Kit for magnetic bead method nucleic acid extraction with large sample loading volume and extraction method
By optimizing the components and processes of the magnetic bead nucleic acid extraction kit, the problem of insufficient nucleic acid concentration in large volume samples is solved, and efficient extraction of high-concentration nucleic acids is achieved to meet the high-sensitivity animal disease pathogen detection needs.
Patent Information
- Application Number
- CN202510355946.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-25
- Publication Date
- 2025-07-22
AI Technical Summary
The existing magnetic bead nucleic acid extraction technology is difficult to meet the high sensitivity requirements in animal disease pathogen detection, especially when loading large-volume samples, the nucleic acid concentration is insufficient and cannot meet the needs of subsequent molecular biology experiments.
A magnetic bead method nucleic acid extraction kit with a large volume loading volume is provided, including lysate, washing solution I, washing solution II and eluent. By optimizing component concentration and pH value, it can achieve efficient extraction of nucleic acids, especially in the absence of adding protease K, to improve the concentration and purity of nucleic acids.
It is achieved that the maximum sample addition amount is 2mL without adding protease K, which significantly improves the nucleic acid concentration, meets the high-sensitivity animal disease pathogen detection needs, and improves the efficiency and quality of nucleic acid extraction.
Smart Images
Figure CN120350091A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of nucleic acid extraction, and specifically relates to a kit and an extraction method for nucleic acid extraction by magnetic bead method with a large sample loading volume. Background Art
[0002] There are many nucleic acid extraction techniques, including SDS alkaline lysis method, phenol-chloroform extraction method, single-phase lysis method, column purification method, and magnetic bead method. With the development of molecular diagnosis, nucleic acids of pathogenic microorganisms can be identified by methods such as fluorescence PCR after extraction for clinical reference, which also continuously raises the technical requirements for nucleic acid extraction. The magnetic bead method for nucleic acid extraction has become the mainstream method due to its high degree of automation, fast extraction speed, high nucleic acid concentration and good purity obtained.
[0003] Currently, for commercial kits of the magnetic bead method for automatic nucleic acid extraction based on the magnetic rod method, the sample addition amount is basically 200 μL or 400 μL, and the nucleic acid concentration and quality obtained by extraction are sufficient to meet subsequent molecular biology experiments. However, currently, in the detection of animal disease pathogens, the requirements for detection sensitivity are getting higher and higher. While optimizing the fluorescence PCR reaction system, further requirements are also put forward for the concentration of nucleic acid extraction. Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a kit and an extraction method for nucleic acid extraction by magnetic bead method with a large sample loading volume, which do not require the addition of proteinase K and can extract nucleic acids with a higher concentration.
[0005] An embodiment of the present invention provides a kit for nucleic acid extraction by magnetic bead method with a large sample loading volume, which includes a lysis solution, a washing solution I, a washing solution II, and an elution solution.
[0006] Optionally, the lysis solution includes components with the following concentrations: guanidine isothiocyanate 4 - 6 M, Tris-HCl 50 - 100 mM, Triton X-100 (Triton, v / v) 5 - 10%, NP-40 (ethyl phenyl polyethylene glycol, v / v) 15 - 25%, SDS (sodium dodecyl sulfate, w / v) 0.1 - 1%, EDTA (ethylenediaminetetraacetic acid) 20 - 50 mM, DTT (dithiothreitol) 10 - 50 mM, sodium chloride 200 - 500 mM, acetate (preferably sodium acetate) 50 - 100 mM, and the pH is 7.5 - 9.0.
[0007] Preferably, the lysis solution comprises components with the following concentrations: guanidine isothiocyanate 5 M, Tris-HCl 50 mM, Triton X-100 (v / v) 5%, NP-40 (v / v) 15%, SDS (w / v) 1%, EDTA 20 mM, DTT 10 mM, sodium chloride 200 mM, acetate 50 mM, and the pH is 7.5 - 8.0.
[0008] The lysis solution of the present invention can denature proteins, inhibit the activity of nucleases, prevent the degradation of nucleic acids, precipitate proteins, and at the same time provide a buffer system for the released nucleic acids, making the obtained nucleic acids more stable without adding and using proteinase K.
[0009] Optionally, the washing solution I comprises components with the following concentrations: guanidine isothiocyanate 4 - 6 M, Tris-HCl 10 - 50 mM, sodium chloride 100 - 250 mM, sodium acetate 10 - 50 mM, and the pH is 5.5 - 7.0.
[0010] Preferably, the washing solution I comprises components with the following concentrations: guanidine isothiocyanate 4 M, Tris-HCl 20 mM, sodium chloride 200 mM, sodium acetate 20 mM, and the pH is 6.0 - 7.0.
[0011] Optionally, the washing solution II comprises components with the following concentrations: absolute ethanol (v / v) 50 - 80%, Tris-HCl 50 - 100 mM, EDTA 0.01 - 0.05 M, magnetic bead suspension (v / v) 2.5 - 7.5%, and the pH is 7.0 - 8.5.
[0012] Preferably, the washing solution II comprises components with the following concentrations: absolute ethanol (v / v) 70%, Tris-HCl 50 mM, EDTA 10 mM, magnetic bead suspension (v / v) 5%, and the pH is 7.0 - 8.0. The magnetic beads in the magnetic bead suspension are carboxyl magnetic beads with a particle size of 50 - 400 nm.
[0013] The washing solution I and washing solution II of the present invention are mainly used to rinse nucleic acids and remove residual guanidine salts, etc. At the same time, the washing solution II also serves as a magnetic bead preservation solution. The magnetic beads mainly adsorb nucleic acids through the groups on the surface of the magnetic beads. The elution solution is used to dissolve nucleic acids, so that the nucleic acids are detached from the magnetic beads and provide a stable environment for storing nucleic acids.
[0014] Optionally, the elution solution comprises Tris-HCl 10 - 50 mM, and the pH is 8.0 - 9.0.
[0015] Preferably, the elution solution comprises Tris-HCl 10 mM, and the pH is 8.5.
[0016] An embodiment of the present invention provides a nucleic acid extraction method with a large sample loading amount. The extraction is carried out using the described kit, and the extracted nucleic acid is DNA or RNA, mainly nucleic acid of pathogenic microorganisms.
[0017] The maximum sample addition amount in this application is 2 mL, which is much more than the 200 μL or 400 μL of the conventional magnetic bead method nucleic acid extraction kit.
[0018] The beneficial effect of the present invention is that the kit of the present invention includes a lysis solution, a washing solution I, a washing solution II, and an elution solution. The washing solution II is a combination of the washing solution and the magnetic bead preservation solution, which increases the sample loading amount, can improve the nucleic acid concentration of pathogenic microorganisms, and provides high-concentration nucleic acid for subsequent detection of animal disease pathogens. Description of the Drawings
[0019] Figure 1 It is the fluorescence PCR amplification curve of the nucleic acid extracted by the magnetic bead method nucleic acid extraction kit with a large sample loading amount of the present invention.
[0020] Figure 2 It is the fluorescence PCR amplification curve of the nucleic acid extracted by the magnetic bead method nucleic acid extraction kit of Company A.
[0021] Figure 3 It is the fluorescence PCR amplification curve of the nucleic acid extracted by the magnetic bead method nucleic acid extraction kit of Company B. Detailed Embodiments
[0022] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the materials, unless otherwise specified, can all be obtained from commercial channels.
[0023] Embodiment 1 Magnetic Bead Method Nucleic Acid Extraction Kit with a Large Sample Loading Amount The magnetic bead method nucleic acid extraction kit with a large sample loading amount includes a lysis solution, a washing solution I, a washing solution II, and an elution solution.
[0024] The lysis solution contains 5 M guanidine isothiocyanate, 50 mM Tris-HCl, 5% (v / v) Triton X-100, 15% (v / v) NP-40, 1% (w / v) SDS, 20 mM EDTA, 10 mM DTT, 200 mM sodium chloride, 50 mM sodium acetate, and the pH is 7.5 - 8.0.
[0025] The washing solution I includes 4M guanidine isothiocyanate, 20 mM Tris-HCl, 200 mM sodium chloride, 20 mM sodium acetate, and has a pH of 6.0 - 7.0.
[0026] The washing solution II includes 70% (v / v) absolute ethanol, 50 mM Tris-HCl, 10 mM EDTA, 5% (v / v) magnetic bead suspension, and has a pH of 7.0 - 8.0. The magnetic beads in the used magnetic bead suspension are carboxyl magnetic beads with a magnetic bead particle size of 50 - 400 nm.
[0027] The elution solution includes 10 mM Tris-HCl and has a pH of 8.5.
[0028] Example 2 Extraction of total DNA / RNA from whole blood samples In this example, the whole blood sample is from a whole blood sample of a pig infected with ASFV. The total DNA / RNA in the whole blood sample is extracted using the kit in Example 1 on the Guoce Bio automatic nucleic acid extraction and purification instrument GT32 as follows: Turn on the instrument power supply, enter the setting interface, and set the instrument parameters according to the steps shown in the following table: Table 1 Nucleic acid extraction running program by magnetic bead method with large volume sample loading
[0029] Take out 1 64-well plate, and pre-add the respective component reagents to the corresponding well positions of the deep well plate according to the following table.
[0030] Table 2 Dispensing amounts and corresponding positions of each component of the nucleic acid extraction kit by magnetic bead method with large volume sample loading
[0031] Add 2000 μL of the sample (the sample needs to be balanced to room temperature) to the 1st / 5th columns of the deep well plate, place it in the card slot of the automatic nucleic acid extraction and purification instrument GT32, insert the magnetic rod sleeve into the instrument magnetic rod sleeve, close the hatch, and start running the program. After the operation ends, take out the 64-well plate, transfer the sample from the 4th / 8th columns to a sterile and enzyme-free centrifuge tube, which is the extracted whole blood DNA / RNA.
[0032] Example 3 Comparative extraction of whole blood samples using the nucleic acid extraction kit by magnetic bead method with large volume sample loading of the present invention and a conventional magnetic bead method nucleic acid extraction kit In this example, the whole blood sample is from a whole blood sample of a pig infected with ASFV, and the fluorescence PCR detection kits are all from Hunan Guoce Biotechnology Co., Ltd.
[0033] The nucleic acid extraction kit using magnetic beads with a large sample loading volume was used according to Example 1 and Example 2, and the conventional nucleic acid extraction kit using magnetic beads was used with reference to the provided instructions. The Ct values of the extracted nucleic acids were detected using a fluorescence PCR kit ( Figures 1-3 ), and the results are as follows: Table 3 Comparative test results of the nucleic acid extraction kit using magnetic beads with a large sample loading volume and the conventional nucleic acid extraction kit using magnetic beads
[0034] As can be seen from Table 3, for the same sample, the average Ct value of the nucleic acid obtained using the nucleic acid extraction kit using magnetic beads with a large sample loading volume provided by the present invention is at least 3 Ct values lower than that of the conventional nucleic acid extraction kit using magnetic beads.
[0035] Those of ordinary skill in the art should understand that the discussion of any of the above embodiments is only exemplary and is not intended to imply that the scope of protection of this application is limited to these examples; under the concept of this application, the technical features in the above embodiments or different embodiments can also be combined, the steps can be implemented in any order, and there are many other variations in different aspects of one or more of the embodiments in the present application as described above, and they are not provided in detail for the sake of brevity.
[0036] One or more embodiments of this application are intended to cover all such substitutions, modifications, and variations that fall within the broad scope of this application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of this application shall be included within the scope of protection of this application.
Claims
1. A kit for nucleic acid extraction by magnetic bead method with a large sample loading volume, characterized in that, It includes a lysis solution, Wash Solution I, Wash Solution II, and an elution solution.
2. The kit according to claim 1, characterized in that, The lysis solution includes components with the following concentrations: guanidine isothiocyanate 4 - 6 M, Tris-HCl 50 - 100 mM, Triton X-100 at a volume concentration of 5 - 10%, NP-40 at a volume concentration of 15 - 25%, SDS at a mass concentration of 0.1 - 1%, EDTA 20 - 50 mM, DTT 10 - 50 mM, sodium chloride 200 - 500 mM, acetate 50 - 100 mM, and the pH is 7.5 - 9.
0.
3. The kit according to claim 2, characterized in that, The lysis solution includes components with the following concentrations: guanidine isothiocyanate 5 M, Tris-HCl 50 mM, Triton X-100 at a volume concentration of 5%, NP-40 at a volume concentration of 15%, SDS at a mass concentration of 1%, EDTA 20 mM, DTT 10 mM, sodium chloride 200 mM, acetate 50 mM, and the pH is 7.5 - 8.
0.
4. The kit according to claim 1, characterized in that, Wash Solution I includes components with the following concentrations: guanidine isothiocyanate 4 - 6 M, Tris-HCl 10 - 50 mM, sodium chloride 100 - 250 mM, sodium acetate 10 - 50 mM, and the pH is 5.5 - 7.
0.
5. The kit according to claim 4, characterized in that, Wash Solution I includes components with the following concentrations: guanidine isothiocyanate 4 M, Tris-HCl 20 mM, sodium chloride 200 mM, sodium acetate 20 mM, and the pH is 6.0 - 7.
0.
6. The kit according to claim 1, characterized in that, Wash Solution II includes components with the following concentrations: absolute ethanol at a volume concentration of 50 - 80%, Tris-HCl 50 - 100 mM, EDTA 10 - 50 mM, magnetic bead suspension at a volume concentration of 2.5 - 7.5%, and the pH is 7.0 - 8.
5. The magnetic beads in the magnetic bead suspension are carboxyl magnetic beads with a particle size of 50 - 400 nm.
7. The kit according to claim 6, characterized in that, Wash Solution II includes components with the following concentrations: absolute ethanol at a volume concentration of 70%, Tris-HCl 50 mM, EDTA 10 mM, magnetic bead suspension at a volume concentration of 5%, and the pH is 7.
5.
8. The kit according to claim 1, characterized in that, The elution solution has the following components: Tris-HCl 10 - 50 mM, and the pH is 8.0 - 9.
0.
9. The kit according to claim 8, characterized in that, The elution solution has the following components: Tris-HCl 10 mM, and the pH is 8.
5.
10. A nucleic acid extraction method, characterized in that, When extracting using the kit according to any one of claims 1 - 9, the maximum sample loading amount is 2 mL, and the extracted nucleic acid is DNA or RNA.