Kit for extracting intact bacterial total RNA using magnetic beads and extraction method thereof

Abstract

The present invention provides a kit for extracting intact bacterial total RNA using a magnetic bead method and an extraction method thereof. The kit includes an extraction reagent set for extracting bacterial total RNA; the extraction reagent set includes a magnetic bead binding solution, a lysis solution, a washing solution I, a washing solution II, an eluent, and a DNase I digestion solution; the lysis solution includes sodium citrate, sodium lauroyl sarcosinate, sodium chloride, sodium metabisulfite, guanidine hydrochloride, polyether alcohol, and ethanol. When the bacteria are lysed by the lysis solution, the rational combination of the various raw materials in the lysis solution enables them to play a synergistic role in the RNA lysis and binding process, especially the combination of polyether alcohol and sodium metabisulfite, so that the lysis solution system has excellent binding force and stable binding properties for bacterial total RNA. The total RNA fragments extracted using the kit are intact, highly concentrated, and not easily degraded. The overall performance is superior to similar kits on the market, and the kit has good application prospects and market value.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology detection, and in particular to a kit for extracting intact bacterial total RNA using a magnetic bead method and an extraction method thereof. Background Art

[0002] RNA is an important genetic material in organisms, and the extraction of high-purity and high-integrity RNA is an important prerequisite for microbial molecular biology research. For downstream molecular biology experiments such as cDNA library construction, in vitro reverse transcription, fluorescent quantitative PCR, RT-PCR, and Northern hybridization analysis, high-quality and high-integrity RNA is required. Compared with eukaryotic cells such as animals and plants, bacterial RNA content is relatively low and its half-life is shorter. Since it does not have a 5' cap structure and a 3' Poly (A) tail, it is easily affected by external RNase (ribonuclease) and degraded. At the same time, the polysaccharides and polyphenols contained in the cells will form insoluble substances with RNA, making RNA isolation difficult, resulting in reduced RNA concentration and purity after extraction. Therefore, researchers often use some improved methods to improve the yield and purity of RNA.

[0003] Common bacterial RNA extraction methods include strong denaturants, Trizol, SDS-Phenol, CTAB, and hot boric acid. These extraction methods effectively inhibit RNases during the extraction process, and the extracted RNA product is of high purity, suitable for subsequent downstream experiments. However, most of these extraction methods use highly toxic organic solvents, which can cause environmental pollution and potential safety issues. Furthermore, most require the use of centrifuges, which significantly reduces RNA extraction efficiency and hinders industrial development. Many reagent companies, both domestically and internationally, have developed magnetic bead-based RNA extraction kits, but these kits suffer from drawbacks such as incomplete extraction, low purity, low concentration, the need for lysozyme pretreatment, and the addition of additional chemical reagents.

[0004] In view of this, it is necessary to design an improved kit for extracting intact bacterial total RNA using magnetic beads and its extraction method to solve the above problems. Summary of the Invention

[0005] The present invention aims to provide a kit for extracting intact bacterial total RNA using a magnetic bead method and an extraction method thereof. The kit uses sodium metabisulfite in combination with polyether alcohol, overcoming the defect that RNA is easily deformed and degraded during cleavage and binding. The bacterial total RNA extracted by this method has the advantages of intact fragments, high concentration, and is not easily degraded. The overall performance is superior to similar kits on the market, and the kit has good application prospects and market value.

[0006] To achieve the above-mentioned object of the invention, the present invention provides a kit for extracting intact bacterial total RNA using a magnetic bead method, comprising an extraction reagent set for extracting bacterial total RNA; the extraction reagent set comprises a magnetic bead binding solution, a lysis solution, a washing solution I, a washing solution II, an eluent, and a DNase I digestion solution; the lysis solution comprises sodium citrate at a concentration of 1 to 100 mM, sodium lauroyl sarcosinate at a concentration of 1 to 100 mM, sodium chloride at a concentration of 0.1 to 10 M, sodium metabisulfite at a concentration of 0.01 to 1 M, guanidine hydrochloride at a concentration of 1.5 to 6 M, polyether alcohol at a total volume ratio of 0.1% to 30% to the total volume of the lysis solution, and ethanol at a volume ratio of 30% to 70%.

[0007] As a further improvement of the present invention, the magnetic bead binding solution includes magnetic beads, sodium chloride and tris(hydroxymethylaminomethane), and is prepared with sterile water; the pH value of the magnetic bead binding solution is 6.8-7.2.

[0008] As a further improvement of the present invention, the particle size of the magnetic beads ranges from 0.3 to 2 μm, and the content is from 10 to 200 mg / mL; and the surface of the magnetic beads is modified with hydroxyl groups or carboxyl groups.

[0009] As a further improvement of the present invention, the washing solution I is a mixed aqueous solution containing 2-6 M guanidine hydrochloride, 0.01-100 mM sodium chloride and 30%-50% ethanol.

[0010] As a further improvement of the present invention, the pH value of the DNase I digestion solution is 6.8-7.2; the DNase I digestion solution is an aqueous solution containing DNase I with a mass concentration of 1wt%-10wt%, glycerol with a mass concentration of 30wt%-50wt%, calcium chloride with a concentration of 1-5mM, and tris(hydroxymethyl)aminomethane with a concentration of 10-100mM.

[0011] As a further improvement of the present invention, the concentration of sodium chloride in the magnetic bead binding solution is 0.01-1M, and the concentration of tris(hydroxymethyl)aminomethane is 0.02-1M.

[0012] As a further improvement of the present invention, the washing liquid II is an isopropyl alcohol aqueous solution or an ethanol aqueous solution with a volume fraction of 70% to 85%; the eluent is one of distilled water, deionized water, and ultrapure water.

[0013] A method for extracting intact bacterial total RNA, using a kit for extracting intact bacterial total RNA using the magnetic bead method described above, comprising the following steps:

[0014] S1. Take 1-4 mL of bacterial culture solution and centrifuge it into a centrifuge tube for 1-5 minutes. Discard the supernatant and add 100-200 μL of physiological saline solution. Vortex the mixture to obtain a homogenized bacterial solution.

[0015] S2. Add 300-600 μL of lysate and 10-100 μL of magnetic bead binding solution to the centrifuge tube containing the mixed bacterial solution obtained in step S1, vortex and let stand for 5-15 minutes, then perform magnetic separation using a magnetic stand and discard the liquid;

[0016] S3. Add 200-700 μL of washing solution II to the centrifuge tube treated in step S2, vortex and let stand, then perform magnetic separation using a magnetic stand, discard the liquid, and let stand at room temperature with the lid open for 3-5 minutes;

[0017] S4. Add 80-200 μL of DNase I digestion solution to the centrifuge tube treated in step S3, vortex and let stand at room temperature for 5-10 minutes; continue to add 200-700 μL of washing solution I to the centrifuge tube, vortex and let stand, then perform magnetic separation using a magnetic stand and discard the liquid;

[0018] S5. Add 200-700 μL of washing solution II to the centrifuge tube treated in step S4, vortex and let it stand, then perform magnetic separation using a magnetic stand, discard the liquid, and let it stand at room temperature with the lid open for 3-5 minutes;

[0019] S6. Add 40-160 μL of eluate to the centrifuge tube treated in step S5, vortex and let stand for 1-3 minutes, then perform magnetic separation using a magnetic stand, and aspirate the supernatant into a new centrifuge tube to obtain complete bacterial total RNA.

[0020] As a further improvement of the present invention, in step S2, during the standing process, the centrifuge tube is inverted 5 to 6 times every 5 minutes to mix; in steps S1 to S6, the vortex time is 10 to 15 seconds; in steps S3 to S5, after adding the washing solution I or the washing solution II, the standing time after vortexing is 10 to 30 seconds.

[0021] As a further improvement of the present invention, in step S1, the rotation speed of the centrifugal treatment is 10,000 to 12,000 rpm; in step S2, the magnetic bead-bound solution should be manually inverted and mixed 10 to 20 times before use; in steps S3 to S5, the centrifuge tube should be kept on the magnetic stand when aspirating the liquid.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention provides a kit and method for extracting intact bacterial total RNA using a magnetic bead method. The kit comprises an extraction reagent set for extracting bacterial total RNA; the extraction reagent set includes a magnetic bead binding buffer, a lysis buffer, a washing buffer I, a washing buffer II, an eluent, and a DNase I digestion buffer; the lysis buffer includes sodium citrate, sodium lauroyl sarcosinate, sodium chloride, sodium metabisulfite, guanidine hydrochloride, polyether alcohol, and ethanol. Total RNA extracted using this kit exhibits intact fragments, high concentration, and resistance to degradation after agarose gel electrophoresis. The overall performance of this kit is superior to similar kits on the market, demonstrating promising application prospects and market value.

[0024] 2. The present invention obtains the optimal ratio and extraction conditions of the lysis solution compound system by rationally compounding the various raw materials of the lysis solution, and exerts a synergistic effect between the various raw materials during the lysis and binding process of RNA, so that the lysis solution system has excellent binding force and stable binding to the total bacterial RNA. When the bacteria are lysed by the lysis solution, components such as protein, RNase and RNA in the bacteria are released. At this time, RNase will have a strong degradative effect on RNA. At the same time, protein and RNA are adsorbed together due to electrostatic interaction, which will cause a certain amount of RNA loss; however, when the appropriate concentration of sodium salt sodium citrate, sodium lauroyl sarcosine, sodium chloride, polyether alcohol, reducing agent sodium metabisulfite, guanidine hydrochloride and hydroxyl or carboxyl magnetic beads of the present invention are added, while sodium ions increase the adsorption amount of RNA by the magnetic beads, citrate can effectively protect the group activity on the surface of the magnetic beads. , enhancing the binding ability of magnetic beads to RNA; high concentrations of guanidine hydrochloride can not only lyse bacteria, but also denature proteins, while inhibiting the degradation of RNA by RNase; at the same time, polyether alcohol can keep RNA active to a certain extent, which is more conducive to the formation of "salt bridges" between it and magnetic beads; sodium metabisulfite can prevent RNA from being oxidized and denatured, ensuring the effective binding of magnetic beads and RNA; sodium lauroyl sarcosinate can maintain the stability of the entire system to prevent the system changes of the lysis solution from causing the extraction effect to deteriorate; after being treated with the lysis solution of this system, the integrity of the total RNA finally extracted can be guaranteed.

[0025] 3. The present method for extracting intact bacterial total RNA is highly effective for both Gram-negative and Gram-positive bacteria. It also eliminates the need for the addition of pungent reagents such as mercaptoethanol or dithiothreitol, and pretreatment with lysozyme, saving costs. Furthermore, the reagents used can be stored at room temperature for long periods of time, pose minimal environmental risk, and can be used to replace most similar products on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1These are the electrophoresis results of Escherichia coli and Staphylococcus aureus extracted by the kits of Example 1 of the present invention and Comparative Examples 1 to 2, respectively; wherein, 1 and 2 are the present invention; 3 and 4 are OMEGA; 5 and 6 are Tiangen; odd numbers are Escherichia coli, and even numbers are Staphylococcus aureus. DETAILED DESCRIPTION

[0027] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] It should also be noted here that, in order to avoid obscuring the present invention due to unnecessary details, only structures and / or processing steps closely related to the solutions of the present invention are shown in the drawings, while other details that are not closely related to the present invention are omitted.

[0029] In addition, it should be noted that the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also includes other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0030] A kit for extracting intact bacterial total RNA using a magnetic bead method comprises an extraction reagent set for extracting bacterial total RNA; the extraction reagent set comprises a magnetic bead binding solution, a lysis solution, a washing solution I, a washing solution II, an eluent, and a DNase I digestion solution; the lysis solution comprises 1-100 mM sodium citrate, 1-100 mM sodium lauroyl sarcosinate, 0.1-10 M sodium chloride, 0.01-1 M sodium metabisulfite, 1.5-6 M guanidine hydrochloride, 0.1%-30% polyether alcohol in a total volume ratio to the lysis solution, and 30%-70% ethanol.

[0031] In particular, after the bacteria are lysed by the lysis solution, components such as proteins, RNase and RNA in the bacteria are released. At this time, RNase will have a strong degradative effect on RNA. At the same time, proteins and RNA are adsorbed together due to electrostatic interactions, which will cause a certain amount of RNA loss. However, the present invention adds appropriate concentrations of sodium salts such as sodium citrate, sodium lauroyl sarcosinate, sodium chloride, polyether alcohol, reducing agent sodium metabisulfite, guanidine hydrochloride, and magnetic beads containing hydroxyl or carboxyl groups on their surfaces. While sodium ions increase the amount of RNA adsorbed by the magnetic beads, citrate can effectively protect the group activity on the surface of the magnetic beads, thereby enhancing the binding ability of the magnetic beads to RNA. High concentrations of guanidine hydrochloride can not only lyse bacteria but also denature proteins, while inhibiting RNA degradation by RNase. At the same time, polyether alcohol can maintain RNA activity to a certain extent, which is more conducive to the formation of "salt bridges" between RNA and the magnetic beads. Sodium metabisulfite can prevent RNA from being oxidized and denatured, ensuring effective binding of the magnetic beads and RNA. Sodium lauroyl sarcosinate can maintain the stability of the entire system to prevent changes in the lysate system from causing a deterioration in the extraction effect, thereby ensuring that the extracted RNA fragments are complete.

[0032] Because during the RNA extraction process, it is very easy to denature and degrade during cleavage and binding, and the addition of polyether alcohol can protect its terminal groups from denaturation; and RNA is a single-stranded structure and is easily denatured by environmental factors, so the requirements for antioxidant properties are also higher. The addition of sodium metabisulfite can protect RNA from oxidative denaturation, thereby cooperating with polyether alcohol to prevent the denaturation and degradation of total RNA during the extraction process. Sodium metabisulfite aqueous solution is generally acidic. If it is directly used in RNA extraction, it will affect the stability of the reagent. Therefore, the present invention avoids the influence of sodium metabisulfite on the reagent by regulating the ratio of the lysis solution system; that is, by rationally compounding the various raw materials of the lysis solution, the optimal ratio and extraction conditions of the lysis solution compound system are obtained, and the various raw materials play a synergistic role, so that the lysis solution system has excellent binding force and stable binding properties for bacterial total RNA.

[0033] Specifically, the magnetic bead binding solution includes magnetic beads, sodium chloride and tris(hydroxymethyl)aminomethane, and is prepared with sterile water; the pH value of the magnetic bead binding solution is 6.8-7.2. The particle size range of the magnetic beads is 0.3-2 μm, and the content is 10-200 mg / mL; the surface of the magnetic beads is modified with hydroxyl or carboxyl groups. The concentration of sodium chloride in the magnetic bead binding solution is 0.01-1 M, and the concentration of tris(hydroxymethyl)aminomethane is 0.02-1 M. During the extraction process, RNA will react with the groups on the surface of the magnetic beads and adsorb on the surface of the magnetic beads; therefore, the particle size, content and surface groups of the magnetic beads must be limited accordingly.

[0034] Wash Solution I is an aqueous solution containing 2-6 M guanidine hydrochloride, 0.01-100 mM sodium chloride, and 30%-50% ethanol. Because the lysis system effectively protects the RNA during total RNA lysis and binding, resulting in a high RNA yield, Wash Solution I only needs to perform a routine washing function, reducing reagent usage and saving costs.

[0035] Specifically, the pH value of the DNase I digestion solution is 6.8-7.2; the DNase I digestion solution is an aqueous solution containing 1-10 wt% DNase I, 30-50 wt% glycerol, 1-5 mM calcium chloride, and 10-100 mM tris(hydroxymethyl)aminomethane. Washing solution II is a 70-85% by volume aqueous isopropanol or ethanol solution; and the eluent is selected from distilled water, deionized water, or ultrapure water.

[0036] A method for extracting intact bacterial total RNA, which uses a kit for extracting intact bacterial total RNA using a magnetic bead method, comprises the following steps:

[0037] S1. Take 1-4 mL of bacterial culture solution and centrifuge it in a centrifuge tube for 1-5 minutes at a speed of 10,000-12,000 rpm; then discard the supernatant and add 100-200 μL of physiological saline solution, vortex for 10-15 seconds to obtain a mixed bacterial solution;

[0038] S2. Add 300-600 μL of lysate and 10-100 μL of magnetic bead binding solution to the centrifuge tube containing the mixed bacterial solution obtained in step S1, vortex for 10-15 seconds, and let it stand for 5-15 minutes. During this period, invert the centrifuge tube 5-6 times every 5 minutes to mix it; then perform magnetic separation using a magnetic stand and discard the liquid;

[0039] S3. Add 200-700 μL of washing solution II to the centrifuge tube treated in step S2, vortex for 10-15 seconds, let it stand for 10-30 seconds, then perform magnetic separation using a magnetic stand, discard the liquid, and let it stand at room temperature with the lid open for 3-5 minutes;

[0040] S4. Add 80-200 μL of DNase I digestion solution to the centrifuge tube treated in step S3, vortex and let stand at room temperature for 5-10 minutes; continue to add 200-700 μL of washing solution I to the centrifuge tube, vortex for 10-15 seconds, let stand for 10-30 seconds, and then perform magnetic separation using a magnetic stand, and discard the liquid;

[0041] S5. Add 200-700 μL of washing solution II to the centrifuge tube treated in step S4, vortex for 10-15 seconds, let it stand for 10-30 seconds, then perform magnetic separation using a magnetic stand, discard the liquid, and let it stand at room temperature with the lid open for 3-5 minutes;

[0042] S6. Add 40-160 μL of eluate to the centrifuge tube treated in step S5, vortex for 10-15 seconds, let stand for 1-3 minutes, and then perform magnetic separation using a magnetic stand. Pipette the supernatant into a new centrifuge tube to obtain complete bacterial total RNA.

[0043] Specifically, in step S1, the mixed bacterial solution is turbid; in steps S3 to S5, the centrifuge tube should be kept on the magnetic stand when aspirating the liquid; in step S2, the magnetic bead binding solution should be manually inverted and mixed 10 to 20 times before use.

[0044] The extraction method of the present invention has excellent extraction effects on both Gram-negative and Gram-positive bacteria. It does not require the addition of pungent reagents such as mercaptoethanol or dithiothreitol, nor does it require pretreatment with lysozyme. Furthermore, the reagents used can be stored at room temperature for long periods of time and have minimal environmental pollution, making them suitable alternatives to most similar products on the market.

[0045] Example 1

[0046] This embodiment provides a kit for extracting intact bacterial total RNA using a magnetic bead method and an extraction method thereof. The kit includes an extraction reagent set for extracting bacterial total RNA; the extraction reagent set consists of a magnetic bead binding solution, a lysis solution, a washing solution I, a washing solution II, an eluent, and a DNase I digestion solution. Among them, the magnetic bead binding solution consists of 80 mg / mL magnetic beads, 0.5M sodium chloride, and 0.2M tris(hydroxymethyl)aminomethane; the lysis solution includes 20mM sodium citrate, 40mM sodium lauroyl sarcosinate, 0.2M sodium chloride, 5% polyether alcohol, 0.1M sodium metabisulfite, 2M guanidine hydrochloride and 45% ethanol; the washing solution I is a mixed aqueous solution containing 3M guanidine hydrochloride, 50mM sodium chloride, and 50% ethanol; the washing solution II is an aqueous solution with a volume fraction of 75% ethanol; the eluent is distilled water; the DNase I digestion solution includes an aqueous solution containing DNase I with a mass concentration of 5wt%, 40wt% glycerol, 2.5mM calcium chloride, and 50mM tris(hydroxymethyl)aminomethane.

[0047] The method for extracting RNA from Escherichia coli / Staphylococcus aureus using the above-mentioned kit for extracting intact bacterial total RNA by magnetic bead method comprises the following steps:

[0048] S1. Take 1-4 mL of fresh overnight bacterial culture medium into a centrifuge tube, centrifuge at 10,000-12,000 rpm for 1-5 minutes, discard the supernatant, add 100-200 μL of physiological saline solution, vortex for 10-15 seconds to mix the bacterial solution;

[0049] S2. Add 300-600 μL of lysis buffer and 10-100 μL of magnetic bead binding solution to the bacterial solution obtained in step S1, vortex for 10-15 seconds, and let it stand for 5-15 minutes. During this period, invert and mix 5-6 times every 5 minutes, then perform magnetic separation using a magnetic stand and discard the liquid;

[0050] S3. Add 200-700 μL of washing solution II to the centrifuge tube in step S2, vortex for 10-15 seconds, let it stand for 10-30 seconds, then perform magnetic separation using a magnetic stand, discard the liquid, and let it stand at room temperature with the lid open for 3-5 minutes;

[0051] S4. Add 80-200 μL of DNase I digestion solution to the centrifuge tube in step S3, vortex for 10-15 seconds, and let stand at room temperature for 5-10 minutes. Then, add 200-700 μL of washing solution I to the centrifuge tube, vortex for 10-15 seconds, and let stand for 10-30 seconds. Then, perform magnetic separation using a magnetic stand and discard the liquid;

[0052] S5. Add 200-700 μL of washing solution II to the centrifuge tube in step S4, vortex for 10-15 seconds, let it stand for 10-30 seconds, then perform magnetic separation using a magnetic stand, discard the liquid, and let it stand at room temperature with the lid open for 3-5 minutes;

[0053] S6. Add 40-160 μL of elution buffer to the centrifuge tube in step S5, vortex for 10-15 seconds, let it stand for 1-3 minutes, and then perform magnetic separation using a magnetic stand. Pipette the supernatant into a new centrifuge tube to obtain total bacterial RNA.

[0054] Comparative Examples 1-2

[0055] Comparative Example 1: The total RNA of Escherichia coli / Staphylococcus aureus was extracted using a commercially available magnetic bead total RNA extraction kit (M6930) from OMEGA.

[0056] Comparative Example 2: The total RNA of Escherichia coli / Staphylococcus aureus was extracted using a commercially available bacterial culture total RNA extraction kit (DP430) from Tiangen Biochemical Technology Co., Ltd.

[0057] See also Figure 1 As shown, Figure 1The electrophoresis results of the kits for Example 1 and Comparative Examples 1-2 are respectively extracted for Escherichia coli and Staphylococcus aureus; wherein 1 and 2 are for Example 1, 3 and 4 are for Comparative Example 1, and 5 and 6 are for Comparative Example 2; odd numbers are for Escherichia coli, and even numbers are for Staphylococcus aureus. Figure 1 As can be seen, the total RNA fragments extracted from E. coli and S. aureus using the kit of Example 1 of the present invention are complete and clear. The M6930 kit of Comparative Example 1 only has a good extraction effect on total RNA from E. coli, but is still inferior to the extraction effect of Example 1 for RNA from E. coli, and its extraction effect on total RNA from S. aureus is poor. The DP430 kit of Comparative Example 2 has poor extraction effects on total RNA from both E. coli and S. aureus.

[0058] Examples 2 to 5 and Comparative Examples 3 to 6

[0059] Examples 2 to 5 and Comparative Examples 3 to 6 provide a kit for extracting intact bacterial total RNA using a magnetic bead method and an extraction method thereof. Compared with Example 1, the difference is that the content of polyether alcohol and the concentration of sodium metabisulfite in the lysate are shown in the table below. The rest is roughly the same as in Example 1 and will not be repeated here.

[0060] Table 1 Parameters of polyether alcohol and sodium metabisulfite in the kits of Examples 2 to 5 and Comparative Examples 3 to 6

[0061] Polyether alcohol / % Sodium metabisulfite / M Example 2 0.1 0.1 Example 3 30 0.1 Example 4 5 0.01 Example 5 5 1 Comparative Example 3 - 0.1 Comparative Example 4 5 - Comparative Example 5 40 0.1 Comparative Example 6 5 3

[0062] The total RNA of Escherichia coli and Staphylococcus aureus (5×10E5 bacteria) extracted in Examples 1 to 5 and Comparative Examples 3 to 6 was subjected to quality testing (purity range of 1.9-2.2). The results are shown in the following table.

[0063] Table 2 Quality test results of total RNA extracted from Examples 2 to 5 and Comparative Examples 3 to 6

[0064]

[0065] As shown in Table 2, when the polyether alcohol content is in the range of 0.1% to 30% and the sodium metabisulfite concentration is in the range of 0.01 to 1M, the concentration and purity of the extracted Escherichia coli and Staphylococcus aureus are both high, indicating that the total bacterial RNA extracted using this lysis solution system has excellent binding capacity and stable binding properties. As can be seen from Comparative Examples 3 to 4, when the lysis solution does not contain polyether alcohol, the terminal groups of the RNA are easily denatured and degraded, resulting in a decrease in its purity; when sodium metabisulfite is not present, the RNA is easily affected by environmental factors and oxidized, resulting in a decrease in nucleic acid concentration. As can be seen from Comparative Examples 5 to 6, when the content of both polyether alcohol and sodium metabisulfite is too high, the nucleic acid content and purity are both low, because sodium metabisulfite aqueous solution is generally acidic, and if added too much, it will affect the stability of the reagent; similarly, excessive polyether alcohol will destroy the composite system of the various raw materials in the lysis solution, which will have an adverse effect on the extraction process of bacterial total RNA.

[0066] In summary, the present invention provides a kit for extracting intact bacterial total RNA using a magnetic bead method and an extraction method thereof. The kit comprises an extraction reagent set for extracting bacterial total RNA; the extraction reagent set comprises a magnetic bead binding solution, a lysis solution, a washing solution I, a washing solution II, an eluent, and a DNase I digestion solution; the lysis solution comprises sodium citrate at a concentration of 1 to 100 mM, sodium lauroyl sarcosinate at a concentration of 1 to 100 mM, sodium chloride at a concentration of 0.1 to 10 M, sodium metabisulfite at a concentration of 0.01 to 1 M, guanidine hydrochloride at a concentration of 1.5 to 6 M, polyether alcohol at a total volume ratio of 0.1% to 30% to the total volume of the lysis solution, and ethanol at a concentration of 30% to 70%. When the bacteria are lysed by the lysis buffer, appropriate concentrations of sodium salts such as sodium citrate, sodium lauroyl sarcosinate, sodium chloride, polyether alcohol, reducing agent sodium metabisulfite, guanidine hydrochloride, and magnetic beads with hydroxyl or carboxyl groups on the surface are added; while sodium ions increase the amount of RNA adsorbed by the magnetic beads, citrate can effectively protect the group activity on the surface of the magnetic beads and enhance the binding ability of the magnetic beads to RNA; high concentrations of guanidine hydrochloride can not only lyse bacteria, but also denature proteins and inhibit RNase degradation of RNA; at the same time, polyether alcohol can maintain the activity of RNA to a certain extent, which is more conducive to the formation of "salt bridges" between it and the magnetic beads; sodium metabisulfite can prevent RNA from being oxidized and denatured, ensuring the effective binding of magnetic beads and RNA; sodium lauroyl sarcosinate can maintain the stability of the entire system to prevent changes in the lysis buffer system from causing poor extraction results, so that the extracted RNA fragments are complete. The present invention rationally compounds the various lysate ingredients to achieve the optimal ratio and extraction conditions for the lysate compound system. This allows the ingredients to synergize during RNA lysis and binding, resulting in a lysate system with excellent and stable binding to bacterial total RNA. Total RNA extracted using this kit exhibits intact fragments, high concentration, and resistance to degradation after agarose gel electrophoresis. The overall performance of this kit is superior to similar kits on the market, demonstrating promising application prospects and market value.

[0067] 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, it should be understood by those skilled in the art 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.

Claims

1. A kit for extracting intact bacterial total RNA using a magnetic bead method, characterized in that: The invention comprises an extraction reagent group for extracting total bacterial RNA; the extraction reagent group comprises a magnetic bead binding solution, a lysis solution, a washing solution I, a washing solution II, an eluent and a DNase I digestion solution; the lysis solution comprises sodium citrate with a concentration of 1 to 100 mM, sodium lauroyl sarcosinate with a concentration of 1 to 100 mM, sodium chloride with a concentration of 0.1 to 10 M, sodium metabisulfite with a concentration of 0.01 to 1 M, guanidine hydrochloride with a concentration of 1.5 to 6 M, polyether alcohol with a total volume ratio of 0.1% to 30% to the total volume of the lysis solution, and ethanol with a concentration of 30% to 70%; the magnetic bead binding solution comprises magnetic beads, sodium chloride and tris(hydroxymethyl)aminomethane, and the surfaces of the magnetic beads are modified with hydroxyl groups or carboxyl groups; the sodium ions of the sodium citrate increase the adsorption capacity of the magnetic beads to RNA, while the citrate radicals protect the group activity on the surface of the magnetic beads.

2. The kit for extracting intact bacterial total RNA by magnetic bead method according to claim 1, characterized in that The magnetic bead binding liquid is prepared from sterile water; the pH value of the magnetic bead binding liquid is 6.8-7.

2.

3. The kit for extracting intact bacterial total RNA by magnetic bead method according to claim 2, characterized in that, The particle size of the magnetic beads ranges from 0.3 to 2 μm, and the content is from 10 to 200 mg / mL.

4. The kit for extracting intact bacterial total RNA by magnetic bead method according to claim 1, characterized in that The washing solution I is a mixed aqueous solution containing 2-6M guanidine hydrochloride, 0.01-100mM sodium chloride and 30%-50% ethanol.

5. The kit for extracting intact bacterial total RNA by magnetic bead method according to claim 1, characterized in that The pH value of the DNase I digestion solution is 6.8-7.2; the DNase I digestion solution is an aqueous solution containing DNase I with a mass concentration of 1wt%-10wt%, glycerol with a mass concentration of 30wt%-50wt%, calcium chloride with a concentration of 1-5mM, and tris(hydroxymethyl)aminomethane with a mass concentration of 10-100mM.

6. The kit for extracting intact bacterial total RNA by magnetic bead method according to claim 2, characterized in that: The concentration of sodium chloride in the magnetic bead binding solution is 0.01-1M, and the concentration of tris(hydroxymethyl)aminomethane is 0.02-1M.

7. The kit for extracting intact bacterial total RNA by magnetic bead method according to claim 1, characterized in that: The washing liquid II is an isopropyl alcohol aqueous solution or an ethanol aqueous solution with a volume fraction of 70% to 85%; the eluent is one of distilled water, deionized water, and ultrapure water.

8. A method for extracting intact bacterial total RNA, characterized in that: A kit for extracting intact bacterial total RNA using the magnetic bead method according to any one of claims 1 to 7, comprising the following steps: S1. Take 1-4 mL of bacterial culture solution and centrifuge it into a centrifuge tube for 1-5 minutes. Discard the supernatant and add 100-200 μL of physiological saline solution. Vortex the mixture to obtain a homogenized bacterial solution. S2. Add 300-600 μL of lysate and 10-100 μL of magnetic bead binding solution to the centrifuge tube containing the mixed bacterial solution obtained in step S1, vortex and let stand for 5-15 minutes, then perform magnetic separation using a magnetic stand and discard the liquid; S3. Add 200-700 μL of washing solution II to the centrifuge tube treated in step S2, vortex and let stand, then perform magnetic separation using a magnetic stand, discard the liquid, and let stand at room temperature with the lid open for 3-5 minutes; S4. Add 80-200 μL of DNase I digestion solution to the centrifuge tube treated in step S3, vortex and let stand at room temperature for 5-10 minutes; continue to add 200-700 μL of washing solution I to the centrifuge tube, vortex and let stand, then perform magnetic separation using a magnetic stand and discard the liquid; S5. Add 200-700 μL of washing solution II to the centrifuge tube treated in step S4, vortex and let it stand, then perform magnetic separation using a magnetic stand, discard the liquid, and let it stand at room temperature with the lid open for 3-5 minutes; S6. Add 40-160 μL of eluate to the centrifuge tube treated in step S5, vortex and let stand for 1-3 minutes, then perform magnetic separation using a magnetic stand, and aspirate the supernatant into a new centrifuge tube to obtain complete bacterial total RNA.

9. The extraction method of the kit for extracting intact bacterial total RNA by magnetic bead method according to claim 8, characterized in that, In step S2, during the standing process, the centrifuge tube is inverted 5 to 6 times every 5 minutes to mix; in steps S1 to S6, the vortex time is 10 to 15 seconds; in steps S3 to S5, after adding the washing solution I or the washing solution II, the standing time after vortexing is 10 to 30 seconds.

10. The extraction method of the kit for extracting intact bacterial total RNA by magnetic bead method according to claim 8, characterized in that, In step S1, the rotation speed of the centrifugation is 10,000 to 12,000 rpm; in step S2, the magnetic bead-bound solution should be manually inverted and mixed 10 to 20 times before use; in steps S3 to S5, the centrifuge tube should be kept on the magnetic stand when aspirating the liquid.

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