Rapid methylation pretreatment kit based on plasma sample and application thereof

Abstract

The invention relates to the technical field of methylation detection, and particularly discloses a plasma sample-based rapid methylation pretreatment kit, which comprises a lysis solution, magnetic beads, a rinsing solution and a conversion solution, the lysis solution is prepared from Tris-HCl, EDTA (Ethylene Diamine Tetraacetic Acid), NaCl, SDS (Sodium Dodecyl Sulfate), guanidine isothiocyanate, sodium acetate, PVP (Polyvinyl Pyrrolidone), PEG200 ( The magnetic beads are polydisperse magnetic microspheres with the particle size of 100-400 nm; the magnetic bead rinsing liquid is an ethanol solution; according to the method, the formula of the extraction reagent is improved, the protease K is successfully removed on the premise that the extraction efficiency is not changed, the specific conversion reagent is adopted, the large volume of the specific conversion reagent serves as the extraction eluent, and conversion can be directly carried out after elution. The innovative design effectively reduces the loss of nucleic acid and obviously improves the detection sensitivity. Meanwhile, the extraction process and the conversion process are integrated, so that the operation steps are greatly simplified, the operation complexity is reduced, and a more efficient and convenient sample pretreatment solution is provided for methylation detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of methylation detection, and particularly relates to a rapid methylation pretreatment kit based on plasma samples and applications thereof. Background Art

[0002] Methylation pretreatment reagent is a special kit used for detecting methylation of free DNA (cfDNA) in plasma samples. Its core function is to provide high-quality samples for downstream methylation analysis (such as PCR and sequencing) through extraction, conversion and purification steps.

[0003] Currently, cfDNA mostly exists as a complex of DNA and proteins, with only a very small amount present as free DNA fragments. The low concentration of cfDNA in blood and the small size of the fragments make it difficult to extract, significantly hindering the genetic testing and various research studies of free nucleic acids.

[0004] Currently available cfDNA extraction methods require the addition of proteinase K, requiring the use of two separate kits for extraction and conversion. This process can easily lead to nucleic acid loss, limiting the sensitivity of downstream testing. Furthermore, existing methods are cumbersome, requiring multiple centrifugation and transfer operations from sample processing to final testing. This is not only time-consuming, but each step can introduce errors, affecting the accuracy of test results.

[0005] In response to the above problems, the present invention provides a rapid methylation pre-treatment kit based on plasma samples.

[0006] This method uses specialized extraction reagents, magnetic beads, and conversion reagents to extract small nucleic acid fragments from plasma. CFDNA in the sample is released by a lysis buffer. In the presence of a binding buffer, the released DNA specifically binds to unique magnetic beads. A wash buffer is then used to remove impurities, and finally, an eluent is used to obtain high-quality cell-free DNA. The entire process is safe and convenient, resulting in a high yield of extracted CFDNA, high purity, and stable and reliable quality.

[0007] 2. DNA denatures into single strands at high temperatures, exposing the bases. The bisulfite ions in the conversion agent sulfonate and hydrolytically deaminate unmethylated cytosines in the denatured DNA, generating uracil sulfonate, which is then desulfonated and ultimately converted to uracil. Cytosine C in methylated DNA selectively adds a methyl group to form 5-methylcytosine, which cannot be converted and remains unchanged. Summary of the Invention

[0008] The purpose of the present invention is to provide a rapid methylation pretreatment kit based on plasma samples and its application to solve the problems raised in the above background technology.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] A rapid methylation pre-treatment kit based on plasma samples, comprising a lysis solution, magnetic beads, a rinsing solution, and a conversion solution;

[0011] Lysis buffer includes Tris-HCl, EDTA, NaCl, SDS, guanidine thiocyanate, sodium acetate, PVP, PEG200, and polyacrylamide;

[0012] The magnetic beads are polydisperse magnetic microspheres with a particle size of 100-400 nm; the magnetic bead rinse solution is ethanol solution; the conversion solution is 3M sodium bisulfite,

[0013] The concentration of Tris-HCl in the lysate is 150-200 mmol / L, pH 7.0-8.0, the concentration of EDTA is 10-20 mmol / L, the concentration of NaCl is 0.8-2.0 mol / L, the concentration of guanidine isothiocyanate is 6 mol / L, the concentration of sodium acetate is 0.5-1.0 mol / L, the volume fraction of SDS is 5-8%, the volume fraction of PVP is 0.5-1%, the volume fraction of PEG200 is 1-2%, and the volume fraction of polyacrylamide is 0.1-0.2%.

[0014] Preferably, the volume fraction of the ethanol solution in the magnetic bead rinsing solution is 70%.

[0015] A rapid methylation pre-treatment kit based on plasma samples is used in the extraction, bisulfite conversion, enrichment and purification of cfDNA from plasma samples.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] The present invention uses special extraction reagents, magnetic beads and conversion reagents to extract small fragments of nucleic acids from plasma. The cfDNA in the sample is released under the action of a lysis solution. In the presence of a binding solution, the released DNA specifically binds to unique magnetic beads, and then a washing solution is used to remove impurities. Finally, high-quality free DNA is obtained under the action of an elution solution.

[0018] The present invention improves the extraction reagent formula, successfully removing proteinase K while maintaining extraction efficiency. Furthermore, it utilizes a specific conversion reagent and uses a large volume as the extraction eluent, allowing for direct conversion after elution. This innovative design effectively reduces nucleic acid loss and significantly improves detection sensitivity. Furthermore, the present invention integrates the extraction and conversion processes, significantly simplifying the operational steps and reducing operational complexity, providing a more efficient and convenient sample pretreatment solution for methylation detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of the qPCR results before optimization of the present invention;

[0020] Figure 2 Schematic diagram of the optimized qPCR results of the present invention. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] A rapid methylation pre-treatment kit based on plasma samples, comprising a lysis solution, magnetic beads, a rinsing solution, and a conversion solution;

[0023] Lysis buffer includes Tris-HCl, EDTA, NaCl, SDS, guanidine thiocyanate, sodium acetate, PVP, PEG200, and polyacrylamide;

[0024] The magnetic beads are polydisperse magnetic microspheres with a particle size of 100-400 nm; the magnetic bead rinse solution is ethanol solution; the conversion solution is 3M sodium bisulfite,

[0025] The concentration of Tris-HCl in the lysate was 170 mmol / L, pH 7.5, the concentration of EDTA was 15 mmol / L, the concentration of NaCl was 1.5 mol / L, the concentration of guanidine isothiocyanate was 6 mol / L, the concentration of sodium acetate was 0.8 mol / L, the volume fraction of SDS was 7%, the volume fraction of PVP was 0.8%, the volume fraction of PEG200 was 1.5%, the volume fraction of polyacrylamide was 0.15%, and the volume fraction of ethanol solution in the magnetic bead rinse was 70%.

[0026] The test principle of this invention is that cfDNA in the sample is released by the action of a lysis solution. The released cfDNA specifically binds to magnetic beads. A wash solution is used to remove impurities. The nucleic acid sample is treated with sulfite, and unmethylated cytosine is deaminated to uracil, while methylated cytosine remains unchanged. Simultaneously, combined with magnetic bead purification, the nucleic acid is converted, enriched, and purified.

[0027] Example 1:

[0028] A rapid methylation pre-treatment kit based on plasma samples: designated model QCL-1

[0029] For 1.5-2mL samples: 48 tests / box

[0030] The difference is that it is designated as model QCL-2

[0031] Applicable to 3.5-4mL samples, 48 tests / box

[0032] The main components of the kit are:

[0033] composition Main ingredients Filling volume Extraction of magnetic bead suspension magnetic beads 1920μL / bottle × 1 bottle Extraction Plate 1 / 24 tests / board × 1 board Extraction Plate 2 Guanidine thiocyanate 24 tests / board × 2 boards Extraction Plate 3 Guanidine hydrochloride, anhydrous ethanol 24 tests / board × 2 boards Extraction Plate 4 Guanidine hydrochloride, anhydrous ethanol 24 tests / board × 2 boards Extraction Plate 5 Anhydrous ethanol 24 tests / board × 2 boards Extraction Plate 6 Anhydrous ethanol 24 tests / board × 2 boards Extraction Plate 7 Bisulfite 24 tests / board × 2 boards 24 integrated magnetic rod sets / 2 pieces / bag × 1 bag 8-link magnetic rod set / 2 pieces / bag × 3 bags Purification Plate Guanidine hydrochloride, anhydrous ethanol 16 tests / board × 3 boards

[0034] The present invention is applicable to instruments such as fully automatic nucleic acid extractors and PCR amplifiers.

[0035] Methylation detection sample pre-treatment method:

[0036] Sample processing

[0037] Collect EDTA anticoagulated whole blood samples and centrifuge them twice in sequence:

[0038] First centrifugation: Centrifuge at 4°C, 1600 rcf for 10 minutes, transfer the upper plasma to a new centrifuge tube, avoiding aspirating the cell buffy coat layer.

[0039] Second centrifugation: Centrifuge at 4°C, 16,000 rcf for 10 minutes, transfer the upper plasma to a new centrifuge tube to avoid aspirating the precipitate.

[0040] The processed plasma samples can be stored at -20°C or -80°C.

[0041] Nucleic acid extraction (taking Jifan Bio Purifier24 as an example)

[0042] Pre-treat the extraction plate: gently shake or centrifuge (500 rpm × 1 min) to concentrate the reagents and tear off the aluminum seal.

[0043] Add sample and magnetic beads:

[0044] QCL-1: Add 1.5-2 mL of plasma sample and 40 μL of magnetic beads to extraction plate 2.

[0045] QCL-2: Add 3.5-4 mL of plasma sample and 80 μL of magnetic beads to extraction plate 2.

[0046] Install the magnetic rod cover and place the extraction plate: Place the 24-in-one magnetic rod cover on extraction plate 1 and place the extraction plates in order.

[0047] Set up the nucleic acid extraction program as follows (using the Jifan Bio fully automatic nucleic acid extractor Purifier24 as an example) and select Run.

[0048]

[0049] Set up the program and run: Set up the extraction program as described above (e.g., combined steps: volume 5000 μL, temperature 60°C, time 600 s, high shaking intensity).

[0050] Collect the product: After the program is completed, transfer the product in the extraction plate 7 to a 0.2 mL 8-tube strip.

[0051] Transformation reaction (using PCR instrument)

[0052] Centrifuge the tube strips from step 8 briefly, then place them in the PCR instrument and set the program:

[0053]

[0054]

[0055] After transformation, the product was immediately removed and centrifuged briefly.

[0056] Nucleic acid purification (taking Jifan Purifier32 as an example)

[0057] Pre-treat the purification plate: gently shake or centrifuge (500 rpm × 1 min) to concentrate the reagents and tear off the aluminum sealing film.

[0058] Sample addition: Add all transformation products to columns 2 and 8 of the purification plate.

[0059] Place the purification plate with the sample in the correct position on the base of the automated nucleic acid extraction instrument;

[0060] Insert the 8-link magnetic rod set into the magnetic rod set holder slot of the automatic nucleic acid extractor.

[0061] Close the hatch, set the nucleic acid purification program according to the table below (using the Jifan fully automatic nucleic acid extraction instrument Purifier32 as an example), and select Run. (Elution step: volume 80μL, temperature 65°C, time 60s).

[0062]

[0063] After the program is finished, remove the magnetic rod cover and deep-well plate, transfer all the products in columns 6 and 12 of the deep-well plate to a centrifuge tube, and test immediately or store at -20°C.

[0064] Reference for the expression of the results of the QCL-1 treatment kit implemented in the present invention Figure 1 and Figure 2 After optimization, the qPCR results were advanced by 1-2 Ct values, indicating that under the same fluorescence threshold, the optimized kit enabled the target nucleic acid to reach the detection signal earlier, reflecting that the sample processing efficiency was improved and the kit could more sensitively identify low-abundance target nuclei.

[0065] It should be understood that in the development of any actual embodiment, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but for those of ordinary skill having the benefit of this disclosure, the development effort will be a routine task of design, fabrication, and production without undue experimentation.

[0066] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A rapid methylation pretreatment kit based on plasma samples, characterized in that: Including lysis buffer, magnetic beads, rinsing buffer, and conversion buffer; Lysis buffer includes Tris-HCl, EDTA, NaCl, SDS, guanidine thiocyanate, sodium acetate, PVP, PEG200, and polyacrylamide; The magnetic beads are polydisperse magnetic microspheres with a particle size of 100-400 nm; the magnetic bead rinse solution is ethanol solution; the conversion solution is 3M sodium bisulfite, The concentration of Tris-HCl in the lysate is 150-200 mmol / L, pH 7.0-8.0, the concentration of EDTA is 10-20 mmol / L, the concentration of NaCl is 0.8-2.0 mol / L, the concentration of guanidine isothiocyanate is 6 mol / L, the concentration of sodium acetate is 0.5-1.0 mol / L, the volume fraction of SDS is 5-8%, the volume fraction of PVP is 0.5-1%, the volume fraction of PEG200 is 1-2%, and the volume fraction of polyacrylamide is 0.1-0.2%.

2. A rapid methylation pre-treatment kit based on plasma samples according to claim 1, characterized in that: The volume fraction of the ethanol solution in the magnetic bead rinsing solution is 70%.

3. Use of a rapid methylation pretreatment kit based on plasma samples according to claim 1 in cfDNA extraction, cfDNA bisulfite conversion, enrichment, and purification of plasma samples.

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