Primer probe group, detection reagent and detection kit for detecting glycogen synthase kinase 3 gene

Abstract

The invention relates to the technical field of biological detection, in particular to a primer probe group, a detection reagent and a detection kit for detecting glycogen synthase kinase 3 genes. The primer probe group of the GSK-3beta gene provided by the invention is high in specificity, the amplification performance of the detection reagent is excellent, the kit provided by the invention has the advantages of high specificity, high sensitivity and high accuracy when being used for rapidly identifying GSK-3beta in a kidney lesion tissue sample or urine, the stable detection lower limit is 0.05 ng / mu L, and the kit can be used for rapidly identifying the GSK-3beta in the kidney lesion tissue sample or urine. In addition, interference of other types of common pathogens on experimental results can be avoided, and a reliable experimental basis is provided for GSK-3beta identification.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a primer probe set, a detection reagent and a detection kit for detecting glycogen synthase kinase 3 gene. Background Art

[0002] Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine / threonine enzyme present in all eukaryotic organisms. GSK-3β plays an important role in physiological processes such as cell differentiation, growth, proliferation, apoptosis, motility, cell cycle continuity, embryonic development, and insulin response. Studies have shown that GSK-3β has dual roles as a tumor suppressor and promoter.

[0003] Given the crucial role of GSK-3β expression in diabetic kidney disease, the development of a nucleic acid assay for GSK-3β expression has significant clinical implications for identifying sensitive biomarkers for early cancer diagnosis and developing better treatments. Currently, there are no commercially available reagents for identifying GSK-3β gene expression related to diabetic kidney disease, and GSK-3β targets are primarily used for regulatory expression and non-diabetic kidney disease applications. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a primer probe set, a detection reagent and a detection kit for detecting the glycogen synthase kinase 3 gene. The primer probe set, the detection reagent and the detection kit provided by the present invention can specifically detect the expression level of the GSK-3β gene related to diabetic kidney disease, and can provide guidance to patients with diabetic kidney disease through the difference in expression levels.

[0005] The present invention provides a primer set for detecting the GSK-3β gene, comprising at least one of primer sets 1 to 3;

[0006] The primer set 1 has:

[0007] (1), the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2; or

[0008] (2) a complementary sequence of the nucleotide sequence shown in (1); or

[0009] (3) A nucleotide sequence obtained by substituting, deleting or adding one or more bases from the nucleotide sequence shown in (1) or (2), and having the same or similar functions as the nucleotide sequence shown in (1) or (2); or

[0010] (4) a nucleotide sequence that is at least 80% identical to the nucleotide sequence of (1), (2) or (3); and / or

[0011] The primer set 2 has:

[0012] (5) the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4; or

[0013] (6) a complementary sequence of the nucleotide sequence shown in (5); or

[0014] (7) A nucleotide sequence obtained by substituting, deleting or adding one or more bases from the nucleotide sequence shown in (5) or (6), and having the same or similar functions as the nucleotide sequence shown in (5) or (6); or

[0015] (8) a nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (5), (6) or (7); and / or

[0016] The primer set 3 has:

[0017] (9) the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; or

[0018] (10), a complementary sequence of the nucleotide sequence shown in (9); or

[0019] (11) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (9) or (10), and having the same or similar functions as the nucleotide sequence shown in (9) or (10); or

[0020] (12) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (9), (10) or (11).

[0021] In some embodiments, a primer set for an internal standard gene is further included, wherein the internal standard gene includes any one of RNaseP, 18S and GAPDH genes;

[0022] Preferably, the internal standard gene is the RNaseP gene, and the primer set for the RNaseP gene has:

[0023] (13), the nucleotide sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8; or

[0024] (14), a complementary sequence of the nucleotide sequence shown in (13); or

[0025] (15) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (13) or (14), and having the same or similar function as the nucleotide sequence shown in (13) or (14); or

[0026] (16) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (13), (14) or (15).

[0027] The plurality is 2 to 5.

[0028] The primer set provided by the present invention using the conserved sequence of the GSK-3β gene as a detection marker can specifically detect the target without non-specific amplification, thereby achieving a more accurate technical effect.

[0029] In some embodiments, the primer set includes primer set 1 and primer set 4.

[0030] Experimental verification shows that, compared with other primer sets, the primer set 1 provided by the present invention has better reaction specificity, higher amplification efficiency, higher sensitivity, and is more suitable for the specific detection of the GSK-3β gene, thereby obtaining a more accurate technical effect.

[0031] The present invention also provides a probe having:

[0032] (17) a nucleotide sequence as shown in any one of SEQ ID NO: 9 to SEQ ID NO: 12; or

[0033] (18), a complementary sequence of the nucleotide sequence shown in (17); or

[0034] (19) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (17) or (18), and having the same or similar function as the nucleotide sequence shown in (17) or (18); or

[0035] (20) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (17), (18) or (19).

[0036] In some specific embodiments of the present invention, the two ends of the probe are modified with a fluorescent group and a quenching group, respectively. The fluorescent group includes at least one of FAM, VIC, ROX, Cy5 and A425, and the quenching group includes at least one of BHQ1, BHQ2, BHQ2 and MGB.

[0037] In some embodiments, the 5' end of the probe of the nucleic acid sequence shown in SEQ ID NO: 9 is modified with a FAM group, and the 3' end is modified with a BHQ1 group.

[0038] In some embodiments, the 5' end of SEQ ID NO: 12 is modified with a CY5 group, and the 3' end is modified with a BHQ2 group.

[0039] The present invention provides a primer-probe set, which comprises the primer set for detecting the GSK-3β gene and the probe.

[0040] The present invention provides the primer set for detecting the GSK-3β gene, the probe and / or the use of the primer probe set in preparing a detection reagent and / or a detection kit for the GSK-3β gene.

[0041] The present invention provides a detection reagent or a detection kit, comprising the primer set for detecting the GSK-3β gene, the probe and / or the primer-probe set, and an acceptable auxiliary agent, carrier and / or device.

[0042] In some embodiments, the method comprises a PCR reaction solution 1 and a PCR reaction solution 2, wherein the PCR reaction solution 1 comprises the primer set for detecting the GSK-3β gene, the probe and / or the primer-probe set;

[0043] Based on a total system volume of 15 μL, the PCR reaction solution 1 includes: 20-100 pmol of Tricine at pH 7.9-8.8, 0.1-1 μmol of the primer set, 0.05-0.3 μmol of the probe, 50-200 pmol of KOAc, 1 vol%-5 vol% of glycerol, 0.1-0.5 mmol of dNTPs, and 2-10 U of a bifunctional DNA polymerase;

[0044] Based on a total system volume of 15 μL, the concentration of manganese acetate in the PCR reaction solution 2 is 1 to 5 mmol.

[0045] In other embodiments, the PCR reaction solution 1 further includes 0.001 vol% to 0.002 vol% of Tween 20 and 1 vol% to 2 vol% of DMSO.

[0046] In some specific embodiments, based on a total system volume of 15 μL, the PCR reaction solution 1 includes: 50 pmol of Tricine at pH 8.3, 0.2 μmol of the primer set, 0.1 μmol of the probe, 100 pmol of KOAc, 2 vol% of glycerol, 0.2 mmol of dNTPs, 2 U of rTth enzyme, 0.0012 vol% of Tween 20, and 1 vol% of DMSO;

[0047] Based on a total system volume of 15 μL, the concentration of manganese acetate in the PCR reaction solution 2 is 2 mmol.

[0048] In some embodiments, the detection reagent or detection kit further includes a negative quality control and a positive quality control.

[0049] The present invention provides a detection method, comprising: taking the primer set for detecting the GSK-3β gene, the probe, the primer-probe set and / or the detection reagent or detection kit to perform PCR amplification on a sample to be tested to obtain a detection result;

[0050] The PCR amplification reaction system includes: based on a total system volume of 15 μL, 14 μL of the PCR reaction solution, 24 μL of the PCR reaction solution, and 7 μL of the nucleic acid of the sample to be tested;

[0051] The PCR amplification program included: 60° C. for 30 min, 95° C. for 2 min, (95° C. for 10 s, 60° C. for 22 s)×45 cycles.

[0052] In some specific embodiments, the test results are judged as follows: the Ct value of the CY5 channel of the negative control product is ≤35, and the FAM channel displays No Ct; the Ct value of the FAM channel of the positive control product is ≤30, and the Ct value of the CY5 channel is ≤30.

[0053] The present invention provides a primer probe set, a detection reagent and a detection kit for detecting the glycogen synthase kinase 3 gene. The primer probe set for the GSK-3β gene provided by the present invention has high specificity, and the detection reagent has excellent amplification performance. The kit provided by the present invention can quickly identify GSK-3β in diseased tissue or urine, and has the advantages of high specificity, high sensitivity and high accuracy. The detection limit that can be stably detected is 0.05ng / μL, and it can avoid the interference of other types of common pathogens on the experimental results, providing a reliable experimental basis for the identification of GSK-3β. The present invention is equipped with an extraction reagent for storage and transportation at room temperature, which is convenient to use. It can be stored for 12 months at 2-8℃, avoiding the influence of repeated freezing and thawing on the performance of the reagent. The experimental results show that the detection performance of the kit provided by the present invention after 21 days of acceleration at 37℃ is basically the same as that at 2-8℃ for 0 days. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Shown is the melting curve peak diagram of Example 4;

[0055] Figure 2 Graph showing comparative amplification results of adding additives to the amplification system in Example 5;

[0056] Figure 3 Schematic diagram of GSK-3β amplification in Example 6;

[0057] Figure 4 A linear graph is shown between the GSK-3β target Ct value and the human genome concentration Log value in Example 9;

[0058] Figure 5 A linear plot is shown between the internal standard Ct value and the human genome concentration Log value in Example 9;

[0059] Figure 6 The figure shows the simulation verification bar graph in Example 10;

[0060] Figure 7 14. A bar chart comparing clinical test data in Example 11 is shown. DETAILED DESCRIPTION

[0061] The present invention provides a primer probe set, a detection reagent, and a detection kit for detecting the glycogen synthase kinase 3 gene. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the desired results. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein without departing from the content, spirit, and scope of the present invention to implement and apply the technology of the present invention.

[0062] The nucleotide sequences of the primers and probes for GSK-3β and human internal reference in the technical solution of the present invention are SEQ ID NOs: 1 to 15.

[0063] In the kit provided by the present invention, the PCR reaction solution 1 contains PCR reaction buffer, 20-100 pmol Tricine (PH7.9-8.8), 50-200 pmol KOAc, 1%-5% Glycerol, 0.001%-0.002% Tween 20, 1%-2% DMSO, 0.1-0.5 mmol dNTPs, 2U-10U bifunctional DNA polymerase, 0.1-1 μmol nucleotide primer sequence, and 0.05-0.3 μmol probe sequence; the PCR reaction solution 2 contains 1-5 mmol Mn(CH3COO)2.

[0064] In the technical solution of the present invention, the total amount of the system refers to PCR reaction solution 1+PCR reaction solution 2+sample amount.

[0065] In some specific embodiments, the total volume of the system is 15 μL.

[0066] The amplification system includes: 14 μL of PCR reaction solution, 24 μL of PCR reaction solution, and 7 μL of nucleic acid of the sample to be tested.

[0067] The amplification program included: 60°C for 30 min, 95°C for 2 min, and (95°C for 10 s, 60°C for 22 s) × 45 cycles.

[0068] The detection method for determining the expression level of GSK-3β in other tumor tissues established based on the methodology of the present invention has the advantages of high throughput, high sensitivity, high specificity, and full automation, provides an efficient solution for identifying GSK-3β targets, and has important clinical significance.

[0069] The present invention adopts the method of fluorescence quantitative PCR. Compared with the method of probe hybridization, the method of fluorescence quantitative PCR is simple to operate, has direct feedback results, and is relatively accurate. The hybridization method requires more equipment, is complex to operate, and is costly. The results are judged by the color depth of the hybridization band, which is subjective.

[0070] The present invention uses the relative expression of the GSK-3β gene and the human internal reference gene to interpret the results. The relative expression of the internal reference gene is constant, and the expression of the target gene GSK-3β relative to the internal reference gene is more direct and reliable. Compared with the method of hybridization of the GSK-3β target, ① there are many hybridization steps, and the amplification product is easily lost in the intermediate steps, resulting in relatively inaccurate results; ② hybridization requires opening the lid after amplification is completed in special equipment, which is very likely to cause contamination of the amplification product in the intermediate step.

[0071] In the prior art, the association between the GSK-3β target and disease is based on a comparison of the expression levels of the same target in the subject and control samples. The selection of the control sample is crucial and greatly affects the determination of the target expression level in the subject. In the present invention, the relative expression level difference between GSK-3β and the internal reference gene is selected. Both the target and the internal reference are from the same person, so the result feedback is direct. The control is the average value of a large number of healthy people, and the result is relatively reliable.

[0072] At the same time, compared with the existing technology of protein biomarkers, the detection method provided by the present invention is different from the immune method. In theory, the gene expression level in the patient cannot be directly inferred based on the protein expression level.

[0073] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention will be further described below with reference to the examples.

[0074] Example 1

[0075] This embodiment provides a fluorescent PCR kit for identifying GSK-3β, which specifically includes the following components:

[0076] (1) PCR reaction solution 1: The base solution is 50 pmol Tricine (pH 8.3), a total of 4 μL, including 0.2 μmol primer, 0.1 μmol probe, 100 pmol KOAc, 2% Glycerol, 0.2 mmol dNTPs, and 2UrTth enzyme.

[0077] The target primers are:

[0078] Primer 1: 5′-TCTTCCGACCCCGAACTCC-3′ (SEQ ID NO: 1);

[0079] Primer 2: 5′-GGGCAGTTGGTGTATACTCCAGC-3′ (SEQ ID NO: 2);

[0080] Primer 3: 5'-GGAAGCGAGGAGGAGCCG-3' (SEQ ID NO: 3);

[0081] Primer 4: 5′-GTGCCCGCTCAGGAAGTGT-3′ (SEQ ID NO: 4);

[0082] Primer 5: 5′-TCTCACTTTGAGGGAGATCCAGG-3′ (SEQ ID NO: 5);

[0083] Primer 6: 5′-AGATTGTATGTTCTAGTGCTCCGCTT-3′ (SEQ ID NO: 6);

[0084] Primer 7: 5′-CAAGGTGACAACAGTGGTGGC-3′ (SEQ ID NO: 13);

[0085] Primer 8: 5′-ACCAAATGATCCATTTCCAATCACT-3′ (SEQ ID NO: 14);

[0086] The internal standard primers are:

[0087] Primer 3: 5′-AGATTTGGACCTGCGAGCG-3′ (SEQ ID NO: 7);

[0088] Primer 4: 5'-GAGCGGCTGTCTCCACAAGT-3' (SEQ ID NO: 8).

[0089] The target probes are:

[0090] Probe 1: 5′-CGGAGGCAATTGCACTGTGTAGCC-3′ (SEQ ID NO: 9);

[0091] Probe 2: 5′-AGGGCTCTGGGCTGGGGCAAA-3′ (SEQ ID NO: 10);

[0092] Probe 3: 5'-TGGGAGGGGTTGTGGGGAGGG-3' (SEQ ID NO: 11);

[0093] Probe 4: 5'-CTGGGCAGGGTCCAGACAGGCC-3' (SEQ ID NO: 15);

[0094] The internal standard probe is:

[0095] Probe 5: 5'-TTCTGACCTGAAGGCTCTGCGCG-3' (SEQ ID NO: 12).

[0096] PCR reaction solution 2: The base solution is H2O, totaling 4 μL, including 2 mmol Mn(CH3COO)2.

[0097] (2) Negative control: The base solution is TE solution, 1000 μL, containing human RNaseP gene plasmid.

[0098] Positive quality control: The basic solution is TE solution (10mM Tris-HCl, 1mM EDTA, pH=8.0), 1000μL, containing GSK-3β plasmid and human RNaseP gene plasmid.

[0099] Example 2

[0100] This example provides the operating steps of the kit of the present invention for identifying GSK-3β:

[0101] 1. Reagent Preparation

[0102] According to the number of samples to be tested (nucleic acid extraction or purification reagents, nucleic acid extraction or purification reagents are from Antu Biotechnology Co., Ltd., Yuzheng Xiebei 20180037) and positive and negative quality control products, prepare the corresponding amount of reaction solution (14 μL / person for PCR reaction solution, 24 μL / person for PCR reaction solution). If the total amount is N, prepare N + 1 PCR reaction solutions and mix them for later use.

[0103] 2. Template extraction

[0104] Follow the instructions of the nucleic acid extraction and purification kit.

[0105] 3. Polymerase chain reaction

[0106] Prepare the following reaction system in a PCR tube: 8 μL of PCR reaction mix (PCR reaction mix 1 + PCR reaction mix 2) and 7 μL of template. Perform amplification on a real-time fluorescence PCR system. The amplification program is: 60°C for 30 min, 95°C for 2 min (95°C for 10 s, 60°C for 22 s, 45 cycles).

[0107] 4. Results and calculations

[0108] Judgment of test results: The Ct value of the CY5 channel of the negative quality control product is ≤35, and the FAM channel shows No Ct; the Ct value of the positive quality control product (a synthetic plasmid containing the GSK-3β target gene segment (SEQ ID NO:16) and the internal standard gene segment (SEQ ID NO:17)) is ≤30 in the FAM channel and ≤30 in the CY5 channel.

[0109] The Ct values ​​of the target and internal standard results were calculated and compared using the 2^-△△Ct analysis method, where △Ct = target - internal standard, and △△Ct = △Ct of the experimental group - △Ct of the control group; the control group is the mean Ct value of normal subjects.

[0110] Example 3: GSK-3β primer probe screening

[0111] The combination of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 9 was used as Scheme 1; the combination of SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 10 was used as Scheme 2; the combination of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 11 was used as Scheme 3; the combination of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15 was used as Scheme 4; the combination of SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 12 was used as internal standard primers and probes, and the human genome was used as the amplification template. Reaction solution 1 and reaction solution 2 were as described in Example 1. The comparison results are shown in Table 1 below.

[0112] Table 1. GSK-3β primer and probe screening

[0113] Solution 1 Option 2 Option 3 Option 4 GSK-3β 22.26 24.36 25.68 NoCt GSK-3β 22.1 24.18 26.01 NoCt GSK-3β 22.08 24.48 25.77 NoCt

[0114] Among them, in schemes 1 to 4, the 5' end of the probe is modified with a FAM group, and the 3' end is modified with a BHQ1 group.

[0115] The internal standard probe was modified with a CY5 group at its 5' end and a BHQ2 group at its 3' end.

[0116] Conclusion: The Ct value of scheme 1 combination is the best compared with other schemes, and the primer-probe pair of scheme 1 combination is preferred.

[0117] The sequences of the amplified fragments corresponding to the primers in Scheme 1 are as follows:

[0118]

[0119] The amplified fragment sequences corresponding to the internal reference gene are as follows:

[0120]

[0121]

[0122] Example 4. Specificity

[0123] Using human genome as template, SYBR-GREEN method was used to make melting curve and amplification detection. The results were as follows: Figure 1 shown.

[0124] Conclusion: The melting curve has a single peak without abnormal protrusions, indicating that the primer pair is specific for amplification.

[0125] Example 5: Additive Scheme

[0126] The amplification system was added with 0.001%-0.002% Tween 20 and 1%-2% DMSO and the amplification without additives was compared. The results are as follows Figure 2 (0.0012% Tween 20 and 1% DMSO were added to the amplification system).

[0127] Conclusion: Compared with the control group, the amplification height signal value of the additive scheme increased by 1000, and the Ct value difference was within ±1.5.

[0128] Example 6. Sensitivity test

[0129] The original concentration of human genome Nanodrop was 69 ng / μL and then tested after gradient dilution. The data are shown in Table 2 and Figure 3 The detection limit verification results are shown in Table 3.

[0130] Table 2 Sensitivity gradient results

[0131] Detection target GSK-3β Internal standard Sample concentration FAMCt Cy5Ct 69 ng / μL 18.45 18.97 69 ng / μL 18.82 19 10 ng / μL 21.8 21.63 10 ng / μL 21.75 21.75 1ng / μL 25.26 25.65 1ng / μL 25.09 24.91 0.5 ng / μL 25.62 26.12 0.5 ng / μL 25.72 26.29 0.1 ng / μL 28.58 28.38 0.1 ng / μL 28.76 28.57 0.05 ng / μL 30.11 30.43 0.05 ng / μL 29.79 30.03 0.01 ng / μL 35.38 34.2 0.01 ng / μL No Ct 33.59

[0132] Conclusion: The detection limit of this kit is 0.01ng / μL, and there are cases of missed detection.

[0133] Table 3. Detection limit verification results

[0134]

[0135] Conclusion: The detection limit of the kit can be stably detected at 0.05ng / μL.

[0136] Example 7. Precision Test

[0137] According to the above method, 10 replicate wells were tested for the median and low values, and the results are shown in Table 4.

[0138] Table 4. Precision test results

[0139]

[0140] Conclusion: The precision CV of this kit is less than 5%.

[0141] Example 8. Stability test

[0142] Human genomic samples were diluted to 0.5 ng / μL, 0.1 ng / μL, and 0.05 ng / μL, respectively. The prepared reagents were placed in a 37°C incubator for accelerated stability verification. The test results were compared with those from the day 0 test. The final Ct deviation was small, indicating stable test results. The results were considered to be indistinguishable, thus meeting the requirements. The results are shown in Table 5.

[0143] Table 5 Stability test results

[0144]

[0145] Conclusion: The test results showed little difference after 21 days of accelerated storage at 37°C. The reagent can be stored for 12 months at 2-8°C, avoiding the effects of repeated freeze-thaw cycles on the performance of the reagent.

[0146] Example 9. Comparison of target and internal standard amplification slopes

[0147] The human genome samples were diluted in a gradient manner, and the target detection Ct value and the internal standard detection Ct value were plotted against the human genome concentration ( Figure 4 It is a linear graph between the GSK-3β target Ct value and the human genome concentration Log value. Figure 5 (a linear plot between the internal standard Ct value and the human genome concentration Log value) and the amplification slopes were compared. The results are shown in Table 6.

[0148] Table 6. Target internal standard Ct values ​​and genome concentrations

[0149]

[0150]

[0151] Conclusion: Based on the above results, the sensitivity of the GSK-3β reagent can detect a human genome concentration of 0.05 ng / μL, the target detection slope k = -0.2824, the internal standard detection slope k = -0.2847, the target and internal standard amplification efficiency k values ​​are basically the same, and the Ct value comparison analysis between the target and internal standard can be performed.

[0152] Example 10. Clinical simulation verification

[0153] Different concentrations of target plasmid and internal standard plasmid were used to simulate the amplification Ct values ​​of the target and internal standard in the sample. The amplified Ct values ​​were analyzed using the 2^-△△Ct method to establish the amplification trend. The Ct values ​​of the target and internal standard in the control group were selected to be closer to the Ct values ​​of the human genome. The data are shown in Table 7. The simulation verification bar chart is shown in Figure 6 shown.

[0154] Table 7.2^-△△Ct method data model verification

[0155]

[0156] Conclusion: According to the above target-internal standard plasmid simulation data, the expression difference between the experimental group and the control group was obvious, and the methodology was feasible.

[0157] Example 11. Clinical Validation

[0158] Fifteen clinical renal puncture lesion tissue samples were used for amplification detection using the above kit. The data are shown in Table 8 and Figure 7 shown.

[0159] Table 8. Clinical test data

[0160]

[0161] Conclusion: In clinically sampled patients, the expression of GSK-3β targets was elevated to varying degrees, and this target has a reference value for detection guidance in patients with diabetic kidney disease.

[0162] The above are only preferred embodiments of the present invention. It should be pointed out that 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 should also be regarded as the scope of protection of the present invention.

Claims

1. A primer set for detecting the GSK-3β gene, characterized in that: including at least one of primer sets 1 to 3; The primer set 1 has: (1), the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2; or (2) a complementary sequence of the nucleotide sequence shown in (1); or (3) A nucleotide sequence obtained by substituting, deleting or adding one or more bases from the nucleotide sequence shown in (1) or (2), and having the same or similar functions as the nucleotide sequence shown in (1) or (2); or (4) a nucleotide sequence that is at least 80% identical to the nucleotide sequence of (1), (2) or (3); and / or The primer set 2 has: (5) the nucleotide sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4; or (6) a complementary sequence of the nucleotide sequence shown in (5); or (7) A nucleotide sequence obtained by substituting, deleting or adding one or more bases from the nucleotide sequence shown in (5) or (6), and having the same or similar functions as the nucleotide sequence shown in (5) or (6); or (8) a nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (5), (6) or (7); and / or The primer set 3 has: (9) the nucleotide sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; or (10), a complementary sequence of the nucleotide sequence shown in (9); or (11) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (9) or (10), and having the same or similar functions as the nucleotide sequence shown in (9) or (10); or (12) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (9), (10) or (11).

2. The primer set according to claim 1, characterized in that Also included is a primer set for an internal standard gene, wherein the internal standard gene includes any one of RNaseP, 18S and GAPDH genes; Preferably, the internal standard gene is the RNaseP gene, and the primer set for the RNaseP gene has: (13), the nucleotide sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8; or (14), a complementary sequence of the nucleotide sequence shown in (13); or (15) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (13) or (14), and having the same or similar function as the nucleotide sequence shown in (13) or (14); or (16) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (13), (14) or (15).

3. A probe, characterized in that It has: (17) a nucleotide sequence as shown in any one of SEQ ID NO: 9 to SEQ ID NO: 12; or (18), a complementary sequence of the nucleotide sequence shown in (17); or (19) A nucleotide sequence obtained by replacing, deleting or adding one or more bases of the nucleotide sequence shown in (17) or (18), and having the same or similar function as the nucleotide sequence shown in (17) or (18); or (20) A nucleotide sequence that is at least 80% identical to the nucleotide sequence shown in (17), (18) or (19).

4. A primer probe set, characterized in that: The method comprises the primer set for detecting the GSK-3β gene according to claim 1 or 2 and the probe according to claim 3.

5. Use of the primer set for detecting the GSK-3β gene according to claim 1 or 2, the probe according to claim 3, and / or the primer-probe set according to claim 4 in preparing a detection reagent and / or a detection kit for the GSK-3β gene.

6. A detection reagent or a detection kit, characterized in that: The method comprises the primer set for detecting the GSK-3β gene according to claim 1 or 2, the probe according to claim 3 and / or the primer-probe set according to claim 4, and acceptable auxiliary agents, carriers and / or devices.

7. The detection reagent or detection kit according to claim 6, characterized in that Comprising PCR reaction solution 1 and PCR reaction solution 2, wherein the PCR reaction solution 1 comprises the primer set for detecting the GSK-3β gene according to claim 1 or 2, the probe according to claim 3 and / or the primer-probe set according to claim 4; The PCR reaction solution 1 comprises: 20-100 pmol of Tricine at pH 7.9-8.8, 0.1-1 μmol of the primer set, 0.05-0.3 μmol of the probe, 50-200 pmol of KOAc, 1 vol%-5 vol% of glycerol, 0.1-0.5 mmol of dNTPs, and 2-10 U of a bifunctional DNA polymerase; The concentration of manganese acetate in the PCR reaction solution 2 is 1-5 mmol.

8. The detection reagent or detection kit according to claim 7, characterized in that The PCR reaction solution 1 further includes 0.001 vol% to 0.002 vol% of Tween 20 and 1 vol% to 2 vol% of DMSO.

9. The detection reagent or detection kit according to any one of claims 6 to 8, characterized in that The detection reagent or detection kit also includes negative quality control and positive quality control.

10. A detection method, characterized in that include: The primer set for detecting the GSK-3β gene according to claim 1 or 2, the probe according to claim 3, the primer-probe set according to claim 4 and / or the detection reagent or detection kit according to any one of claims 6 to 9 are used to perform PCR amplification on the sample to be tested to obtain the test result.

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