Primer pair and kit for detecting methylation of kidney tissue GRP78 gene promoter and application

Abstract

The invention belongs to the technical field of biological diagnosis preparations, and particularly discloses a methyl specific PCR (Polymerase Chain Reaction) primer pair for detecting methylation of a kidney tissue GRP78 gene promoter, application of the methyl specific PCR primer pair in a kit and a kit for evaluating the disease progress of diabetic nephropathy. The kit is suitable for researching the DNA methylation state of the diabetic nephropathy kidney tissue slice, has the advantages of high universality, convenience, rapidness, high sensitivity and low cost, and can be used as a diagnostic kit for indicating the disease progress of diabetic nephropathy; the kit is used for auxiliary diagnosis of diabetic nephropathy and early warning prompt of the progress of the diabetic nephropathy, and has important clinical significance.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological diagnostic preparations, and more specifically, relates to a primer pair, a kit and an application thereof for detecting methylation of the GRP78 gene promoter in renal tissue. Background Art

[0002] Diabetic nephropathy is a common cause of chronic renal failure in my country and worldwide. Due to the lack of clarity in early diagnosis and difficulty in determining the correct time to initiate intensive therapeutic intervention, patients with diabetic nephropathy have poor treatment outcomes. Therefore, the urgent need to develop biomarkers for clinical diagnosis and disease progression assessment of diabetic nephropathy has important clinical guidance significance, thereby establishing a more precise diagnosis and treatment model for diabetic nephropathy.

[0003] DNA methylation is one of the most widely studied epigenetic mechanisms. Under the action of DNA methyltransferases, the 5th carbon atom of cytosine in cytosine-guanine (CpG) dinucleotides accepts a methyl group, transforming it into 5-methylcytosine. DNA methylation can alter chromatin structure, DNA conformation and stability, and DNA-protein interactions, thereby regulating gene expression.

[0004] Previous studies have shown that overall genomic methylation levels differ in patients with diabetic nephropathy compared to those without nephropathy. Altered methylation levels at certain CpG sites are closely associated with diabetic nephropathy, suggesting that DNA methylation plays a crucial role in the development and progression of diabetic nephropathy. However, current studies investigating the DNA methylation mechanisms in diabetic nephropathy have primarily used DNA from saliva or peripheral blood mononuclear cells. Because epigenetic mechanisms are clearly cell-specific, studies of methylation in diabetic nephropathy renal tissue and renal resident cells will be more effective in uncovering the mechanisms by which DNA methylation contributes to the development and progression of diabetic nephropathy. Summary of the Invention

[0005] To solve the above problems, the present invention discloses a methyl-specific PCR primer pair for detecting methylation of the GRP78 gene promoter in renal tissue and its application in a kit, as well as a kit for evaluating the progression of diabetic nephropathy, which are used for auxiliary diagnosis of diabetic nephropathy and early warning of disease progression, and have important clinical significance.

[0006] To achieve the above object, the technical solution of the present invention is as follows: The present invention provides a use of a primer pair for amplifying methylation of the GRP78 gene promoter in renal tissue in the preparation of a kit for assessing diabetic nephropathy progression. The primer pair comprises a pair of methylated primers and a pair of unmethylated primers. The nucleotide sequences of the methylated primers are shown in SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the unmethylated primers are shown in SEQ ID NO.3 and SEQ ID NO.4, as specifically shown in Table 1.

[0007] Table 1

[0008] Furthermore, the methylated region of the GRP78 gene promoter is from 3000 bp upstream to 200 bp downstream of the GRP78 gene promoter sequence, as shown in SEQ ID NO.5.

[0009] Furthermore, the sequences of the methylated sites of the GRP78 gene promoter are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8.

[0010] The present invention also provides a methyl-specific PCR primer pair for detecting methylation of the GRP78 gene promoter in renal tissue, comprising a pair of methylated primers and a pair of unmethylated primers, the nucleotide sequences of the methylated primers being shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the unmethylated primers being shown as SEQ ID NO.3 and SEQ ID NO.4.

[0011] The present invention also provides a kit for evaluating the progression of diabetic nephropathy, comprising a pair of methylated primers and a pair of unmethylated primers, the nucleotide sequences of the methylated primers being shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the unmethylated primers being shown as SEQ ID NO.3 and SEQ ID NO.4.

[0012] Furthermore, the methylation primer and the non-methylation primer are a PCR primer combination for methylation-specific and non-methylation-specific reactions for detecting methylation of the GRP78 gene promoter in renal tissue.

[0013] As a specific embodiment of the present invention, it also contains a kidney tissue paraffin section DNA extraction reagent, a bisulfite DNA conversion reagent, a desulfonation spin column, a DNA purification reagent, and a methylation-specific PCR reaction reagent.

[0014] The renal tissue paraffin section DNA extraction reagent contains: protease, protease digestion buffer, nucleic acid binding beads, DNA enzyme, and anhydrous ethanol.

[0015] The bisulfite DNA conversion reagent contains: CT conversion reagent, Tris-saturated phenol, isopropanol, and ddH2O.

[0016] The DNA purification reagent contains: a DNA binding column, RNA enzyme, and protease.

[0017] The methylation-specific PCR reaction reagent contains: DNA polymerase, DNase / RNase-Free ddH2O, dNTP Mixture, and Reaction Buffer.

[0018] The reaction system of methylation-specific PCR reagent is: DNA polymerase 0.5 μL; DNase / RNase-Free ddH2O 1 μL; dNTP mixture 0.5 μL; upstream primer 1 μL; downstream primer 1 μL; test sample DNA 1 μL; Reaction Buffer 5 μL; total reaction system 10 μL, of which the concentration of upstream primer and downstream primer is 0.5 μM.

[0019] The methylation-specific PCR reaction procedure is as follows: The annealing temperature of the methylated primers was 65°C. The amplification reaction program for the methylated primers was as follows: pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 45 seconds, 35 cycles, and a final extension at 72°C for 7 minutes.

[0020] The annealing temperature of the unmethylated primer was 65°C. The amplification reaction program for the unmethylated primer was as follows: pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 45 seconds, 35 cycles, and final extension at 72°C for 7 minutes.

[0021] The principle of methylation-specific PCR is simple and effective. By treating DNA with sulfite, unmethylated cytosine is converted to uracil, while methylated cytosine remains unchanged. Subsequently, PCR amplification of both methylated and unmethylated DNA is performed using specific primer pairs, allowing the methylation status of DNA to be detected at the molecular level.

[0022] The beneficial effects of the present invention are: The diabetic nephropathy disease progression assessment kit of the present invention, based on detecting methylation of the GRP78 gene promoter in renal tissue, is suitable for studying the DNA methylation status of renal tissue sections of diabetic nephropathy. It has the advantages of strong universality, convenience, high sensitivity, and low cost. The detection can achieve high sensitivity and is applicable to various pathological sections that are relatively easy to obtain. Therefore, it can be used as a diagnostic kit to indicate the disease progression of diabetic nephropathy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the GRP78 gene promoter sequence from 3000 bp upstream to 200 bp downstream, and the locations of three CpG islands; Figure 2 The results are based on the detection of GRP78 mRNA in healthy subjects with early diabetic nephropathy and patients with late diabetic nephropathy; Figure 3 These are the results of a diabetic nephropathy progression assessment kit based on the detection of GRP78 gene promoter methylation in healthy subjects with early-stage diabetic nephropathy and patients with late-stage diabetic nephropathy. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Example 1

[0025] Design of methylation-specific PCR primers for detecting GRP78 gene promoter methylation: Based on the GRP78 gene promoter sequence, Figure 1 As shown, the CpG island (CpGIsland 3) was analyzed and a set of primers (M, methylated primer; U, unmethylated primer) were designed, as shown in Table 2 below: Table 2

[0026] The methylated region of GRP78 gene promoter methylation is from 3000 bp upstream to 200 bp downstream of the GRP78 gene promoter sequence, e.g. Figure 1 The sequence is shown in SEQ ID NO.5.

[0027] The three CpG islands methylated in the GRP78 gene promoter are located at Figure 1 The sequences of the sites are shown in SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8. SEQ ID NO.5: CCTGGGCAAGTTTCTTTACCTCTTGGGGCTTCAGATTCCTTCACTCTAAA ATAGGGGTAAGAAAAAAATGCTTTTTTGTTTGTTTCTTTTTGTTTTTGTT TTTGTTTTTGTTTTGAGACAGAGTCTCGTTCTGTGGCCAGGCTGGAGTGC AGTGGTGCGATCTCAGCTCACTGAACCTCCGCCTCCCAGGTTCAAGCGAT TTTCCTGCCTCATCCTCTGAGTAGCAGTAGCTGGGACTATAGGCGCGCGC CACTACACCCAACTGATTTTTGTATTTTTAGTAGAGACTGGGCACCATGT TGGCCGGGATGGTCTCGATCTCTTGACCTCGTGATCCGCCCACCTCGGCC TCCCAAAATACTGGGATTACAAAAAACTGCTTTCTTAAAGATAAAAAGAG ATAATGCACGTACGTCTTTAGCACAGTGTCTAACAGTTAAGTATTGATAA CTTGACCTGGATATTATTTTTGGCATTTAATGTTCAGCAAGCTAGTTTTG ACCATGTACTTTCCTATTCGTAAGTGACTGTGCTTTGGAATATACTAGAA AATGTATGTAAAATGTCTGGTTTTAAAAAGATGAAACCACAATATATTCA ATTCCCTTTGGAAGTTAGCCATGTATCGCTTTTCTAAATTTTACAAAAAG GAGAGTTACAAAACGTTACTCTGTTAATCAAGGATAAGGCTGAAATGGAG AGTTTTTTTTTTTAATGGAAATGGACTGCACTATAAAAACATTTGATTGA AATGAAAGAAGCTGTAATGTGGTCAAATGATGATTTCTATCTTAATGCTG GACTGATGCAAGCCAAAGAAGGGTCAATGTTGCCGCAGGATTACAAGCCC TGCTGAAAGAGATGCATGATGCAACCTCTGTAGTGAACAGAGTCAACTCT TGAGTCACCAGTCTAACCTTTGAGAAGCACTAAGGAAAAAGAAAATTCTT GAGTACTGAGTCACAGGCTCAACATCTTAATAAAGTGTGTTTAGGATGTT TTAGTGGTATCTGGACACAGTATACCCATGAATCTCAAATATTTCATATC AGAGCTTTGCCCTTTCCATAGTAGGTCCTCCAAAAACATTCCTCAAACAA ATGAATTTTTTTTTTTTTTTTGAGATGGAGTCTAGCTCTGCTGCCCAGGC TGGAGTGTAGTGACGTGATCTCGGCTCACTGCAACCTCCACCTCCCGGGT TCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCAT GCACCACCACGCCCAGCTAATTTTTGGATTTTTATAGAGATGGGGTTTCA TCGTGTTACCCCGGATGGTCTTGATCTCCTGACCCCGTGATCCGCCCACC TCGGCCTCCCAAAGTGTTGGGATTACAGGCGTGAGCCACCACGCCCGGCC ACAGATGAATTTTTTGTTTTAAAGTTAAGAGATCAGGCACAGTGTCTCAC ACCTGTAATCCCAGCACTTTGGGAGGCCGAGGTGGGCAGATCGCTTGAGG TTGGGAGTTCGAAACCAGCCTGGCTAAAATGGTGAAACCTCATCTCCACA AAAAAAAAAAAAAAAAAATTAGCCAGGCATGGTGGTGCGTGCCTGTAATC CCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCCCGGAAGCG GAGGTTGCAGTGAGCTGAGATTGCACTACTGAACTCCAGCCTGGGCGTGA CAGAGTGATACTCCGTCTTGAAAAAAAAAAAAGTATTGGCAATAGAGTGA ACTCCACCTGGTTATCAATCCTAACTCCATCACTCACCAGCTCTGTGACT TGTTTAGTTATTTAATCTTTTTCAGTTTCAATGTCCTCATCTGGAAAACG GGAAATGCCTACCGAGTATTGAACTGTCAAGACTAAATGACACATGAAAA GCACACTGAACAATGCTTAATCCACGGTAGGCTTTCAGCAAATAGTGGTC TCTATTATTTTTTGCAACAGGATGTTATATAAGAATAAGGCATTATCAAG ACGATTTTCGCCTATCAAATGTGTTGTCTGCGTGTACATCAGACCTTTTC CTGGAATGAAGGTAGAAAAGTATCAACAATAAAGGATGGAGAAAGCCCCG TAAATGATAACCTTTGCCCCAGAGGGAAAGGATGGGCAGCGGAGGGGAAA GTAAAGGGTGGCAAGCTAGAAAAATTAGACATCCAGTGTGGGAGGGCTGG CACTGGAGTGGGTTGCCACAGTAGGGAGGGGACTCAGAGCTGGAGGCAAT TCCTTTGGCCGGGCTTGTCCTGCGACTTACCGTGGGGCAGCGCAATGTGG AGAGGCCTGGTAAAATGGCTGGGCAAGGGTGCGGAGGGGACATAACTGGC AGGAAGGAGTCATGATTCGTGGTCGAACAGAGTCCAGACCAGCTCGACCT GTGAGCAACGAACGGCCCTGAGACTCGCATACCCCAATACCGGTAGTGGC CGTGAAGGGCAAAGAAATGTGTTCTGAGGCGATCCCAGCATCTAAGCTGC GACTGGTCTACTCAGAGACTGGATGGAAGCTGGGAAGAGAAAGCTGCTTC CCGCTTCGGGGTGAGGGATGGAGGAAGGGAGAACAAGCAGTAGAGAAGAA AAAGTTTCAGATCCCACAGCCCCGGGGGGTCACTCCTGCTGGACCTACTC CGACCCCCTAGGGCCGGGAGTGAAGGCGGGACTTGTGCGGTTACCAGCGG AAATGCCTCGGGGTCAGAAGTCGCAGGAGAGATAGACAGCTGCTGAACCA ATGGGACCAGCGGATGGGGCGGATGTTATCTACCATTGGTGAACGTTAGA AACGAATAGCAGCCAATGAATCAGCTGGGGGGGGCGGAGCAGTGACGTTT ATTGCGGAGGGGGCCGCTTCGAATCGGCGGCGGCCAGCTTGGTGGCCTGG GCCAATGAACGGCCTCCAACGAGCAGGGCCTTCACCAATCGGCGGCCTCC ACGACGGGGCTGGGGGAGGGTATATAAGCCGAGTAGGCGACGGTGAGGTC GACGCCGGCCAAGACAGCACAGACAGATTGACCTATTGGGGTGTTTCGCG AGTGTGAGAGGGAAGCGCCGCGGCCTGTATTTCTAGACCTGCCCTTCGCC TGGTTCGTGGCGCCTTGTGACCCCGGGCCCCTGCCGCCTGCAAGTCGGAA ATTGCGCTGTGCTCCTGTGCTACGGCCTGTGGCTGGACTGCCTGCTGCTG SEQ ID NO.6: TTTTATAGAGATGGGGTTTTATCGTGTTATTTCGGATGGTTTTGATCTCCTGACCCCGTGATCCGCCCACCTCGGCCTCCCAAAGTGTTGGGATTACAGGCTTGATTTTTTGATTTCGTGATTCGTTTATTTCGGTTTTTTAAAGTGTTGGGATTATAGGCGTGAGCCACCACGCCCGGCCACAGATGAATTTTTTGTTTTAAAGTTAAGAGATCAGGCACGTGAGTTATTACGTTCGGTT SEQ ID NO.7: AGGAAGGAGTCATGATTCGTGGTCGAACAGAGTCCAGACCAGCTCGACCTTATAATTGGTAGGAAGGAGTTATGATTCGTGGTCGAATAGAGTTTAGATTAGTTCGATTTGTGAGCAACGAACGGCCCTGAGACTCGCATACCCCAATACCGGTAGTGGCCGTGAAGGGTGAGTAACGAACGGTTTTGAGATTCGTATATTTTAATATCGGTAGTGGTCGTGAAGGGTAAAGAAATGTGTTCTGAGGCGATCC SEQ ID NO.8: . Example 2

[0028] A diabetic nephropathy progression assessment kit based on detecting GRP78 gene promoter methylation The methylation-specific PCR primer combination for detecting GRP78 gene promoter methylation of Example 1, renal tissue paraffin section DNA extraction reagents including: protease, protease digestion buffer, nucleic acid binding beads, DNase, anhydrous ethanol; bisulfite DNA conversion reagent including: CT conversion reagent, Tris-saturated phenol, isopropanol, ddH2O; desulfonated spin column; DNA purification reagent including: DNA binding column, RNase, protease; methylation-specific PCR reagents including: DNA polymerase, DNase / RNase-Free ddH2O, dNTP Mixture, Reaction Buffer.

[0029] The method for detecting GRP78 promoter methylation in paraffin sections of human kidney tissue using the kit of the present invention comprises the following steps: 1. DNA template extraction and preparation (1) DNA extraction from paraffin sections of renal tissue DNA extraction from paraffin sections of renal tissue can be performed using conventional methods in the art.

[0030] This example provides an option: using the MagMAX™ FFPE DNA Extraction and Purification Kit. The specific process is as follows: a. Immerse the patient's paraffin sections in Citrisolv Clearing Agent or equivalent (xylene, other solvents) for 5 minutes, then in absolute ethanol for 5 minutes, air-dry for 15 minutes, and then add 4 µL of protease digestion buffer. Use a scalpel to transfer the tissue block to a sterile 1.5 mL collection tube and add 100 µL of protease solution.

[0031] b. Mix well and submerge the tissue, then centrifuge briefly. Incubate at 55°C for 1 hour, then centrifuge briefly. Incubate at 90°C for 1 hour, then centrifuge briefly to collect condensate droplets.

[0032] c. Add 150 µL of DNA magnetic bead binding buffer and mix for 5 minutes, then spin at 1000 rpm for 5 minutes. Place on a magnetic stand until the solution is clear and discard the supernatant.

[0033] d. Add 200µL DNA Wash Buffer to wash the magnetic beads.

[0034] e. Wash the beads by adding 200µL Wash Solution 2. Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0035] f. Add 50 µL of elution solution to the magnetic beads and shake at 1000 rpm for 5 minutes until the mixture turns completely chocolate brown. Place the sample on a magnetic stand for 2 minutes, or until the solution is clear and the beads rest against the magnet. Collect the supernatant, which contains the purified DNA. The purified DNA can be used immediately. Store long-term at -20°C or -80°C.

[0036] (2) Bisulfite DNA conversion a. Add 5µl of M-dilution buffer to 1µg of DNA sample and adjust the total volume to 50µl with water.

[0037] b. Incubate the sample at 37°C for 15 minutes, then add 100µl of CT conversion reagent.

[0038] c. Incubate the samples at 50°C in the dark for 12 hours.

[0039] d. Incubate the sample at 4°C for 10 minutes.

[0040] e. Add 400 µl of M-Binding Buffer to the Zymo Spin™ IC column and place the column in the provided collection tube.

[0041] f. Load the sample (from step d) into the Zymo Spin™ IC column, close the cap, and mix by inverting several times.

[0042] g. Centrifuge at full speed (10000g) for 30 seconds. Discard the supernatant. h. Add 100 µl of M-Wash buffer to the column. Centrifuge at full speed (10,000 g) for 30 seconds.

[0043] i. Add 200 µl of M-desulfurization buffer to the column and incubate at room temperature (20-30°C) for 20 minutes. After incubation, centrifuge at full speed (10,000 g) for 30 seconds.

[0044] j. Place the column in a 1.5 ml microcentrifuge tube. Add 10 µl of M Elution Buffer 1 and transfer directly to the column matrix. Centrifuge at full speed (10,000 g) for 30 seconds to elute the DNA. The DNA can be analyzed immediately or stored at -20°C or below for later use.

[0045] 2. Methylation-specific PCR The reaction system of the methylation-specific PCR reagent is as follows: DNA polymerase 0.5μL; DNase / RNase-Free ddH2O 1μL; dNTP mixture 0.5μL; upstream primer 1μL; downstream primer 1uL; sample DNA 1μL, Reaction Buffer 5μL; the total reaction system is 10uL, of which the concentration of upstream primer and downstream primer is 0.5μM.

[0046] The methylation-specific PCR reaction procedure is as follows: The annealing temperature of the methylated primers was 65°C. The amplification reaction program for the methylated primers was as follows: pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 45 seconds, 35 cycles, and a final extension at 72°C for 7 minutes.

[0047] The annealing temperature of the unmethylated primer was 65°C. The amplification reaction program for the unmethylated primer was as follows: pre-denaturation at 95°C for 10 minutes, denaturation at 95°C for 30 seconds, annealing at 65°C for 30 seconds, extension at 72°C for 45 seconds, 35 cycles, and final extension at 72°C for 7 minutes.

[0048] 3. Agarose gel electrophoresis Prepare 300 ml of 0.5x TBE electrophoresis buffer; gel concentration 2% (50 mL of 0.5x TBE plus 2.5 g of gel); dissolve the gel in a microwave oven on medium heat for 2 minutes; cool to 60°C and add 2 μl of ethidium bromide; place a comb, pour the plate, wait for solidification, and add 10 μL of PCR reaction product; electrophoresis at 130 V for 30 minutes; observe PCR product bands using a gel imager.

[0049] 4. Result judgment If the gene promoter is methylated, a band of corresponding size will appear when amplified with methylation-specific primers; if not, a band of corresponding size will appear when amplified with non-methylation-specific primers. Example 3

[0050] Application example: A diabetic nephropathy progression assessment kit based on the detection of GRP78 gene promoter methylation is used to detect early-stage diabetic nephropathy and late-stage diabetic nephropathy in healthy subjects.

[0051] Samples from 5 healthy individuals, 5 patients with early-stage diabetic nephropathy, and 5 patients with late-stage diabetic nephropathy were tested using the kit and method of Example 2. Samples 1 to 5 were normal controls, samples 6 to 10 were early-stage diabetic nephropathy, and samples 11 to 15 were late-stage diabetic nephropathy.

[0052] Each sample was also tested by RT-PCR in parallel. The specific operation steps were carried out according to the standard steps of the selected kit. The primers for human GRP78 mRNA amplification are shown in Table 3.

[0053] Table 3

[0054] The primers for amplifying the human housekeeping gene GAPDH are shown in Table 4.

[0055] Table 4

[0056] The results showed (see Figure 2 and Figure 3 In the normal control group, GRP78 expression was low, while the GRP78 promoter methylation rate was high. However, GRP78 expression was high, while the GRP78 promoter methylation rate was low, in renal tissues of patients with early-stage and late-stage diabetic nephropathy. This suggests that as diabetic nephropathy progresses, GRP78 promoter methylation decreases and promoter activity increases, leading to elevated GRP78 transcriptional expression. Therefore, the methylation status of the GRP78 gene promoter methylation island in renal tissue DNA can serve as a marker for diabetic nephropathy progression, providing aided diagnosis and early warning of disease progression, and possesses important clinical significance.

[0057] It should be noted that the above content merely illustrates the technical idea of ​​the present invention and cannot be used to limit the scope of protection of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications all fall within the scope of protection of the claims of the present invention.

Claims

1. A methyl-specific PCR primer pair for detecting methylation of the GRP78 gene promoter in renal tissue, characterized in that: It comprises a pair of methylated primers and a pair of non-methylated primers, the nucleotide sequences of the methylated primers are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the non-methylated primers are shown as SEQ ID NO.3 and SEQ ID NO.

4.

2. A kit for evaluating the progression of diabetic nephropathy, characterized in that: The method comprises a pair of methylated primers and a pair of non-methylated primers as claimed in claim 1.

3. A kit for evaluating the progression of diabetic nephropathy according to claim 2, characterized in that: The methylation primer and the non-methylation primer are a PCR primer combination for detecting the methylation of the GRP78 gene promoter in renal tissues in a methylation-specific and non-methylation-specific reaction.

4. Use of a methyl-specific PCR primer pair for detecting methylation of the GRP78 gene promoter in renal tissue in the preparation of a kit for evaluating the progression of diabetic nephropathy, characterized in that: The primer pair comprises a pair of methylated primers and a pair of unmethylated primers as claimed in claim 1.

5. Use of a methyl-specific PCR primer pair for detecting methylation of the GRP78 gene promoter in renal tissue according to claim 4 in preparing a kit for evaluating the progression of diabetic nephropathy, characterized in that: The methylated region of the GRP78 gene promoter is from 3000 bp upstream to 200 bp downstream of the GRP78 gene promoter sequence, as shown in the sequence of SEQ ID NO.

5.

6. Use of a methyl-specific PCR primer pair for detecting methylation of the GRP78 gene promoter in renal tissue according to claim 5 in preparing a kit for evaluating the progression of diabetic nephropathy, characterized in that: The sequences of the methylated sites of the GRP78 gene promoter are shown in SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8.

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