DNA quantitative detection method based on real methylation level and application of DNA quantitative detection method in preparation of bladder cancer detection kit

Abstract

The invention provides a DNA quantitative detection method based on a real methylation level and application of the DNA quantitative detection method in preparation of a bladder cancer detection kit. The method comprises the following steps: (1) DNA purification: purifying a DNA sample by adopting a sodium acetate method; (2) DNA oxidation: oxidizing the DNA purified in the step (1) by using a potassium perruthenate solution and a neutralizing oxidant; (3) DNA transformation: transforming the DNA oxidized in the step (2) by using a methylation detection sample pretreatment kit; and (4) qRT-PCR detection of the DNA: detecting the DNA converted in the step (3) by using a DNA methylation qRT-PCR mixed system to complete quantitative detection of the DNA. The method provided by the invention is simple and convenient to operate and high in specificity, can effectively remove interference of hydroxymethylation and truly reflect DNA methylation filling, and is suitable for early diagnosis, recurrence prediction and the like of bladder cancer.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and in particular to a method for quantitative detection of DNA based on true methylation levels and its application in the preparation of bladder cancer detection kits. Background Technology

[0002] Urinary bladder cancer (UBC) is one of the most common malignant tumors of the urinary system, causing a large number of cancer-related deaths worldwide. Approximately 75% of bladder cancers are non-muscle-invasive (NMIBC). Although early intervention is possible through transurethral resection of bladder tumor (TURBT), the recurrence rate is as high as 60-80%, requiring lifelong follow-up. Currently, clinical practice mainly relies on cystoscopy for recurrence monitoring. While it is the gold standard, it suffers from problems such as high invasiveness, poor patient compliance, and high cost, and its sensitivity is insufficient, with a missed diagnosis rate of 10-40%. Urine cytology, while an auxiliary tool, has poor sensitivity for low-grade tumors. Therefore, developing an accurate, efficient, and non-invasive method for bladder cancer screening and diagnosis is an urgent clinical problem to be solved.

[0003] DNA methylation is a common epigenetic modification that primarily occurs on cytosine (C) bases, particularly in the CpG dinucleotide region, playing a crucial role in regulating gene expression, cell differentiation, and tumorigenesis and development. In contrast, DNA hydroxymethylation, an intermediate product of DNA demethylation, is often considered to activate gene expression; its biological function in regulating gene transcription shows an opposite trend to DNA methylation. Recent studies have shown that abnormal DNA methylation patterns are widespread in various tumor tissues and body fluids, including bladder cancer, and are closely related to tumor occurrence, development, and prognosis. Therefore, accurate DNA methylation levels have significant potential as biomarkers for early tumor diagnosis, subtyping, and prediction.

[0004] Quantitative real-time PCR (qRT-PCR), a highly sensitive and specific nucleic acid quantification technique, has wide applications in gene expression analysis and pathogen detection. While qRT-PCR can be used for DNA methylation detection, its application has certain limitations. Traditional methods convert unmethylated cytosine to uracil through sulfite treatment, followed by PCR amplification using methylation-specific primers, enabling the detection of methylation status at specific CpG sites. However, this method cannot distinguish between methylation (5mC) and hydroxymethylation (5hmC), and it requires high sample quality and primer design, limiting its widespread clinical application.

[0005] However, a detection technology that can accurately remove hydroxymethylation interference and achieve true quantification of methylation levels is currently lacking. Pyrosequencing is one of the most widely used methods for quantifying DNA methylation, but it has several limitations, such as high requirements for template DNA quality, limited sensitivity, and the inability to effectively distinguish between methylation and hydroxymethylation signals, leading to overestimation or underestimation of methylation levels and resulting in biased results. Furthermore, pyrosequencing instruments are expensive, the operation is complex, the throughput is low, and the cycle time is long, hindering its widespread clinical application.

[0006] In summary, there is currently no quantitative detection technology for true DNA methylation that can effectively remove hydroxymethylation interference, while also possessing high sensitivity, ease of operation, and suitability for clinical application. Existing detection methods, such as pyrosequencing, have significant shortcomings in terms of sensitivity, specificity, and clinical applicability. Therefore, developing a simple, accurate detection method that truly reflects DNA methylation levels is of significant research importance and has broad clinical application prospects, especially in the early screening and precise diagnosis of tumors such as bladder cancer. Summary of the Invention

[0007] The technical problem that this invention aims to solve is that existing detection technologies have significant shortcomings in terms of sensitivity, specificity, and clinical applicability.

[0008] To address the aforementioned technical problems, in a first aspect, the present invention provides a method for quantitative detection of DNA based on true methylation levels, the method comprising the following steps:

[0009] (1) DNA purification: The DNA sample was purified using the sodium acetate method;

[0010] (2) DNA oxidation: The DNA purified in step (1) was oxidized using potassium perruthenate solution and a neutralizing oxidant;

[0011] (3) DNA conversion: The DNA oxidized in step (2) was converted using a methylation detection sample pretreatment kit;

[0012] (4) qRT-PCR detection of DNA: The DNA transformed in step (3) was detected using a DNA methylation qRT-PCR mixture system to complete the DNA quantification.

[0013] The method provided by this invention first extracts DNA, then purifies the extracted DNA using the sodium acetate method. Next, a specific oxidation step is used to selectively remove hydroxymethylation modifications from the DNA, and residual oxidants are neutralized with Na₂SO₃ to protect the target methylation sites from damage. The treated DNA sample is then subjected to sulfite conversion to further distinguish between methylated and unmethylated cytosine. Finally, the treated sample is detected by quantitative real-time PCR (qRT-PCR) to achieve quantitative analysis of the true methylated DNA level. The method provided by this invention is simple to operate, highly specific, and can effectively remove the interference of hydroxymethylation, accurately reflecting DNA methylation loading. It is suitable for early diagnosis, efficacy evaluation, and recurrence prediction of bladder cancer, and has broad clinical application prospects.

[0014] Preferably, the DNA is derived from urine DNA.

[0015] In this invention, urinary DNA is generally obtained from the morning urine sample of the relevant patient, using Quick-DNA. TM Extraction was performed using the Urine Kit. The specific procedure followed the kit's instructions. The procedure was as follows: Centrifuge the urine sample at 1500g for 15 min, discard the supernatant, and retain 100-400 μl. Add an equal volume of Urine Pellet Digestion Buffer and resuspend the precipitate. Add 5% Proteinase K and vortex or pipette to mix, then briefly incubate in a metal bath at 55°C for 30 min. Add an equal volume of Genomic Lysis Buffer and vortex to mix. Transfer all liquid to a Zymo-Spin bath. TM Centrifuge at 16000g for 1 min in an IC-S column. Add 200 μl of Urine DNA Prep Buffer to the column and centrifuge at 16000g for 1 min. Add 700 μl of Urine DNA Wash Buffer to the column and centrifuge at 16000g for 1 min. Add 200 μl of Urine DNA Wash Buffer to the column and centrifuge at 16000g for 1 min. After drying the column, add 25 μl of preheated DNA Elution Buffer and centrifuge at 16000g for 2 min to obtain urine DNA. Quantify and analyze the DNA concentration and OD 260 / 280 values ​​using a quorum analyzer.

[0016] Before DNA purification, this invention groups and records the DNA concentrations separately. Samples with a total DNA mass of no more than 1 μg are recorded as low-concentration DNA samples, while those with a total mass of more than 1 μg are recorded as medium- to high-concentration DNA samples.

[0017] Preferably, the oxidation in step (2) is as follows: DNA is added to enzyme-free sterile water, sodium hydroxide is added, the DNA is heated and then cooled, potassium perruthenate and a neutralizing oxidant are added and mixed for incubation.

[0018] This invention introduces potassium perruthenate oxidation for selective removal of 5hmC, solving the key problem of traditional methods' inability to distinguish between 5mC and 5hmC. Combined with an optimized DNA purification process and qRT-PCR detection system, the accuracy and sensitivity of methylation detection are significantly improved. This makes the technology particularly suitable for clinical applications, especially demonstrating advantages in the early non-invasive diagnosis of bladder cancer. Although the application of qRT-PCR in methylation detection is not yet fully widespread, this invention, through technological innovation, effectively expands its practical value in this field.

[0019] Preferably, the neutralizing oxidant is sodium sulfite.

[0020] In this invention, Na₂SO₃ is added to the oxidation reaction system to react with potassium perruthenate, neutralizing its strong oxidizing properties and thus reducing damage to the DNA structure. When only an oxidant is added to the system, the DNA recovery rate decreases significantly, while the introduction of Na₂SO₃ significantly improves the DNA recovery efficiency, ensuring the accuracy of downstream analysis.

[0021] Preferably, the mass ratio of the DNA to sodium hydroxide, potassium perruthenate, and neutralizing oxidant is (7-10):(1-3):(4-6), and more preferably 8:2:5.

[0022] In this invention, the concentration of potassium perruthenate is correlated with the efficiency and degree of DNA oxidation. When the concentration of potassium perruthenate oxidant reaches 10×, the DNA sample shows the most complete oxidation effect, with clear enzyme digestion bands, indicating a sufficient oxidation reaction.

[0023] The concentration of Na2SO3 has a significant impact on DNA bisulfite transformation and the sensitivity of qRT-PCR detection. When the sodium sulfite concentration is set at 10 mM, the Ct value of the Actin gene is consistently below 30, which is within the effective detection range of the qRT-PCR internal control, indicating that this concentration is most suitable.

[0024] Preferably, the mixing system in step (4) includes primer probes, DNA templates, water, DNA polymerase, and a PCR reaction system.

[0025] Preferably, the DNA template includes any one or a combination of at least two of VIM, ONECUT2, SIX6, TBX15, RSPH9, or H4C6.

[0026] Preferably, the detection in step (4) involves placing the mixed system on a real-time PCR instrument for qRT-PCR reaction, recording and calculating the Ct value after the reaction, and completing the DNA quantification detection.

[0027] Preferably, the qRT-PCR reaction is performed as follows: pre-deformation at 95°C for 1 min; deformation at 95°C for 20 s and annealing and extension at 57°C for 30 s, with 45 cycles of deformation and annealing / extension.

[0028] In this invention, the pre-deformation cycle is 1 time, and the deformation and annealing extension are performed as a whole, with 45 cycles.

[0029] Secondly, the present invention provides an application of the method described in the first aspect in the preparation of a bladder cancer detection kit.

[0030] The method provided by this invention, based on biomarkers for bladder cancer detection, can determine whether a urine sample comes from a bladder cancer patient or a non-bladder cancer patient. It is applicable to the early diagnosis, efficacy evaluation, and recurrence prediction of bladder cancer and has broad clinical application prospects.

[0031] Implementing this invention has the following beneficial effects:

[0032] The method provided by this invention can effectively remove hydroxymethylation interference and truly reflect the DNA methylation status, thereby providing reliable technical means and data support for in-depth research on the role of DNA methylation in the occurrence and development of bladder cancer, as well as for achieving early screening and accurate diagnosis of tumors. Attached Figure Description

[0033] Figure 1 This is a comparison chart of the redox experimental results of this invention.

[0034] Figure 2 This is a comparative graph showing the relationship between potassium perruthenate concentration and DNA oxidation efficiency and damage level according to the present invention.

[0035] Figure 3 This is a graph showing the experimental results of the effect of sodium sulfite concentration on DNA bisulfite conversion and qRT-PCR detection sensitivity in this invention.

[0036] Figure 4 This is a graph showing the detection level of VIM gene DNA methylation in urine samples according to the present invention. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] After obtaining informed consent from the study participants, morning urine samples were collected from UBC patients and placed in 50 mL centrifuge tubes. Quick-DNA was then added. TM After centrifuging the urine preservation solution in the Urine Kit, discard the supernatant and collect the precipitate.

[0039] Urine DNA Extraction

[0040] Using Quick-DNA TM Using the Urine Kit (Zymo), extract DNA according to the kit instructions. Centrifuge urine samples at 1500g for 15 min, discard the supernatant, and retain 100-400 μl. Add an equal volume of Urine Pellet Digestion Buffer and resuspend the precipitate. Add 5% Proteinase K and vortex or pipette to mix, briefly incubate in a metal bath at 55°C for 30 min. Add an equal volume of Genomic Lysis Buffer and vortex to mix. Transfer all liquid to Zymo-Spin. TM Centrifuge at 16000g for 1 min in an IC-S column. Add 200 μl of Urine DNA Prep Buffer to the column and centrifuge at 16000g for 1 min. Add 700 μl of Urine DNA Wash Buffer to the column and centrifuge at 16000g for 1 min. Add 200 μl of Urine DNA Wash Buffer to the column and centrifuge at 16000g for 1 min. After drying the column, add 25 μl of preheated DNAElution Buffer and centrifuge at 16000g for 2 min to obtain urine DNA. Quantify and analyze the DNA concentration and OD 260 / 280 values ​​using a quorum analyzer.

[0041] Example

[0042] (1) DNA purification

[0043] Two DNA purification methods were used to purify DNA.

[0044] DNA sample purification using the sodium acetate method: A portion of the extracted DNA sample was added to 0.1 volumes of 3M sodium acetate, mixed thoroughly, and then 2.5 volumes of anhydrous ethanol were added. The mixture was further mixed and incubated at -20°C for 1 hour to promote DNA precipitation. Subsequently, the sample was centrifuged at 16000g for 20 minutes, the supernatant was discarded, and the precipitate was washed with pre-cooled 70% ethanol. The precipitate was then centrifuged again at 16000g for 5 minutes to completely remove residual ethanol. Finally, 30 μL of Milli-Q ultrapure water was added to dissolve the DNA precipitate. The resulting solution was used for subsequent DNA concentration detection.

[0045] (2) DNA oxidation

[0046] Take 500 ng of DNA and bring the volume to 21.75 μl with enzyme-free sterile water (RNase-free water). Add 1.25 μl of 1M NaOH and incubate in a metal bath at 37°C for 30 min. Place the sample on ice for 3 min to cool rapidly. Then add 2 μl of oxidizing reagent (150 mM potassium perruthenate solution) to the tube, gently tap to mix, and briefly centrifuge. Incubate at 40°C for 10 min.

[0047] Next, add 7.5 μl of 0.1 M Na2SO3, a DNA neutralizing agent, to the DNA sample, mix well, centrifuge at 16000 r for 5 minutes, and transfer the supernatant to a new EP tube.

[0048] (3) DNA transformation

[0049] DNA samples were transformed using a methylation detection sample pretreatment kit (CWY 122M, Jiangsu Kangwei Century Biotechnology Co., Ltd.) according to the kit instructions. Before processing each DNA sample, 100 μL of transformation buffer CR, 5 μL of buffer ND, and 25 μL of buffer NP were added to a new EP tube. The tube was gently inverted to mix and then briefly centrifuged. Next, 20 μL of the DNA sample to be tested was added to a new PCR tube, followed by 130 μL of pre-mixed transformation buffer. After mixing, the tube was placed in a PCR instrument for transformation. The program was set as follows: incubation at 90°C for 10 minutes, followed by reaction at 54°C for 1 hour, and finally held at 4°C. After transformation, the product was purified by column purification: 200 μL of column equilibration buffer PS was added to the adsorption column already loaded into the collection tube, and the column was centrifuged at 12000 rpm for 1 minute, discarding the waste liquid. Then, 600 μL of buffer CL was added, and all the transformation product was transferred to this buffer. After mixing, the column was allowed to stand at room temperature for 10 minutes, then centrifuged at 12000 rpm for 1 minute, discarding the waste liquid. Add 500 μL of wash buffer 1, 200 μL of buffer DB, 500 μL of wash buffer 2, and 200 μL of wash buffer WB sequentially, centrifuging at 12000 rpm for 1 minute each time. Except for the DB elution step, no waste liquid needs to be discarded. Then, centrifuge the adsorption column and collection tube again at 12000 rpm for 2 minutes to remove residual ethanol. Transfer the adsorption column to a new clean collection tube, open the cap, and let it air dry at room temperature for 3 minutes. Finally, carefully add 30 μL of elution buffer to the center of the adsorption column membrane (avoid touching the membrane surface with the pipette tip; replace the pipette tip for each sample), incubate at room temperature for 2 minutes, then centrifuge at 12000 rpm for 1 minute to collect the transformed DNA for later use. It is recommended to preheat the elution buffer at 65-70℃ to improve recovery rate. The modified DNA should be used as soon as possible. If storage is necessary, it should be kept below -20℃ and the storage time should not exceed one month. For long-term storage, it is recommended to keep it at -70℃ or lower and avoid repeated freeze-thaw cycles.

[0050] (4) qRT-PCR detection of DNA

[0051] The system was prepared according to the instructions of Kangwei Century Biotechnology Co., Ltd.'s DNA methylation qRT-PCR Mix (CW3332M) as shown in Table 1 below:

[0052] Table 1

[0053]

[0054]

[0055] After preparation, mix well and centrifuge briefly. Then, place the mixture on an ABI 7500 real-time PCR instrument for qRT-PCR reaction. The reaction program is shown in Table 2 below:

[0056] Table 2

[0057]

[0058] After the qRT-PCR reaction, the Ct values ​​of each sample were recorded and calculated. If the Ct value of the target gene was not higher than 43, the gene was considered to be methylated positive in the sample, and the ΔCt value of each gene was calculated as Ct(target) - Ct(mACTB), and then normalized.

[0059] Comparative experiment

[0060] This invention includes comparative experiments. During the DNA oxidation process, additional comparative experiments were conducted, including no oxidation and no reduction, no oxidation but only reduction, and oxidation but no reduction. The experimental results are as follows: Figure 1 As shown.

[0061] This invention includes a comparative experiment to clarify the relationship between potassium perruthenate concentration and DNA oxidation efficiency and damage level. Different concentration gradients were used for comparative experiments, and the results were verified by Taq I enzyme digestion. The experimental results are as follows: Figure 2 As shown.

[0062] Depend on Figure 2 It can be seen that when the concentration of oxidant reaches 10×, the oxidation effect of DNA sample is the most complete, and the enzyme digestion bands are clear, indicating that the oxidation reaction is sufficient.

[0063] Considering the significant impact of sodium sulfite concentration on DNA bisulfite conversion and qRT-PCR detection sensitivity, this study further optimized the neutralizing agent concentration and used an Actin methylation-specific probe to detect its Ct value. Specific experimental results are as follows: Figure 3 As shown.

[0064] Figure 3 Experimental results show that when the sodium sulfite concentration is set at 10 mM, the Ct value of the Actin gene is consistently below 30, which is within the effective detection range of the qRT-PCR internal reference, indicating that this concentration is the most suitable.

[0065] This invention further investigates the applications of this detection. The methylation level of the VIM gene DNA was detected in urine samples from healthy individuals, patients with benign urinary tract diseases (inflammation or benign tumors), and patients with bladder cancer (UBC). Specific results are as follows... Figure 4 As shown.

[0066] Figure 4 The results show that the true DNA methylation of this invention can effectively detect bladder cancer.

[0067] Experimental Example

[0068] Urine samples were collected from 15 healthy individuals, 15 patients with benign urinary tract diseases (such as cystitis), and 61 patients with bladder cancer from a hospital in Xuzhou. The resulting DNA was extracted, purified, oxidized, and transformed. The methylation levels of the VIM (sequence 1), ONECUT2 (sequence 2), SIX6 (sequence 3), TBX15 (sequence 4), RSPH9 (sequence 5), and H4C6 (sequence 6) genes were then detected. The PCR positive / negative counts for each of the six genes in the four sample types were statistically analyzed. The area under the lines (AUC), sensitivity, and specificity were calculated using SPSS software. The results are shown in Table 3. The sensitivity and specificity for all six genes were greater than 80%.

[0069] Table 3

[0070]

[0071]

[0072]

[0073] Furthermore, the methylation levels of the six genes obtained from the three sets of primers for each subject were summed, and the sensitivity and specificity of each primer combination were calculated. The results are shown in Table 4.

[0074] Table 4

[0075] Primer combination Gene AUC value Sensitivity Specificity Primer combination 1 VIM, ONECUT2, SIX6, TBX15, RSPH9, H4C6 0.967 0.901 0.912 Primer combination two VIM, ONECUT2, SIX6, TBX15, RSPH9, H4C6 0.953 0.916 0.895 Primer combination three VIM, ONECUT2, SIX6, TBX15, RSPH9, H4C6 0.942 0.891 0.872

[0076] The results in Table 4 show that the detection sensitivity and specificity are both high.

[0077] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for DNA quantification based on real methylation level, characterized in that, The method comprises the following steps: (1) DNA purification: purifying the sample of DNA by sodium acetate method; (2) DNA oxidation: oxidizing the DNA purified in step (1) by using potassium perruthenate solution and neutralizing oxidant; (3) DNA transformation: transforming the DNA oxidized in step (2) by using methylation detection sample pretreatment kit; (4) qRT-PCR detection of DNA: detecting the DNA transformed in step (3) by using DNA methylation qRT-PCR mixed system, and completing DNA quantitative detection.

2. The method of claim 1, wherein, The source of the DNA is urine DNA.

3. The method of claim 1, wherein, In step (2), the oxidation is: adding DNA into enzyme-free sterile water, then adding sodium hydroxide, heating, then cooling the DNA, and then adding potassium perruthenate and neutralizing oxidant for mixed incubation.

4. The method of claim 3, wherein, The neutralizing oxidant is sodium sulfite.

5. The method of claim 3, wherein, The mass ratio of the DNA, sodium hydroxide, potassium perruthenate and neutralizing oxidant is (7-10):(1-3):(4-6).

6. The method of claim 1, wherein, The mixed system in step (4) comprises primer probe, DNA template, water, DNA polymerase and PCR reaction system.

7. The method of claim 1, wherein, The DNA template comprises any one or a combination of at least two of VIM, ONECUT2, SIX6, TBX15, RSPH9 or H4C6.

8. The method of claim 1, wherein, The detection in step (4) is: placing the mixed system on a fluorescence quantitative PCR instrument to perform qRT-PCR reaction, recording Ct value and calculating after the reaction, and completing DNA quantitative detection. The procedure of the qRT-PCR reaction is: 95℃ pre-denaturation for 1 min, 95℃ denaturation for 20 s and 57℃ annealing and extension for 30 s, denaturation and annealing and extension cycle for 45 times.

9. Application of the method according to any one of claims 1-8 in preparing a bladder cancer detection kit.

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