Kit for carrying out sulfite conversion on nucleic acid

Abstract

The invention discloses a kit for carrying out sulfite conversion on nucleic acid. The invention provides a reagent for carrying out sulfite conversion on nucleic acid. The reagent comprises sulfite conversion liquid; the sulfite conversion liquid contains sodium metabisulfite with the final concentration of 0.4-0.8 g / L and ammonium hydrogen sulfite with the mass percent of 10%-20%; the pH value of the sulfite conversion liquid is 5-6. Sodium metabisulfite and ammonium bisulfite are selected as sulfite, the cost is low, the research and development cost of an enterprise can be effectively reduced, large-scale industrial production is promoted, and the method is more suitable for the development requirement of the market. Besides, the temperature of the conversion procedure step in the kit using method is adjusted, so that the whole conversion reaction time is only 70 minutes, the preparation efficiency is high, the preparation time is shorter, and the product is long in preservation time, low in price and stable in supply.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a kit for the sulfite conversion of nucleic acids. Background Technology

[0002] DNA methylation refers to the covalent bonding of a methyl group to the 5' carbon position of the cytosine in the 5'-CpG-3' dinucleotide of the genome under the action of DNA methyltransferases, forming 5-methylcytosine (5-mC), which accounts for 2-5% of all cytosine residues. DNA methylation is closely related to human development, gene transcription regulation, genetic imprinting, and tumor diseases. In particular, abnormal methylation of CpG islands is associated with the transcriptional inactivation of tumor suppressor genes. A CpG-rich segment of DNA in the genome is called a CpG island, and CpGs often exist in clusters, with a length of about 1-2 kb. To date, studies have found that CpG hypermethylation in more than 1,000 genes is associated with cancer. Therefore, methylation of specific genes in cancer cells can serve as a biomarker for early cancer diagnosis. Methylation can serve as a biomarker and prognostic indicator for early diagnosis of tumors, and is of great significance for tumor screening and risk assessment, early diagnosis, staging and typing, prognosis, and treatment monitoring.

[0003] DNA methylation analysis typically requires the modification of DNA by sulfurization. Extracted free DNA is treated with a sulfurizing agent, causing unmethylated cytosine to deaminate into uracil, while methylated cytosine remains unchanged. PCR using specific primers and probes then yields the methylation status and level of the target gene.

[0004] Most commercially available methylation sulfidation methods currently use high-concentration sodium bisulfite as the sulfiding agent. High-concentration sodium bisulfite is extremely difficult to dissolve, inconvenient to prepare, and time-consuming. Furthermore, its sulfidation strength is weak, requiring a long sulfidation time, generally more than 3 hours or even overnight. Chinese patent application CN116445582A discloses a nucleic acid sulfidation conversion kit that uses extremely low-cost sodium metabisulfite and adjusts the temperature of the conversion procedure steps in the kit's usage, shortening the entire conversion reaction time to 130 minutes. However, the conversion reaction time is still somewhat long. Further shortening the conversion time while maintaining conversion efficiency without increasing costs would be of great significance. Summary of the Invention

[0005] The purpose of this invention is to provide a reagent for the sulfite conversion of nucleic acids.

[0006] In a first aspect, the present invention claims a reagent for the sulfite conversion of nucleic acids.

[0007] The reagents for sulfite conversion of nucleic acids claimed in this invention include sulfite conversion solutions; The sulfite conversion solution contains sodium metabisulfite at a final concentration of 0.4-0.8 g / L and ammonium bisulfite at a mass percentage of 10-20%. The pH value of the sulfite conversion solution is 5-6.

[0008] Furthermore, the solvent of the sulfite conversion solution is water, and the solute and concentration are as follows: 0.4-0.8 g / L sodium metabisulfite, 10-20% ammonium bisulfite.

[0009] Specifically, in the first embodiment of the present invention, the solvent of the sulfite conversion solution is water, and the solute and concentration are as follows: 0.4 g / L sodium metabisulfite and 10% ammonium bisulfite by mass; pH is 5. In the second embodiment of the present invention, the solvent of the sulfite conversion solution is water, and the solute and concentration are as follows: 0.6 g / L sodium metabisulfite and 15% ammonium bisulfite by mass; pH is 5.5. In the third embodiment of the present invention, the solvent of the sulfite conversion solution is water, and the solute and concentration are as follows: 0.8 g / L sodium metabisulfite and 20% ammonium bisulfite by mass; pH is 6.

[0010] Furthermore, the reagent may also include a binding solution. The binding solution contains 0.01-0.05 M Tris-HCl, 0.06-0.08 M EDTA, 2-4 M guanidine hydrochloride, 0.5%-1% CTAB by mass, and 5%-15% sodium chloride by mass; the pH of the binding solution is 6-8.

[0011] Furthermore, the solvent of the binding solution is water, and the solutes and concentrations are as follows: 0.01-0.05M Tris-HCl, 0.06-0.08M EDTA, 2-4M guanidine hydrochloride, 0.5%-1% CTAB by mass, and 5%-15% sodium chloride by mass.

[0012] Specifically, in the first embodiment of the present invention, the solvent of the binding solution is water, and the solutes and concentrations are as follows: 0.01M Tris-HCl, 0.06M EDTA, 2M guanidine hydrochloride, 0.5% CTAB by mass, and 5% sodium chloride by mass; pH is 6. In the second embodiment of the present invention, the solvent of the binding solution is water, and the solutes and concentrations are as follows: 0.03M Tris-HCl, 0.07M EDTA, 3M guanidine hydrochloride, 0.75% CTAB by mass, and 10% sodium chloride by mass; pH is 7. In the third embodiment of the present invention, the solvent of the binding solution is water, and the solutes and concentrations are as follows: 0.05M Tris-HCl, 0.08M EDTA, 4M guanidine hydrochloride, 1% CTAB by mass, and 15% sodium chloride by mass; pH is 8.

[0013] Furthermore, the reagent may also include an incubation solution. The incubation solution is a NaOH solution, specifically an aqueous solution containing 1-5% NaOH by mass; the pH value of the incubation solution is 7-9.

[0014] Specifically, in the first embodiment of the present invention, the incubation solution is a NaOH solution, specifically a 1% (by mass) aqueous solution of NaOH, with a pH of 7. In the second embodiment of the present invention, the incubation solution is a NaOH solution, specifically a 3% (by mass) aqueous solution of NaOH, with a pH of 8. In the third embodiment of the present invention, the incubation solution is a NaOH solution, specifically a 5% (by mass) aqueous solution of NaOH, with a pH of 9.

[0015] Furthermore, the reagent may also include a rinsing solution. The rinsing solution is an aqueous solution of ethanol with a volume percentage of 60-80%.

[0016] Specifically, in the first embodiment of the present invention, the rinsing solution is a 60% (v / v) aqueous ethanol solution. In the second embodiment of the present invention, the rinsing solution is a 70% (v / v) aqueous ethanol solution. In the third embodiment of the present invention, the rinsing solution is an 80% (v / v) aqueous ethanol solution.

[0017] Furthermore, the reagent may also include an eluent; the eluent is prepared by mixing the rinsing solution and anhydrous ethanol in a volume ratio of 1:3.

[0018] Secondly, the present invention claims a kit for the sulfite conversion of nucleic acids.

[0019] The kit for sulfite conversion of nucleic acids claimed in this invention may include the reagents described in the first aspect above.

[0020] Furthermore, the kit may also contain a purification column.

[0021] Thirdly, the present invention claims protection for the use of the reagents described in the first aspect above or the kits described in the second aspect above in any of the following: (A1) Sulfite conversion of nucleic acids; (A2) Perform methylation analysis on nucleic acids.

[0022] Fourthly, the present invention claims a method for sulfite conversion of nucleic acids.

[0023] The method for sulfite conversion of nucleic acids claimed in this invention is to use the reagents described in the first aspect above or the kits described in the second aspect above to perform sulfite conversion of nucleic acids, characterized in that: the reaction procedure for conversion is: 90-100℃, 10 min; 58.5-60.5℃, 1 h; and incubation at 2-5℃.

[0024] Furthermore, the method may include the following steps: Prepare the eluent before use by adding anhydrous ethanol to the rinsing solution.

[0025] S1. Add 6-8 times the volume of deionized water to the sulfite conversion solution described in the first aspect above, and shake at room temperature for 10 minutes; S2. Add the solution prepared in S1 to the nucleic acid sample to be transformed; S3. After shaking and mixing the system treated in S2, carry out the conversion reaction according to the following procedure: 90-100℃, 10min; 58.5-60.5℃, 1h; keep warm at 2-5℃. S4. Place the purification column in the collection tube and add the binding solution described in the first aspect above to the purification column; S5. Transfer the reaction solution obtained in S3 to the purification column after adding the binding solution in S4, mix the reaction solution and the binding solution, then centrifuge and discard the filtrate, and put the purification column back into the collection tube. S6. Add the washing solution described in the first aspect above to the purification column after S5 treatment, then centrifuge and discard the filtrate, and put the purification column back into the collection tube; S7. Add the incubation solution described in the first aspect above to the purification column after S6 treatment, let it stand at room temperature for 10 minutes, centrifuge and discard the filtrate, and put the purification column back into the collection tube. S8. Add the washing solution described in the first aspect above to the purification column after S7 treatment, centrifuge and discard the filtrate, and put the purification column back into the collection tube. S9. Add the washing solution described in the first aspect above to the purification column after S8 treatment. After centrifugation, take the purification column and put it into a new centrifuge tube. Let it stand at room temperature for 2 minutes. S10. Add the elution buffer described in the first aspect above to the purification column after S9 treatment, incubate at room temperature for 5 min, centrifuge and discard the purification column to obtain the transformed sample.

[0026] Furthermore, in S3, the procedure for carrying out the conversion reaction is as follows: 95°C for 10 min; 59.5°C for 1 h; and holding at 3°C.

[0027] Furthermore, in S5 to S10, the centrifugation is performed at 13800g for 1-2 minutes.

[0028] More specifically, the above method includes the following steps: Prepare the eluent before use by adding 75 mL of anhydrous ethanol to 25 mL of rinsing solution.

[0029] S1. Take one vial of the sulfite conversion solution (150 μL) described in the first aspect above from the kit, add 6-8 times the amount of deionized water, and shake at room temperature for 10 min; S2. Equilibrate the nucleic acid sample to be transformed to room temperature in advance, then add it to a 0.2 mL PCR tube, and then add 120 µL of the solution prepared in S1 to the tube; S3. After mixing the reagents in S2, vortex to mix thoroughly and place in a 96-well PCR instrument. The transformation programs are as follows: Transformation Program 1: 90-100℃ (e.g., 95℃), 10 min; Transformation Program 2: 58.5-60.5℃ (e.g., 59.5℃), 1 h; Transformation Program 3: 2-5℃ (e.g., 3℃), incubate. After the programs are completed, proceed with the subsequent steps or store at 4℃ for no more than 6 h. S4. Place the purification column in the collection tube and add 400µL of the binding solution described in the first aspect above to the purification column; S5. Transfer the reaction solution obtained in step S3 to the purification column after adding the binding solution in step S4, and mix the reaction solution and the binding solution by pipetting or inverting 8-10 times. Centrifuge at 12000 rpm (equivalent to 13800 g) for 1 min, discard the filtrate, and put the purification column back into the collection tube; S6. Add 100µL of the washing solution described in the first aspect above to the purification column, centrifuge at 12000rpm (equivalent to 13800g) for 1min, discard the filtrate, and put the purification column back into the collection tube; S7. Add 200µL of the incubation solution described in the first aspect above to the purification column, let it stand at room temperature for 10 min, centrifuge at 12000 rpm (equivalent to 13800 g) for 1 min, discard the filtrate, and put the purification column back into the collection tube; S8. Add 200µL of the washing solution described in the first aspect above to the purification column, centrifuge at 12000rpm (equivalent to 13800g) for 1min, discard the filtrate, and put the purification column back into the collection tube; S9. Add 200µL of the washing solution described in the first aspect above to the purification column, centrifuge at 12000rpm (equivalent to 13800g) for 2min, and place the purification column into a new 1.5mL centrifuge tube and let it stand at room temperature for 2min. S10. Add 100µL of the elution buffer described in the first aspect above to the purification column, incubate at room temperature for 5 min, centrifuge at 12000 rpm (equivalent to 13800 g) for 1 min, discard the purification column, and obtain the transformed sample, which is stored at -20℃±5℃.

[0030] Furthermore, in some embodiments of the present invention, the reagents in the nucleic acid conversion kit in step S1 can convert 10 samples at a time. If not used up, they can be stored at -20℃±5℃ and can continue to be used within one month without affecting the effectiveness of the kit.

[0031] Furthermore, in some embodiments of the present invention, the volume of the nucleic acid sample to be transformed added in step S2 ranges from 20 to 50 µL. If the sample DNA concentration is low, the maximum sample volume should not exceed 50 µL. If the sample DNA concentration is high, less than 20 µL should be made up with enzyme-free purified water.

[0032] Furthermore, in some embodiments of the present invention, the nucleic acid sample to be converted is derived from cells, bacteria, blood, saliva, urine, or pleural or peritoneal fluid.

[0033] Furthermore, in some embodiments of the present invention, the nucleic acid is preferably deoxyribonucleic acid (DNA).

[0034] The beneficial effects of this invention are: (1) In this invention, sodium metabisulfite and ammonium bisulfite are selected as sulfites, which are low in cost and can effectively reduce the enterprise's R&D costs and promote large-scale industrial production. (2) The present invention has adjusted the temperature and solution ratio of the conversion procedure steps in the method of using the kit. Compared with similar products on the market, it has greatly shortened the reaction time of the whole experiment, so that the whole conversion reaction time is only 70 minutes and the preparation efficiency is high. Attached Figure Description

[0035] Figure 1 The results are obtained by gel electrophoresis detection of DNA transformed by the kits of the present invention (Example 2), Comparative Example 1, and Comparative Example 2. A shows the PCR amplification results of unconverted genomic DNA (gDNA) using gGAPDH_245 as primer (the limit of detection is 0.01 ng); B shows the PCR amplification results of BT DNA (converted using the kit of this invention) using gGAPDH_245 as primer; C shows the PCR amplification results of BT DNA (converted using the ZYMO kit) using gGAPDH_245 as primer; D shows the PCR amplification results of BT DNA (converted using the Novizan kit) using gGAPDH_245 as primer; E shows the PCR amplification results of unconverted genomic DNA (gDNA) using ACTB_334 as primer; F shows the PCR amplification results of BT DNA (converted using the kit of this invention) using ACTB_334 as primer; G shows the PCR amplification results of BT DNA (converted using the ZYMO kit) using ACTB_334 as primer; H shows the PCR amplification results of BT DNA (converted using the Novizan kit) using gGAPDH_245 as primer. PCR amplification results of DNA using ACTB_334 as primer. The values ​​marked in each figure represent the amount of DNA loaded. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] Example 1: Sulfidation conversion treatment of nucleic acid samples using the kit and operating method of the present invention. The nucleic acid sulfidation conversion reagent in this embodiment includes sulfite conversion solution, binding solution, incubation solution and rinsing solution.

[0039] The solvent for the sulfite conversion solution is water, and the solutes and their concentrations are as follows: 0.4 g / L sodium metabisulfite and 10% ammonium bisulfite by mass; the pH is 5.

[0040] The solvent of the binding solution is water, and the solutes and concentrations are as follows: 0.01M Tris-HCl, 0.06M EDTA, 2M guanidine hydrochloride, 0.5% CTAB by mass, and 5% sodium chloride by mass; pH is 6.

[0041] The incubation solution is a NaOH solution, which is an aqueous solution with a NaOH mass percentage of 1% and a pH of 7.

[0042] The rinsing solution is an aqueous solution of ethanol with a volume percentage of 60%.

[0043] The above-mentioned nucleic acid sulfurization conversion reagent was used to transform 0% and 100% quality control samples (0% quality control sample refers to cell line DNA with a theoretical methylation level of 0; 100% quality control sample refers to cell line DNA with a theoretical methylation level of 100; source of quality control sample: 0% quality control sample, EpiScope® Non-Methylated HCT116 gDNA; 100% quality control sample, EpiScope® Methylated HCT116 gDNA; quality control sample mentioned below has the same meaning as here) into nucleic acids. The specific operation steps are as follows: Prepare the eluent before use by adding 75 mL of anhydrous ethanol to 25 mL of rinsing solution.

[0044] S1. Take one vial of the sulfite conversion solution (150 μL) from the kit, add 900 µL of deionized water, and shake at room temperature for 10 min; S2. Equilibrate the 0% and 100% quality control samples to room temperature beforehand, then add them to a 0.2 mL PCR tube, and then add 120 µL of the solution prepared in step S1 to the tube; S3. After mixing the reagents in S2, vortex to mix thoroughly and place in a 96-well PCR instrument. The transformation program is as follows: 95℃, 10 min; 59.5℃, 1 h; 3℃, incubate. After the program is complete, proceed with subsequent steps or store at 4℃ for no more than 6 h. S4. Place the purification column in the collection tube and add 400µL of the binding solution to the purification column.

[0045] S5. Transfer the reaction solution obtained in step S3 to the purification column in S4, and use a pipette to mix the reaction solution and binding solution by agitation or inverting the container 8-10 times. Centrifuge at 12000 rpm (equivalent to 13800 g) for 1 min, discard the filtrate, and return the purification column to the collection tube.

[0046] S6. Add 100µL of the wash solution to the purification column, centrifuge at 12000rpm (equivalent to 13800g) for 1min, discard the filtrate, and put the purification column back into the collection tube.

[0047] S7. Add 200µL of the incubation solution to the purification column, let it stand at room temperature for 10 min, then centrifuge at 12000rpm (equivalent to 13800g) for 1 min, discard the filtrate, and put the purification column back into the collection tube.

[0048] S8. Add 200µL of the wash solution to the purification column, centrifuge at 12000rpm (equivalent to 13800g) for 1min, discard the filtrate, and put the purification column back into the collection tube.

[0049] S9. Add 200µL of the wash solution to the purification column, centrifuge at 12000rpm (equivalent to 13800g) for 2min, transfer the purification column to a new 1.5mL centrifuge tube, and incubate at room temperature for 2min.

[0050] S10. Add 100µL of the elution buffer to the purification column, incubate at room temperature for 5 min, centrifuge at 12000rpm (equivalent to 13800g) for 1 min, discard the purification column, and obtain the transformed sample. Store at -20℃±5℃.

[0051] Example 2: Sulfidation conversion treatment of nucleic acid samples using the kit and operating method of the present invention. The nucleic acid sulfidation conversion reagent in this embodiment includes sulfite conversion solution, binding solution, incubation solution and rinsing solution.

[0052] The solvent for the sulfite conversion solution is water, and the solutes and their concentrations are as follows: 0.6 g / L sodium metabisulfite and 15% ammonium bisulfite by mass; the pH is 5.5.

[0053] The solvent of the binding solution is water, and the solutes and concentrations are as follows: 0.03M Tris-HCl, 0.07M EDTA, 3M guanidine hydrochloride, 0.75% CTAB by mass, and 10% sodium chloride by mass; the pH is 7.

[0054] The incubation solution is a NaOH solution, which is an aqueous solution with a NaOH mass percentage of 3% and a pH of 8.

[0055] The rinsing solution is an aqueous solution of ethanol with a volume percentage of 70%.

[0056] The DNA sample and DNA transformation process were performed in accordance with Example 1, with the only difference being that 1050 μL of deionized water was added in step S1.

[0057] Example 3: Using the kit and operating method of the present invention to perform sulfurization conversion treatment on nucleic acid samples. The nucleic acid sulfidation conversion reagent in this embodiment includes sulfite conversion solution, binding solution, incubation solution and rinsing solution.

[0058] The solvent for the sulfite conversion solution is water, and the solutes and their concentrations are as follows: 0.8 g / L sodium metabisulfite and 20% ammonium bisulfite by mass; the pH is 6.

[0059] The solvent of the binding solution is water, and the solutes and concentrations are as follows: 0.05M Tris-HCl, 0.08M EDTA, 4M guanidine hydrochloride, 1% CTAB by mass, and 15% sodium chloride by mass; pH is 8.

[0060] The incubation solution is a NaOH solution, which is an aqueous solution with a NaOH mass percentage of 5% and a pH of 9.

[0061] The rinsing solution is an ethanol aqueous solution with a volume percentage of 80%.

[0062] The DNA sample and DNA transformation process were performed in accordance with Example 1, with the only difference being that 1200 μL of deionized water was added in step S1.

[0063] Comparative Example 1 The 0% and 100% quality control samples (as above) were converted using the commercially available ZYMO conversion kit, and the operation method was performed according to the instructions.

[0064] Comparative Example 2 The 0% and 100% quality control samples (as above) were transformed using the commercially available Novizan transformation kit, following the instructions.

[0065] Comparative Example 3 The reagent formulation ratio, the DNA sample used, the DNA transformation process and other procedures are the same as in Example 2. The difference is that the transformation procedure in step S3 is: 95℃, 10min; 56℃, 3h; 3℃, keep warm.

[0066] Example 4: Detection of nucleic acid transformation results using a flight mass spectrometry platform. I. Primer Design The company's primer codes are for gene K (ABCG1), gene B (SLC22A18), gene C (MGRN1), and gene L (ACTB). The specific primer sequences for these genes are as follows: The primer sequences used to detect the K gene are as follows: Upstream primer: 5'-aggaagagagATTTTGGAATTGGGTATTTTTTTGT-3'; Downstream primer: 5'-cagtaatacgactcactatagggagaaggctACAATTCTATTCCCTCACAAATCAC-3'.

[0067] The primer sequences used to detect the B gene are as follows: Upstream primer: 5'-aggaagagagTTTTTAGGTAAGGGTTGGGTATAGG-3'; Downstream primer: 5'-cagtaatacgactcactatagggagaaggctAAACCACCTCACAAAAAACAAATTA-3'.

[0068] The primer sequences used to detect the C gene are as follows: Upstream primer: 5'-aggaagagagAATTTTAGTTATTGGGGAGGTTGAG-3'; Downstream primer: 5'-cagtaatacgactcactatagggagaaggctCCCCCTCTAAAATAACTCCCTAAAT-3'.

[0069] The primer sequences used to detect the L gene are as follows: Upstream primer: 5'-aggaagagagATTATTTTTTTGTGTGGATTTGGG-3'; Downstream primer: 5'-cagtaatacgactcactatagggagaaggctCACCACCATATACCCTAACATTACC-3'.

[0070] II. Mass Spectrometry Detection Step 1: Using the 0% and 100% quality control samples treated in Examples 1-3 and Comparative Examples 1-3 as templates, DNA sequences were obtained by PCR amplification using the primer pairs for the four genes K, B, L, and C from Step 1, following standard PCR reaction requirements. All primers were used in a standard PCR reaction system. The specific PCR amplification procedure is as follows: PCR reaction program: 95℃, 4 min; (95℃, 20 s; 56℃, 30 s; 72℃, 2 min) 45 cycles; 72℃, 5 min; 4℃, 1 h.

[0071] Step 2: Add the prepared SAP to each PCR product well, adding 2 μL of SAP to each well, and follow the reaction procedure of "37℃, 20 min; 85℃, 5 min; 4℃, keep warm".

[0072] The SAP system components are as follows (the SAP reagent is an Agena product, catalog number 10129.A / 10129.1): SAP (1.7U) 0.4μL; H2O 1.6μL.

[0073] Step 3: Add the prepared T-cleavage system to a new 384-well PCR plate, adding 2 μL to each well. Then add 2 μL of the product obtained in Step 2 to the corresponding well to form the T-cleavage reaction system. Follow the reaction procedure of "37℃ for 3 hours, then incubate at 4℃" to obtain the final amplification results.

[0074] The T-cleavage system consists of the following components (the corresponding reagents for T-cleavage are from Agena, catalog number 10129A / 10129.1): RNase Free-ddH2O 3.5 μL; 5×T & Polymerase Buffer 0.95 μL; TClevage Mix 0.27 μL; DTT (100 mM) 0.25 μL; T7 RNA & DNA Polymerase 0.48 μL; RNase A 0.08 μL; SAP product 1.5 μL.

[0075] Step 4: Use the CPM system to sample, edit the board, and sample.

[0076] (I) Preliminary preparations 1. Open the lid and add deionized water to the water tank to ensure the tank is full; 2. Add a certain amount of water to the resin, cover it, shake it up and down to clean the resin, repeat the operation several times until the supernatant is clear and there are no floating objects. 3. Click "Add Resin" and pour the resin solution into the resin tank as instructed; (ii) Editing the board 1. Create a board; 2. Add amplicons information; 3. Add sample information, edit and save batch information, and select the Export to RT-Workstation option in File to import the information into the SpectroACQUIRE software; (iii) Spotting 1. Click the "Chip perp module Deck In / out" option on the SpectroACQUIRE main page to send out the sample chamber. Place the chip and PCR plate in the positions shown in the image. 2. Apply resin: Select the edited board, check the resin application option, and click Start chip prep module to begin applying resin; 3. Spotting PCR Samples: Select the spotting sample option and click Start chip prep module to start spotting the resin.

[0077] Step 5: Mass spectrometry detection and data analysis to obtain the final amplification results. 1. Check "Transfer Chip to Analyzer" and then click "Start chip prep module" to transfer the chip containing the sample to the vacuum chamber for evacuation. 2. When the pressure drops to 2.0 × 10⁻⁶ -6 In the following steps, select the Analyze Chips option, click Start chip prepmodule to begin mass spectrometry, and the data will be automatically saved to the computer to obtain the detection results, as shown in Table 1.

[0078] The detection instrument used for the above time-of-flight mass spectrometry detection was a MassARRAY® Analyzer Chip PrepModule 384, model number 41243.

[0079] Table 1. Results of nucleic acid detection after conversion in each example and comparative example using a flight mass spectrometry platform.

[0080] The results are shown in Table 1. As can be seen from the table, the test results of Examples 1-3 for 0% and 100% quality control samples were significantly better. Compared with Comparative Examples 1-3, especially Example 2, the detection results are closer to the actual values.

[0081] Example 5: Detection of nucleic acid transformation results using PCR The DNA transformed by the kits of this invention (Example 2), ZYMO, and Novizan kits was detected by PCR using an Abi384-well thermal cycler PCR instrument. The operation steps are as follows: 1. Primer design and synthesis Primer gPrimer for amplifying genomic DNA (without sulfur conversion): gGAPDH_245.

[0082] Upstream primer: 5'-aggaagagagTCAGTCGTTCCCAAAGTCC-3'; Downstream primer: 5'-cagtaatacgactcactatagggagaaggctGTAAAACCGCTAGTAGCCG-3'.

[0083] Primer BTPrimer:ACTB_334, used to amplify DNA after sulfurization transformation.

[0084] Upstream primer: 5'-aggaagagagATTATTTTTTTGTGTGGATTTGGG-3'; Downstream primer: 5'-cagtaatacgactcactatagggagaaggctCACCACCATATACCCTAACATTACC-3'.

[0085] 2. PCR detection Human genomic DNA quantitative standard material (National Institute of Metrology, China, Fudan University NIM-RM4035-1) was treated with the kits in Example 2 and Comparative Examples 1-2 respectively, and then amplified simultaneously using gPrimer and BTPrimer. The PCR system was prepared according to the amount of experimental reaction. Table 2 shows the general system.

[0086] Table 2. PCR System

[0087] Note: BT DNA is DNA obtained after sulfur conversion. Each kit has a different concentration gradient for BT DNA in the reaction system; see details below. Figure 1 .

[0088] PCR reaction program: 95℃, 5 min; (94℃, 30 sec; 60℃, 30 sec; 72℃, 1 min) 5 cycles; (94℃, 30 sec; 58℃, 30 sec; 72℃, 1 min) 5 cycles; (94℃, 5 sec; 56℃, 30 sec; 72℃, 1 min) 5 cycles; (94℃, 5 sec; 54℃, 30 sec; 72℃, 1 min) 30 cycles; 72℃, 5 min; incubate at 4℃.

[0089] After the reaction was completed, agarose gel electrophoresis was performed for detection.

[0090] The results are as follows Figure 1 As shown: The transformation results using gGAPDH_245 as primers were as follows: The kit of this invention failed to amplify a specific band, indicating complete transformation without containing genomic DNA, with a transformation efficiency of 100%. In Comparative Example 1, the kit amplified a band at 5 ng, indicating that the kit contained at least 0.01 ng of genomic DNA in the 5 ng of sulfite-treated sample (using PCR amplification results of unsulfite-converted genomic DNA (gDNA) with gGAPDH_245 as primers as a reference; the reference results show that to amplify the target band of gGAPDH_245, the sample size should not be less than 0.01 ng of genomic DNA, i.e., 0.01 ng is the minimum detection limit), with a transformation efficiency of 99.8%. In Comparative Example 2, the kit amplified a band at 1 ng, indicating that the kit contained at least 0.01 ng of genomic DNA in the 1 ng sample, with a transformation efficiency of 99%. Therefore, the transformation efficiency of the kit of this invention is higher than that of the two kits in Comparative Examples 1 and 2.

[0091] The transformation results using ACTB_334 as primers: The kit of this invention amplified a specific band at 0.001 ng, indicating that the kit provides excellent protection for the DNA sample during transformation, preventing excessive fragmentation. In Comparative Example 1, the kit amplified a specific band at 0.001 ng, but with a few empty deletions, yet it did not cause excessive fragmentation. In Comparative Example 2, the kit amplified fewer and weaker bands, indicating that the kit in Comparative Example 2 did not provide good protection for the DNA sample during transformation, leading to excessive fragmentation. The comparison of these results shows that the kit of this invention provides better protection for DNA.

[0092] This invention adjusts the temperature in the conversion process to maximize efficiency and significantly shorten extraction time (the entire conversion reaction time is only 70 minutes). Furthermore, the kit's components are simple, and the operation is easy to learn. The kit also uses low-cost raw materials, alleviating financial constraints for businesses and facilitating large-scale nucleic acid conversion processing, effectively meeting the needs of large-scale industrial production in the market.

[0093] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A reagent for the sulfite conversion of nucleic acids, characterized in that: The reagent includes a sulfite conversion solution; The sulfite conversion solution contains sodium metabisulfite at a final concentration of 0.4-0.8 g / L and ammonium bisulfite at a mass percentage of 10-20%. The pH value of the sulfite conversion solution is 5-6.

2. The reagent according to claim 1, characterized in that: The solvent for the sulfite conversion solution is water, and the solutes and concentrations are as follows: 0.4-0.8 g / L sodium metabisulfite and 10-20% ammonium bisulfite.

3. The reagent according to claim 1 or 2, characterized in that: The reagent also includes a binding solution; The binding solution contains Tris-HCl at a concentration of 0.01-0.05M, EDTA at a concentration of 0.06-0.08M, guanidine hydrochloride at a concentration of 2-4M, CTAB at a mass percentage of 0.5%-1%, and sodium chloride at a mass percentage of 5%-15%; the pH value of the binding solution is 6-8. Furthermore, the solvent of the binding solution is water, and the solutes and concentrations are as follows: 0.01-0.05M Tris-HCl, 0.06-0.08M EDTA, 2-4M guanidine hydrochloride, 0.5%-1% CTAB by mass, and 5%-15% sodium chloride by mass.

4. The reagent according to any one of claims 1-3, characterized in that: The reagent also includes an incubation solution; The incubation solution is a NaOH solution, which is an aqueous solution with a NaOH mass percentage of 1-5%; the pH value of the incubation solution is 7-9.

5. The reagent according to any one of claims 1-4, characterized in that: The reagent also includes a rinsing solution; the rinsing solution is an aqueous ethanol solution with a volume percentage of 60-80%.

6. The reagent according to any one of claims 1-5, characterized in that: The reagent also includes an elution buffer; the elution buffer is prepared by mixing the rinsing solution and anhydrous ethanol in a volume ratio of 1:

3.

7. A kit for sulfite conversion of nucleic acids, characterized in that: The kit comprises the reagents described in any one of claims 1-6; Furthermore, the kit contains a purification column.

8. The use of the reagent according to any one of claims 1-6 or the kit according to claim 7 in any of the following: (A1) Sulfite conversion of nucleic acids; (A2) Perform methylation analysis on nucleic acids.

9. A method for sulfite conversion of nucleic acids, comprising using the reagents described in any one of claims 1-6 or the kit described in claim 7 to perform sulfite conversion of nucleic acids, characterized in that: The reaction procedure for the conversion is as follows: 90-100℃ for 10 min; 58.5-60.5℃ for 1 h; and hold at 2-5℃. Furthermore, the method includes the following steps: S1. Add 6-8 times the volume of deionized water to the sulfite conversion solution described in claim 1 or 2, and shake at room temperature for 10 minutes. S2. Add the solution prepared in S1 to the nucleic acid sample to be transformed; S3. After shaking and mixing the system treated in S2, carry out the conversion reaction according to the following procedure: 90-100℃, 10min; 58.5-60.5℃, 1h; keep warm at 2-5℃. S4. Place the purification column in the collection tube and add the binding solution described in claim 3 to the purification column; S5. Transfer the reaction solution obtained in S3 to the purification column after adding the binding solution in S4, mix the reaction solution and the binding solution, then centrifuge and discard the filtrate, and put the purification column back into the collection tube. S6. Add the washing solution described in claim 5 to the purification column after S5 treatment, then centrifuge and discard the filtrate, and put the purification column back into the collection tube; S7. Add the incubation solution described in claim 4 to the purification column after S6 treatment, let it stand at room temperature for 10 min, centrifuge and discard the filtrate, and put the purification column back into the collection tube. S8. Add the washing solution described in claim 5 to the purification column after S7 treatment, centrifuge and discard the filtrate, and put the purification column back into the collection tube; S9. Add the washing solution described in claim 5 to the purification column after S8 treatment, centrifuge, and then place the purification column into a new centrifuge tube and let it stand at room temperature for 2 minutes. S10. Add the elution buffer as described in claim 6 to the purification column after S9 treatment, incubate at room temperature for 5 min, centrifuge, discard the purification column, and obtain the transformed sample.

10. The method according to claim 9, characterized in that: In S3, the procedure for the conversion reaction is as follows: 95°C for 10 min; 59.5°C for 1 h; hold at 3°C; and / or In S5 to S10, the centrifugation is performed at 13800g for 1-2 minutes.

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