the SYTL3 gene is labeled with a FAM fluorescent group, and the 3' end is labeled with a TAMRA quenching group;

[0020] The 5' end of the probe for detecting the GBX2 gene is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ-2 quenching group;

[0021] The 5' end of the probe for detecting the FLRT2 gene is labeled with a TET fluorescent group, and the 3' end is labeled with a BHQ-1 quenching group.

[0022] The present invention provides application of the primer-probe combination in preparing a kit for detecting kidney cancer.

[0023] The present invention also provides a kit for detecting renal cancer, wherein the kit contains the primer-probe combination.

[0024] Furthermore, the kit also contains dNTPs.

[0025] Furthermore, the kit also contains MgCl2.

[0026] Furthermore, the kit also contains ROX dye.

[0027] Furthermore, the kit also contains enzyme-free nucleic acid water.

[0028] Furthermore, the kit also contains a negative control, a positive control and a no-template control.

[0029] Furthermore, the negative control is human leukocyte DNA, the positive control is renal cancer cell DNA, and the template-free control is enzyme-free nucleic acid water.

[0030] The method of using the kit is as follows:

[0031] 1. Extract the DNA of the sample to be tested. Take 30 ng of the DNA of the sample to be tested and perform bisulfite conversion to obtain a bisulfite-converted DNA sample. In a specific embodiment of the present invention, the DNA of the sample to be tested can be extracted using a QIAamp Circulating Nucleic Acid Kit (manufacturer: QIAGEN, catalog number: No. 55114). Ultrafast Bisulfite Kit was used for bisulfite conversion to obtain bisulfite-converted DNA samples.

[0032] 2. Configure the PCR reaction system:

[0033] The PCR reaction system in each well was composed of 10 μL of PCR reaction mixture and 10 μL of DNA sample converted by bisulfite, for a total of 20 μL.

[0034] 10 μL PCR reaction mixture: 200-400 μM dNTPs, 2.5-6 mM MgCl2, 1-3 U AmpliTaq Gold DNA polymerase, 1-3× PCR reaction buffer, 25-60 nM ROX dye, 650-800 nM primers such as the nucleotide sequence shown in SEQ ID NO: 1, 650-800 nM primers such as the nucleotide sequence shown in SEQ ID NO: 2, 300-500 nM probes such as the nucleotide sequence shown in SEQ ID NO: 3, 150-250 nM primers such as the nucleotide sequence shown in SEQ ID NO: 4, 150-250 nM primers such as the nucleotide sequence shown in SEQ ID NO: 5, 50-150 nM probes such as the nucleotide sequence shown in SEQ ID NO: 6, 250-450 nM primers such as the nucleotide sequence shown in SEQ ID NO: 7, 250-450 nM primers such as the nucleotide sequence shown in SEQ ID NO: 8 The primers shown in NO: 8 and the probe with 100-250 nM nucleotide sequence shown in SEQ ID NO: 9 were added with enzyme-free nucleic acid water to make the total volume of the PCR reaction mixture 10 μL. The concentration of each component in the above PCR reaction mixture is the final concentration in a 20 μL PCR reaction system.

[0035] Preferably, a 10 μL PCR reaction mixture comprises: 300 μM dNTPs, 3 mM MgCl2, 2 U AmpliTaq Gold DNA polymerase, 1× PCR reaction buffer, 30 nM ROX dye, 700 nM primers such as the nucleotide sequence shown in SEQ ID NO: 1, 700 nM primers such as the nucleotide sequence shown in SEQ ID NO: 2, 350 nM probes such as the nucleotide sequence shown in SEQ ID NO: 3, 200 nM primers such as the nucleotide sequence shown in SEQ ID NO: 4, 200 nM primers such as the nucleotide sequence shown in SEQ ID NO: 5, 100 nM probes such as the nucleotide sequence shown in SEQ ID NO: 6, 300 nM primers such as the nucleotide sequence shown in SEQ ID NO: 7, 300 nM primers such as the nucleotide sequence shown in SEQ ID NO: 8 and 150 nM probes such as the nucleotide sequence shown in SEQ ID NO: 9, and the total volume of the PCR reaction mixture is made up to 10 μL with enzyme-free nucleic acid water. The concentration of each component in the above PCR reaction mixture is the final concentration in a 20 μL PCR reaction system.

[0036] The manufacturer of AmpliTaq Gold DNA polymerase is Thermo, the product number is N8080010, and the PCR reaction buffer is the matching buffer in AmpliTaq Gold DNA polymerase.

[0037] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing and extension at 63°C for 30 s, and 45 cycles.

[0038] After the PCR reaction was completed, the Ct threshold was set in the linear amplification range (in this experiment, ΔRn was set to 0.15).

[0039] 3. Judgment criteria

[0040] The Ct value of target 1 (SYTL3 gene) was less than 38.7, the Ct value of target 2 (GBX2 gene) was less than 38.4, and the Ct value of target 3 (FLRT2 gene) was less than 38.9.

[0041] If at least one of the above three conditions is met, the sample test is positive; if the sample does not meet the conditions that the Ct value of target 1 is less than 38.7, the Ct value of target 3 is less than 38.4, and the Ct value of target 2 is less than 38.9, the sample test is negative.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The present invention designs a primer-probe combination for methylated renal cancer samples, performs qPCR amplification, and thus detects renal cancer. The primer-probe combination of the present invention can accurately identify the target sequence, effectively avoid the interference of non-target sequences, and has high specificity and sensitivity. A kit for detecting renal cancer is developed through the designed primer-probe combination, and the operation steps are simple and intuitive. Compared with the cumbersome detection process required for the construction of a high-throughput sequencing library, the operation of this kit is easier for laboratory technicians to quickly master and implement. Compared with high-throughput sequencing, the kit not only reduces the expensive sequencing costs, but also greatly shortens the detection time, effectively reducing the detection cost while ensuring the detection quality. This improvement in cost-effectiveness enables more patients to obtain timely and accurate renal cancer detection, which helps in the early detection and treatment of renal cancer, thereby improving the accessibility and effectiveness of treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The Ct value distribution of 182 true positive samples from 300 randomly collected urine samples detected by the kit of the present invention, only the Ct values ​​of the targets that reached the positive judgment were calculated, and the Ct values ​​of the targets that did not meet the positive standard were not included in the statistics.

[0045] Figure 2 The Ct value distribution of 5 false positive samples of 300 randomly collected urine samples detected by the kit of the present invention, only the Ct values ​​of the targets that reached the positive judgment were calculated, and the Ct values ​​of the targets that did not meet the positive standard were not included in the statistics. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0047] Example 1 Site and panel determination

[0048] 1. Following the principle of balanced enrollment of the national population, a total of 300 FFPE samples (Formalin-Fixed and Parrffin-Embedded, formalin-fixed and paraffin-embedded tissue samples) and their corresponding adjacent tissues from renal cancer patients were collected. In addition, urine samples from 30 patients with advanced severe renal cancer and 30 corresponding oral pharyngeal swab samples from renal cancer patients were included. QIAGEN's QIAwave DNA Blood&Tissue Kit (Cat.no.69554), QIAamp Circulating Nucleic Acid Kit (Cat.No.55114), and QIAamp DNAMicrobiome Kit (Cat.No.51704) were used to extract nucleic acids from the above 300 FFPE samples and adjacent tissue samples, 30 urine samples, and 30 oral pharyngeal swab samples to obtain sample DNA.

[0049] 2. Use The Ultrafast Bisulfite Kit performs bisulfite (BS) conversion on sample DNA, converting unmethylated cytosine (C) in the DNA sample into uracil (U), while methylated cytosine (5-mC) remains unchanged, thereby distinguishing methylated and unmethylated cytosine sites and obtaining bisulfite-converted DNA.

[0050] 3. Use the methylation library construction kit (manufacturer: Novozymes, catalog number: NE103) to connect the sequencing adapter to the bisulfite-converted DNA, purify it with QIAseq Beads magnetic beads (manufacturer: QIAGEN, catalog number: 333923), PCR amplification and hybridization capture (manufacturer: Yisheng, catalog number: 12245ES), and obtain a high-throughput sequencing library.

[0051] 4. Sequencing and data analysis

[0052] The library was sequenced at high throughput, and then the sequencing data was analyzed in depth using bioinformatics methods. Factors such as sequencing coverage, data consistency, accuracy of variant detection, and comparability between samples were considered to quantify the quality and reliability of the sequencing data, and the sequencing results were comprehensively evaluated based on multiple parameters to ensure the scientific validity of the obtained data and the accuracy of subsequent analysis. Through strict scoring of the sequencing data and comprehensive evaluation, the top 50 markers were selected.

[0053] 5. Screening of markers and determination of the final panel (primer-probe combination)

[0054] For the 50 markers selected in advance (Table 1 and Table 2), primers and probes were designed using Beacon Designer 8.14 (Table 2). The chromosome loci refer to the human universal genome GRCh37 / hg19 on UCSC. The chromosome fragment sequence recorded in the marker column in Table 2 is the amplification product sequence obtained by PCR reaction of the corresponding primer probe.

[0055] Table 1

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070] Table 2

[0071]

[0072]

[0073]

[0074]

[0075]

[0076] 200 urine samples were collected, including 136 renal cancer-positive samples and 64 renal cancer-negative samples. DNA was extracted using QIAamp Circulating Nucleic Acid Kit (manufacturer: QIAGEN, catalog number: No.55114) to obtain sample DNA. The Ultrafast Bisulfite Kit was used to convert the sample DNA with bisulfite (BS) to obtain bisulfite-converted DNA. The primers and probes in Table 1 were used to perform qPCR detection on the bisulfite-converted DNA. The 7500 Real-Time PCR System instrument of Applied Biosystems was used for qPCR amplification. Through comprehensive analysis and screening of the test results, 16 markers with potential clinical application value were identified for testing and comparison. Therefore, after comprehensive consideration, a small panel for detecting 3 sites was finally constructed, and the site characteristics of the corresponding primer target sequence combination are shown in Tables 3 and 4. Among them, the 5' end of the probe in primer probe set 1 is labeled with a FAM fluorescent group, and the 3' end is labeled with a TAMRA quenching group; the 5' end of the probe in primer probe set 2 is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ-2 quenching group; the 5' end of the probe in primer probe set 3 is labeled with a TET fluorescent group, and the 3' end is labeled with a BHQ-1 quenching group. The Gene ID of SYTL3 gene in NCBI is: 84958, the Gene ID of GBX2 gene in NCBI is: 2637, and the Gene ID of FLRT2 gene in NCBI is: 23768.

[0077] When comparing panels detecting different numbers of sites, the small panel of the present invention detecting 3 sites has higher accuracy and specificity, which is comparable to the performance of the panel detecting 4-6 sites. However, the cost of the panel detecting 7 or more sites is significantly increased, and the operational complexity is also greatly increased.

[0078] Table 3

[0079]

[0080]

[0081] Table 4

[0082]

[0083] Example 2 A kit for detecting renal cancer

[0084] 1. Composition

[0085] Primer probe (panel): Primer probe in Table 1 of Example 1, nucleotide sequences are shown in SEQ ID NO: 1-9.

[0086] Other reagents: enzyme-free nuclease water, dNTPs, MgCl2 and ROX dye.

[0087] Negative control: human leukocyte DNA.

[0088] Positive control: renal cell carcinoma cell DNA. DNA from the ACHN cell line, ATCC number CRL-1611. The ACHN cell line was derived from a 22-year-old female patient's transplanted renal cell carcinoma and is widely used in renal cancer research.

[0089] 2. Usage

[0090] 1. Extract the DNA of the sample to be tested, and add 30 ng of the extracted DNA for bisulfite conversion to obtain a bisulfite-converted DNA sample. The DNA of the sample to be tested can be extracted using the QIAamp Circulating Nucleic Acid Kit (manufacturer: QIAGEN, catalog number: No.55114). Ultrafast Bisulfite Kit was used for bisulfite conversion to obtain bisulfite-converted DNA samples.

[0091] 2. Prepare the PCR reaction mixture as shown in Table 5.

[0092] Table 5

[0093]

[0094] The manufacturer of AmpliTaq Gold DNA polymerase is Thermo, the product number is N8080010, and the 10×PCR reaction buffer is the matching buffer in AmpliTaq Gold DNA polymerase.

[0095] Aliquot the PCR reaction mixture into a 96-well plate (10 μL / well), add 10 μL of bisulfite-converted DNA sample to each well (do 3 replicates for each test sample) to obtain a PCR reaction system (i.e., 10 μL of PCR reaction mixture plus 10 μL of bisulfite-converted DNA sample to each well), use human leukocyte DNA as a negative control, renal cancer cell DNA as a positive control, and enzyme-free nucleic acid water as a no-template control (NTC).

[0096] The final concentrations in Table 5 are the final concentrations of each reagent in a 20 μL PCR reaction system.

[0097] The PCR amplification conditions were as follows: pre-denaturation at 95°C for 10 min, denaturation at 95°C for 15 s, annealing and extension at 63°C for 30 s, and 45 cycles.

[0098] After the PCR reaction was completed, the Ct threshold was set in the linear amplification range (in this experiment, ΔRn was set to 0.15).

[0099] 3. Judgment Criteria

[0100] The Ct values ​​of the no-template control (NTC) and the negative control were not detected or >40, and both had no obvious amplification curves; while the Ct value of the positive control was between 15 and 35, and there was an obvious amplification curve, which means that the experimental data is reliable. On this basis, the samples tested can be judged according to the following standards.

[0101] The Ct value of target 1 (SYTL3 gene) was less than 38.7, the Ct value of target 2 (GBX2 gene) was less than 38.4, and the Ct value of target 3 (FLRT2 gene) was less than 38.9.

[0102] If at least one of the above three conditions is met, the sample test is positive; if the sample does not meet the conditions that the Ct value of target 1 is less than 38.7, the Ct value of target 3 is less than 38.4, and the Ct value of target 2 is less than 38.9, the sample test is negative.

[0103] Example 3 Sensitivity and specificity of the kit for detecting renal cancer of the present invention

[0104] 1. Experimental Methods

[0105] The kit of Example 2 was used to detect 300 randomly collected urine samples, including 223 positive samples and 77 negative samples.

[0106] 2. Experimental Results

[0107] The results of Example 3 of the present invention are shown in Table 6:

[0108] Table 6

[0109] True Positive False Positive True Negative False Negative 182 5 72 41

[0110] There were 187 samples with positive test results in this test, of which 182 were true positive samples. The detection results of each target were as follows: Figure 1 (Only the data of the targets that were judged as positive are counted) As shown in the figure, the detection of each target in the 5 false positive samples is as follows Figure 2 (Only the data of targets judged as positive are counted) is shown.

[0111] Calculated from the test results, the sensitivity of this kit is 82% (calculated as: true positive / (true positive + false negative)), and the specificity is as high as 93% (calculated as: true negative / (true negative + false positive)).

[0112] Example 4 Effect of different primer-probe combinations on PCR amplification efficiency

[0113] 1. Experimental Methods

[0114] According to the three sites determined in Example 1, multiple sets of primer probe combinations were designed by moving the primers at specific positions forward and backward, as shown in Table 7. The fluorescent groups and quenching groups at the 5' and 3' ends of the probes in primer probe sets 1-1 and 1-2 are the same as those in primer probe set 1; the fluorescent groups and quenching groups at the 5' and 3' ends of the probes in primer probe set 2-1 are the same as those in primer probe set 2; the fluorescent groups and quenching groups at the 5' and 3' ends of the probes in primer probe sets 3-1 and 3-2 are the same as those in primer probe set 3.

[0115] Table 7

[0116]

[0117]

[0118] The concentrations were 10 8 copies / μL, 10 7 copies / μL, 10 6 copies / μL, 10 5 copies / μL, 10 4 The positive control (kit in Example 2) with 100 copies / μL was used as a template, and the other reaction conditions and reaction system were the same as those in Example 2. qPCR reaction was performed to detect the amplification efficiency of each set of primer probes. The standard curve was drawn by the Ct value, the amplification efficiency was calculated, and the final result was obtained by combining the amplification efficiency and the primer-probe combination that can obtain the maximum discrimination.

[0119] 2. Experimental Results

[0120] The amplification efficiency of primer probe set 1 in Example 1 is 96.42%, which is higher than primer probe set 1-1 (85.30%) and primer probe set 1-2 (92.15%); the amplification efficiency of primer probe set 2 in Example 1 is 98.23%, which is higher than primer probe set 2-1 (90.59%); the amplification efficiency of primer probe set 3 in Example 1 is 95.97%, which is higher than primer probe set 3-1 (80.72%) and higher than primer probe set 3-2 (93.05%).

[0121] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A primer-probe combination for detecting renal cancer, characterized in that: The primer-probe combination is a primer-probe combination for detecting SYTL3 gene, GBX2 gene and FLRT2 gene; The nucleotide sequences of the primers for detecting the SYTL3 gene are shown in SEQ ID NOs: 1 and 2, and the nucleotide sequence of the probe for detecting the SYTL3 gene is shown in SEQ ID NO: 3; The nucleotide sequences of the primers for detecting the GBX2 gene are shown in SEQ ID NOs: 4 and 5, and the nucleotide sequence of the probe for detecting the GBX2 gene is shown in SEQ ID NO: 6; The nucleotide sequences of the primers for detecting the FLRT2 gene are shown in SEQ ID NOs: 7 and 8, and the nucleotide sequence of the probe for detecting FLRT2 is shown in SEQ ID NO:

9.

2. The primer-probe combination according to claim 1, characterized in that: The 5' end of the probe for detecting the SYTL3 gene is labeled with a FAM fluorescent group, and the 3' end is labeled with a TAMRA quenching group; The 5' end of the probe for detecting the GBX2 gene is labeled with a VIC fluorescent group, and the 3' end is labeled with a BHQ-2 quenching group; The 5' end of the probe for detecting the FLRT2 gene is labeled with a TET fluorescent group, and the 3' end is labeled with a BHQ-1 quenching group.

3. Use of the primer-probe combination according to claim 1 or 2 in preparing a kit for detecting renal cancer.

4. A kit for detecting renal cancer, characterized in that: The kit contains the primer-probe combination according to claim 1 or 2.

5. The kit according to claim 4, characterized in that The kit also contains dNTPs.

6. The kit according to claim 4, characterized in that The kit also contains MgCl2.

7. The kit according to claim 4, characterized in that The kit also contains ROX dye.

8. The kit according to claim 4, characterized in that The kit also contains enzyme-free nucleic acid water.

9. The kit according to claim 4, characterized in that The kit also contains a negative control, a positive control and a no-template control.

10. The kit according to claim 9, characterized in that The negative control is human leukocyte DNA, the positive control is renal cancer cell DNA, and the no-template control is enzyme-free nucleic acid water.

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