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CN-121994567-A - Sample pretreatment method, detection method of 5-methylcytosine to cytosine ratio and kit

CN121994567ACN 121994567 ACN121994567 ACN 121994567ACN-121994567-A

Abstract

The application relates to the technical field of biochemical detection, in particular to a sample pretreatment method, a detection method of a 5-methylcytosine-cytosine ratio and a kit. The sample pretreatment method comprises the steps of providing a sample containing an object to be tested, wherein the object to be tested comprises 5-methylcytosine and cytosine, mixing magnetic beads containing sulfonic acid groups with the sample for adsorption treatment, then leaching the mixture by using an alcohol solution to obtain an object to be tested-magnetic bead compound, eluting the object to be tested-magnetic bead compound by using an eluent to obtain the sample to be tested, and the eluent comprises at least one of a formic acid-alcohol solution and an ammonia water-alcohol solution. The sample pretreatment method provided by the application has the advantages that the flow is simple, the cost is low, the sample to be detected containing 5-methylcytosine and cytosine can be efficiently obtained, the ratio of 5-methylcytosine to cytosine can be precisely and sensitively quantified on the basis of an electrospray efficient ion mobility spectrometry technology, and the method has a good application prospect.

Inventors

  • ZHU FEI
  • JIANG YUXIANG
  • WU CHAO
  • HAN LIANG

Assignees

  • 联影越质科学仪器(上海)有限公司

Dates

Publication Date
20260508
Application Date
20260226

Claims (12)

  1. 1. A method of sample pretreatment comprising: providing a sample comprising an analyte, the analyte comprising 5-methylcytosine and cytosine; Mixing magnetic beads containing sulfonic acid groups with the sample for adsorption treatment, and then leaching with an alcohol solution to obtain an object to be detected-magnetic bead compound; and eluting the object to be detected-magnetic bead compound by using an eluent to obtain a sample to be detected, wherein the eluent comprises at least one of formic acid-alcohol solution and ammonia water-alcohol solution.
  2. 2. The method for sample pretreatment according to claim 1, wherein the magnetic beads comprise polystyrene-divinylbenzene copolymer bonded with sulfonic acid groups, wherein the magnetic core is coated on the surface of the magnetic core; and/or the magnetic beads comprise a mixed strong cation exchange adsorbent with a magnetic core coated on the surface of the magnetic core, wherein the mixed strong cation exchange adsorbent comprises a benzenesulfonic acid group and a hydrophobic group; and/or the average particle size of the magnetic beads is 1-15 mu m.
  3. 3. The method for sample pretreatment according to claim 1, wherein the alcohol solution comprises a methanol solution having a volume percentage of 5% to 10%; And/or the formic acid-alcohol solution in the eluent comprises 0.00001% -1% formic acid-methanol solution by volume percent; And/or the ammonia water-alcohol solution in the eluent comprises 0.001% -1% ammonia water methanol solution by volume percent.
  4. 4. The sample pretreatment method according to any one of claims 1 to 3, wherein the eluting treatment with an alcohol solution comprises eluting 1 to 2min with a 5 to 7% by volume methanol solution, and then eluting 1 to 2min with a 7 to 10% by volume methanol solution; And/or eluting the to-be-detected object-magnetic bead compound by using an eluent, wherein the eluting treatment comprises eluting 1-2 min by using an ammonia water methanol solution with the volume percentage of 0.001% -0.1%.
  5. 5. A sample pretreatment method according to any one of claims 1 to 3, wherein said magnetic beads are subjected to an activation treatment before being mixed with said sample, and then water is added to perform an equilibration treatment; and/or mixing the magnetic beads with the sample in 0.01% -1% formic acid water by volume percent for adsorption treatment.
  6. 6. A sample pretreatment method according to any one of claims 1 to 3, wherein the analyte is derived from free bases released upon DNA degradation; and/or the sample containing the to-be-detected object comprises a standard curve sample, a quality control sample and an analysis sample which are arranged in parallel.
  7. 7. A method for detecting the ratio of 5-methylcytosine to cytosine comprising: Treating the sample containing the analyte with the sample pretreatment method according to any one of claims 1 to 6 to obtain a sample to be tested; and detecting the sample to be detected by adopting an ion mobility spectrometry to obtain the ratio of 5-methylcytosine to cytosine.
  8. 8. The method according to claim 7, wherein the ion mobility spectrometry comprises electrospray ionization source ion mobility spectrometry, and the detection conditions comprise a positive ion mode, an ion source voltage of 1800-2100V, a mobility tube voltage of 8000-10000V, an air inlet temperature of 170-240 ℃, a mobility tube temperature of 170-240 ℃, an ion gate voltage of 50-60V, an ion gate voltage pulse width of 80-120 mu s, and a mobility gas and exhaust gas flow rate of 1.0-1.5L/min; and/or, the lower limit of quantification of the ratio of 5-methylcytosine to cytosine by the detection method is 1%.
  9. 9. A kit for pretreatment of a sample containing 5-methylcytosine and cytosine, comprising: the magnetic beads contain sulfonic acid groups on the surfaces; Eluent, namely alcohol solution; and eluent, namely at least one of formic acid-alcohol solution and ammonia water-alcohol solution.
  10. 10. The kit of claim 9, wherein the magnetic beads comprise polystyrene-divinylbenzene copolymer bonded with sulfonic acid groups, wherein the magnetic core is coated on the surface of the magnetic core; and/or the magnetic beads comprise a mixed strong cation exchange adsorbent with a magnetic core coated on the surface of the magnetic core, wherein the mixed strong cation exchange adsorbent comprises a benzenesulfonic acid group and a hydrophobic group; and/or the average particle size of the magnetic beads is 1-15 mu m.
  11. 11. The kit of claim 9, wherein the alcohol solution comprises 5% -10% by volume of a methanol solution; And/or the formic acid-alcohol solution in the eluent comprises 0.00001% -1% formic acid-methanol solution by volume percent; And/or the ammonia water-alcohol solution in the eluent comprises 0.001% -1% ammonia water methanol solution by volume percent.
  12. 12. The kit of any one of claims 9-11, further comprising 0.01% -1% formic acid water by volume for diluting the magnetic beads to be mixed and adsorbed with the sample; and/or the kit further comprises an alcoholic solution for dispersing and activating the magnetic beads.

Description

Sample pretreatment method, detection method of 5-methylcytosine to cytosine ratio and kit Technical Field The application belongs to the technical field of biochemical detection, and particularly relates to a sample pretreatment method, a detection method of a 5-methylcytosine-cytosine ratio and a kit. Background DNA methylation (DNA methylation) refers to an epigenetic modification that covalently links a methyl group to a DNA molecule, which can occur in cytosine (Cytosine, abbreviated C), adenine (Adenine, abbreviated a) or guanine (Guanine, abbreviated G). DNA methylation is an important epigenetic modification in eukaryotes, whose abnormal expression is closely related to the occurrence and progression of a variety of diseases, including, for example, cancer, diabetes, neurological disorders, autoimmune diseases, and the like. Among them, 5-methylcytosine (5-mC) is the most important epigenetic modification in DNA, usually occurring at the carbon atom 5 of cytosine in CpG dinucleotide sequences. As a normal component of DNA, 5-mC only accounts for about 3% of all cytosines, and in human DNA, it accounts for about 2.6-4.8%. As a key regulator of gene expression, 5-mC plays an important role in maintaining chromatin structure, guaranteeing DNA stability and protein interaction. The detection of the global level is of great importance for understanding the epigenetic state of cells, mining disease biomarkers and assessing the influence of the environment on the epigenetic genome. Currently, common methods for detecting the overall methylation level of a genome include High Performance Capillary Electrophoresis (HPCE), high Performance Liquid Chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS/MS), enzyme-linked immunosorbent assay (ELISA), and the like. These methods, while capable of quantifying the average DNA methylation level at all CpG sites in the genome of interest, have certain limitations. For example, HPCE has low sensitivity, long analysis time, poor stability, complicated HPLC flow, long analysis time, expensive LC-MS/MS instrument, complex operation, long analysis time, high maintenance requirement, professional operation, easy cross reaction of ELISA, strong antibody dependence, insufficient specificity and large development difficulty. In general, the existing detection method is difficult to consider among detection speed, sensitivity and cost effectiveness, and is difficult to meet the clinical rapid detection requirement. Disclosure of Invention The application aims to provide a sample pretreatment method, a detection method and a kit for the ratio of 5-methylcytosine to cytosine, and aims to solve the problem of how to accurately and accurately quantify the ratio of 5-methylcytosine to cytosine by pretreatment of a sample containing 5-methylcytosine and cytosine with low cost, high efficiency and high sensitivity. In order to achieve the purposes of the application, the technical scheme adopted by the application is as follows: in a first aspect, the present application provides a sample pretreatment method comprising: providing a sample comprising an analyte, the analyte comprising 5-methylcytosine and cytosine; Mixing magnetic beads containing sulfonic acid groups with the sample for adsorption treatment, and then leaching with an alcohol solution to obtain an object to be detected-magnetic bead compound; and eluting the object to be detected-magnetic bead compound by using an eluent to obtain a sample to be detected, wherein the eluent comprises at least one of formic acid-alcohol solution and ammonia water-alcohol solution. In some embodiments, the magnetic beads comprise polystyrene-divinylbenzene copolymer bonded with sulfonic acid groups, wherein the polystyrene-divinylbenzene copolymer is coated on the surface of the magnetic core by the magnetic core; and/or the magnetic beads comprise a mixed strong cation exchange adsorbent with a magnetic core coated on the surface of the magnetic core, wherein the mixed strong cation exchange adsorbent comprises a benzenesulfonic acid group and a hydrophobic group; and/or the average particle size of the magnetic beads is 1-15 mu m. In some embodiments, the alcohol solution comprises 5% -10% by volume of methanol solution; And/or the formic acid-alcohol solution in the eluent comprises 0.00001% -1% formic acid-methanol solution by volume percent; And/or the ammonia water-alcohol solution in the eluent comprises 0.001% -1% ammonia water methanol solution by volume percent. In some embodiments, the leaching treatment with the alcohol solution comprises leaching 1 to 2min with 5 to 7 volume percent of methanol solution, and leaching 1 to 2min with 7 to 10 volume percent of methanol solution; And/or eluting the to-be-detected object-magnetic bead compound by using an eluent, wherein the eluting treatment comprises eluting 1-2 min by using an ammonia water methanol solution with the volume percentage of 0.001% -0.1%. In