A lysis binding solution and kit for rapid RNA extraction using magnetic beads and their application

Abstract

The invention discloses a lysis binding solution, a kit and its application for rapid RNA extraction by a magnetic bead method, wherein the lysis binding solution comprises 0.1-1 mmol / L dodine, 5-1000 mol / L sodium chloride, 10-50 mmol / L sodium citrate, 10-60 mmol / L dithiothreitol, 1-30 mmol / L disodium ethylenediaminetetraacetic acid, 10%-30% (v / v) 50% polyethylene glycol 8000, 0.1%-1% (v / v) 100% Proclin300. Relative to the prior art, the lysis binding solution or the kit provided by the present invention, when applied to RNA extraction by a magnetic bead method, can effectively lyse cells in a short time and release nucleic acids. The lysis solution has stable properties and is used in conjunction with magnetic beads to adsorb and recover nucleic acids.

Description

Technical Field

[0001] The invention belongs to the technical field of RNA extraction, and particularly relates to a lysis binding solution for rapid RNA extraction using a magnetic bead method, a kit and applications thereof. Background Art

[0002] Nucleic acid is the general term for deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). It is a biological macromolecular compound formed by the polymerization of many nucleotide monomers and is one of the most basic substances of life. Nucleic acid is a class of biological polymers, an indispensable component of all known life forms, the most important substance among all biological molecules, and widely present in all animal and plant cells and microorganisms. Nucleic acid is composed of nucleotides, and nucleotide monomers are composed of pentose, phosphate group and nitrogenous base. If the pentose is ribose, the polymer formed is RNA; if the pentose is deoxyribose, the polymer formed is DNA.

[0003] Nucleic acid extraction is a necessary basic technology for modern molecular biology research, and the preparation of high-quality nucleic acids is the key to the success of subsequent research. For example, at present, the global nucleic acid detection for the new coronavirus is based on the premise that the nucleic acid in the new coronavirus particles can be released or extracted to successfully carry out nucleic acid detection and meet the requirements of epidemic prevention and control. The principle of nucleic acid extraction is to first ensure the integrity of the primary structure of nucleic acid, and secondly to exclude contamination by other molecules. The extraction method of nucleic acid has a long history and has formed a variety of extraction methods. The methods for extracting deoxyribonucleic acid (DNA) include the SDS method, CTAB method, enzyme lysis method, etc., which are classic methods that first lyse and release DNA and then purify it by chromatography, as well as a variety of commercial kits based on these basic DNA extraction methods. The most important ones are column extraction method, magnetic bead method, etc. For the extraction of ribonucleic acid (RNA), such as extracting RNA of the new coronavirus, the classic methods include Trizol method, as well as commercial kits such as column extraction method and magnetic bead method.

[0004] RNA is one of the most important basic substances in biological molecules and has become one of the research objects in this field. Nucleic acid (RNA) extraction is particularly important in the experimental process. Nucleic acid extraction has also been continuously developed and improved from traditional time-consuming extraction methods to simple and efficient extraction methods. In order to make the extracted nucleic acid meet the requirements of purity and quality, the extraction technology is also constantly updated.

[0005] Magnetic bead extraction has become a commonly used extraction method in the molecular field. The magnetic bead method uses nanotechnology to improve and modify the surface of superparamagnetic nanoparticles to prepare superparamagnetic nanoparticles. The magnetic beads can specifically identify and efficiently bind to nucleic acid molecules at the microscopic interface. Using the superparamagnetism of nanomagnetic microspheres, DNA and RNA can be separated from samples such as blood and pathogenic microorganisms under certain buffer conditions and external magnetic fields. At present, the demand for nucleic acid extraction in our major company hospitals has greatly increased. The magnetic bead extraction operation is simple, takes a short time, and can be automated in large quantities.

[0006] The main components of the commonly used lysis buffer are guanidine salts, such as guanidine hydrochloride and guanidine isothiocyanate. They can denature proteins, dissolve proteins, break cells, and release nucleic acids from cells. However, guanidine salts are easily affected by conditions such as temperature and humidity, which affect the effect of the lysis buffer. These shortcomings need to be continuously improved through technology. Therefore, a lysis buffer with stable components that is not easily affected by other conditions is very important. Summary of the invention

[0007] Purpose of the invention: In view of the defects in the prior art, the present invention provides a lysis and binding solution, a kit and its application for rapid extraction of RNA by magnetic beads. The components of the lysis and binding solution and the kit are stable and suitable for extracting viral RNA from saliva and throat swab samples with high speed and efficiency.

[0008] Technical solution: In order to achieve the above-mentioned invention object, the present invention adopts the following technical solution:

[0009] The invention discloses a lysis binding solution for rapidly extracting RNA by a magnetic bead method. The components contained in the lysis binding solution include 0.1-1 mmol / L dodecylguanidine acetate, 5-1000 mmol / L sodium chloride (NaCl), 10-50 mmol / L sodium citrate, 10-60 mmol / L dithiothreitol (DTT), 1-30 mmol / L disodium ethylenediaminetetraacetic acid (EDTA-2Na), 10%-30% (v / v) 50% polyethylene glycol (PEG8000), and 0.1%-1% (v / v) 100% Proclin300.

[0010] In the lysis binding solution, the component stability of dodecanol is better, which can play the role of lysing cells and a part of detergent. Sodium chloride provides certain salt ions in the reagent, and sodium chloride of suitable concentration plays the role of promoting the binding of magnetic beads to nucleic acids. Sodium citrate can control the ionic strength in the reaction system, play the role of regulating pH, and maintain a stable buffer system. EDTA-2Na can play the role of protecting nucleic acids. Polyethylene glycol 8000, as the main component in the binding solution, can make magnetic beads and nucleic acids effectively combined.

[0011] The 50% polyethylene glycol (PEG8000) is prepared by mixing polyethylene glycol powder with deionized water in a ratio of 1:2.

[0012] Preferably, the lysis and binding solution comprises:

[0013] 0.1-0.5mmol / L, more preferably 0.1-0.4mmol / L of choline;

[0014] 5-500mmol / L, more preferably 50-200mmol / L sodium chloride;

[0015] 10-50mmol / L, more preferably 10-30mmol / L sodium citrate;

[0016] 10-50mmol / L, more preferably 15-30mmol / L dithiothreitol;

[0017] 10-30mmol / L, more preferably 15-30mmol / L of disodium edetate;

[0018] 10%-25% (v / v), further preferably 15%-25% (v / v) 50% polyethylene glycol 8000;

[0019] 0.1%-0.6% (v / v), more preferably 0.3%-0.6% (v / v) 100% Proclin300.

[0020] Proclin300 is added after the above reagents are prepared. It should be added according to the prepared amount to protect the stability of the reagents.

[0021] A kit for rapidly extracting RNA by a magnetic bead method comprises the lysis binding solution, a washing solution, an eluting solution and a magnetic bead mixed solution.

[0022] As a preference:

[0023] The washing liquid may contain 75%-80% ethanol.

[0024] The elution buffer may be enzyme-free water or low TE buffer.

[0025] In the magnetic bead mixture, the recovery effect is best when the magnetic bead particle size is in the range of 300-500 nm, and the selected magnetic bead modification groups can be carboxyl or silanol magnetic beads.

[0026] Finally, the present invention provides application of the lysis binding solution in extracting RNA by magnetic bead method.

[0027] And the application of the kit in extracting RNA using a magnetic bead method.

[0028] Beneficial effects: Compared with the prior art, the lysis binding solution or kit provided by the present invention can effectively lyse cells and release nucleic acids in a short time when used for RNA extraction by magnetic bead method. The lysis solution has stable properties and can be used in conjunction with magnetic beads to adsorb and recover nucleic acids. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a PCR amplification curve diagram after pseudovirus nucleic acid is extracted by the nucleic acid extraction kit of formula 1 of the present invention.

[0030] Figure 2 This is a PCR amplification curve diagram after pseudovirus nucleic acid is extracted by the nucleic acid extraction kit of formula 2 of the present invention.

[0031] Figure 3 This is the PCR amplification curve after pseudovirus nucleic acid was extracted using the Sinobright nucleic acid extraction kit.

[0032] Figure 4 This is an overlapping analysis diagram of the PCR amplification curves after pseudovirus nucleic acid is extracted by the nucleic acid extraction kit of formula 2 of the present invention and the nucleic acid extraction kit of Zhongkebaier. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The experimental methods in the examples described below are conventional methods unless otherwise specified; the materials, reagents, instruments, etc. used are all commercially available unless otherwise specified.

[0034] Example 1: Preparation of the reagent of the present invention at two different levels

[0035] 1. Prepare dodecan (sigma, product number 45466, powder), sodium chloride (sigma, product number S3014, granules), sodium citrate (sigma, product number C8532, granules), DTT (Solarbia, product number 578517, granules), EDTA-2Na (sigma, product number E7889, liquid, 0.5M), polyethylene glycol 8000 (PEG8000, sigma, product number: P5413, powder), proclin300 (Solarbio, liquid, 100%), and other H2O, utensils or consumables required for configuration;

[0036] 2. According to a certain ratio, prepare the above reagents into corresponding mother solutions:

[0037] Reagents Mother liquor concentration Dogodendron 20mM Sodium chloride 5M Sodium Citrate 0.1M DTT 1M EDTA-2Na 0.5M (original concentration) PEG8000 50%(w / v)

[0038] 3. Prepare lysis solution: add reagents according to the following formula table

[0039] Reagent name (stock solution) Lysis buffer recipe 1 Lysis buffer recipe 2 Dodine (20mM) 5mL 1mL Sodium chloride (5M) 20mL 2mL Sodium citrate (0.1M) 20mL 20mL DTT(1M) 2mL 4mL EDTA-2Na(0.5M) 0.4mL 0.5mL PEG8000 (50%) 20mL 17mL Proclin 300 (100%) 0.5mL 0.5mL water Make up to 100ml Make up to 100ml

[0040] 4. Prepare detergent: prepare anhydrous ethanol and deionized water to prepare 80% detergent.

[0041] 5. Prepare magnetic beads: Prepare magnetic beads with a magnetic solid content of 10 mg / ml and a particle size range of 300-500 nm. The magnetic beads can be diluted 30-50 times with water.

[0042] 6. Prepare elution buffer: deionized water or 0.1x TE buffer.

[0043] 7. Reagent packaging: The reagents are packaged in a 96-well deep-well plate, with the lysis and binding solution in the first column, the magnetic bead solution in the second column, the washing reagent in the third to fifth columns, and the elution reagent in the sixth column.

[0044] 8. The reagent plate that is packaged using Lysis Buffer Formula 1 is marked as Formula 1 reaction plate; the reagent plate that is packaged using Lysis Buffer Formula 2 is marked as Formula 2 reaction plate.

[0045] Example 2: Performance verification of the reagent of the present invention

[0046] 1. Sample preparation: Prepare pseudovirus for extraction and inactivate the pseudovirus at 56°C for 30 minutes. After inactivation, dilute the pseudovirus to 200 copies / ul and prepare for RNA extraction.

[0047] 2. Nucleic acid extraction: Take 200ul of the inactivated pseudovirus and add it to the pre-packaged reaction plate of formula 1 and the reaction plate of formula 2 respectively. According to the "extraction procedure" described in the table below, use an automated extractor to extract. After the extraction is completed, collect the eluate for subsequent PCR reaction.

[0048] The extraction procedure is

[0049] name time Module temperature Magnetic bead mixing 20s Room temperature Transferring magnetic beads 40s Room temperature Splitting and binding 360s 90℃ washing 40s 80℃ washing 40s 80℃ washing 40s 80℃ Elution recovery 300s 85℃ Discard magnetic beads 20s Room temperature

[0050] 3. The nucleic acid extracted from the reaction plate of formula 1 is labeled as sample 1; the nucleic acid extracted from the reaction plate of formula 2 is labeled as sample 2.

[0051] 4. PCR reaction: Take 5ul of sample 1 and sample 2 respectively, add 15ul of amplification reagent for PCR reaction, repeat each reaction 3 times, and carry out PCR amplification reaction according to the reaction program of 55℃10min, 95℃1min, (95℃10S, 60℃1min45cycles).

[0052] 5. After amplification, the amplification curve obtained from sample 1 is Figure 1 ; The amplification curve obtained from sample 2 is Figure 2 , see attached figure for details.

[0053] Example 3: Competitive product performance verification

[0054] 1. Sample preparation: Prepare pseudovirus for extraction and inactivate the pseudovirus at 56°C for 30 minutes. After inactivation, dilute the pseudovirus to 200 copies / ul and prepare for RNA extraction.

[0055] 2. Nucleic acid extraction: Take 200ul of the inactivated pseudovirus and add it to the pre-packaged Zhongkebaier reaction plate. Use an automated extractor to extract according to the "Extraction Procedure" described in the table below. After the extraction, collect the eluate for subsequent PCR reaction.

[0056] The extraction procedure is

[0057] name time Module temperature Magnetic bead mixing 20s Room temperature Transferring magnetic beads 40s Room temperature Splitting and binding 360s 90℃ washing 40s 80℃ washing 40s 80℃ washing 40s 80℃ Elution recovery 300s 85℃ Discard magnetic beads 20s Room temperature

[0058] 3. The extracted nucleic acid is labeled as sample zk.

[0059] 4. PCR reaction: Take zk5ul of sample and add 15ul of amplification reagent for PCR reaction. Repeat each reaction 3 times. Perform PCR amplification reaction according to the reaction program of 55℃10min, 95℃1min, (95℃10S, 60℃1min 45cycles).

[0060] 5. After the amplification is completed, the amplification curve obtained from sample zk is Figure 3 , see attached figure for details.

[0061] 6. Data result analysis: In the above two embodiments of the present invention, in Example 2, the amplification curve obtained by Sample 2 is compared with the amplification curve obtained by Sample 1, and the amplification curve obtained by Sample 2 performs better in terms of parallelism, stability, and amplification efficiency. Compared with the Zhongkebaier extraction kit, the kit containing the lysate formula 2 of the present invention (Example 2) has better stability and small data differences between parallel samples ( Figure 4 ).

[0062] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the attached claims.

Claims

1. A kit for rapid RNA extraction using a magnetic bead method, characterized in that: The kit comprises a lysis binding solution, a washing solution, an eluting solution and a magnetic bead mixture; the lysis binding solution comprises 0.1-1 mmol / L dodecanedione, 5-1000 mmol / L sodium chloride, 10-50 mmol / L sodium citrate, 10-60 mmol / L dithiothreitol, 1-30 mmol / L disodium ethylenediaminetetraacetate, 10%-30% (v / v) 50% polyethylene glycol 8000, and 0.1%-1% (v / v) 100% Proclin300.

2. The kit for rapid RNA extraction using magnetic beads according to claim 1, characterized in that: The lysis and binding solution contains 0.1-0.5mmol / L dodecanedione, 5-500mmol / L sodium chloride, 10-50mmol / L sodium citrate, 10-50mmol / L dithiothreitol, 10-30mmol / L disodium ethylenediaminetetraacetic acid, 10%-25% (v / v) 50% polyethylene glycol 8000, and 0.1%-0.6% (v / v) 100% Proclin300.

3. The kit for rapid RNA extraction using magnetic beads according to claim 1, characterized in that: The washing liquid is selected from 75%-80% ethanol.

4. The kit for rapid RNA extraction using magnetic beads according to claim 1, characterized in that: The eluent is selected from enzyme-free water or low TE buffer.

5. The kit for rapid RNA extraction using magnetic beads according to claim 1, characterized in that: In the magnetic bead mixture, the particle size of the magnetic beads is in the range of 300-500 nm, and the magnetic beads are selected from carboxyl-modified or silanol-modified magnetic beads.

6. Use of the kit according to any one of claims 1 to 5 in extracting RNA using a magnetic bead method.

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