CN115305246A - Kit for extracting complete bacteria total RNA by paramagnetic particle method and extraction method thereof - Google Patents
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Abstract
The invention provides a kit for extracting complete bacteria total RNA by a paramagnetic particle method and an extraction method thereof, wherein the kit comprises an extraction reagent group for extracting the bacteria total RNA; the extraction reagent group comprises magnetic bead binding solution, lysis solution, washing solution I, washing solution II, eluent and DNaseI digestive juice; the lysis solution comprises sodium citrate, sodium lauroyl sarcosinate, sodium chloride, sodium metabisulfite, guanidine hydrochloride, polyether alcohol and ethanol. When bacteria are cracked by the lysate, the raw materials of the lysate are reasonably compounded to play a synergistic role in the RNA cracking and combining process, and particularly, the polyether alcohol and the sodium metabisulfite are matched, so that the lysate system has excellent binding force and stable binding property on the total RNA of the bacteria. The total RNA fragment extracted by the kit is complete, high in concentration and not easy to degrade, has overall performance superior to that of the kits of the same type in the market, and has better application prospect and market value.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a kit for extracting complete bacteria total RNA by a paramagnetic particle method and an extraction method thereof.
Background
RNA is important genetic material in organisms, and the extraction of RNA with high purity and good integrity is an important prerequisite for the research of microbial molecular biology. For downstream molecular biology experiments such as cDNA library construction, in vitro reverse transcription, fluorescent quantitative PCR, RT-PCR, northern hybridization analysis and the like, RNA with good quality and high integrity is required. Compared with eukaryotic cells such as animals and plants, the bacteria have relatively low RNA content and shorter half-life period, and are easily degraded by external RNase (ribonuclease) due to no 5 'cap structure and 3' Poly (A) tail. Meanwhile, polysaccharide polyphenol contained in cells and RNA form insoluble substances, so that the RNA is difficult to separate, and the concentration and purity of the extracted RNA are reduced. Therefore, researchers often employ improved methods to increase the yield and purity of RNA.
Common methods for extracting bacterial RNA include a strong denaturing agent method, a Trizol method, an SDS-Phenol method, a CTAB method, a hot boric acid method, and the like. The extraction method has good inhibition effect on the RNase in the extraction process, and the purity of the extracted RNA product is high, so that the subsequent downstream experiments can be met. On one hand, most of the extraction methods use organic solvents with strong toxicity, so that environmental pollution and potential safety problems are caused; on the other hand, most of the methods require a centrifuge, which seriously reduces the extraction efficiency of RNA and is not favorable for the development of industrialization. At present, many reagent companies at home and abroad develop RNA extraction kits by a magnetic bead method, but the kits have the defects of incomplete extraction fragments, low purity, low concentration, requirement for lysozyme pretreatment, requirement for additional addition of chemical reagents and the like.
In view of the above, there is a need to design an improved kit for extracting total RNA from bacteria by the magnetic bead method and an extraction method thereof, so as to solve the above problems.
Disclosure of Invention
The invention aims to provide a kit for extracting total RNA of complete bacteria by a paramagnetic particle method and an extraction method thereof, wherein the kit adopts sodium metabisulfite and polyether alcohol, so that the defect that the RNA is easily deformed and degraded in the cracking and combining process is overcome; the total RNA of bacteria extracted by the method has the advantages of complete fragments, high concentration and difficult degradation, has overall performance superior to that of the kits of the same type in the market, and has better application prospect and market value.
In order to realize the purpose, the invention provides a kit for extracting complete bacteria total RNA by a magnetic bead method, which comprises an extraction reagent group for extracting bacteria total RNA; the extraction reagent group comprises magnetic bead binding solution, lysis solution, washing solution I, washing solution II, eluent and DNase I digestive juice; the lysate comprises 1-100 mM of sodium citrate, 1-100 mM of sodium lauroyl sarcosinate, 0.1-10M of sodium chloride, 0.01-1M of sodium metabisulfite, 1.5-6M of guanidine hydrochloride, 0.1-30% of polyether alcohol and 30-70% of ethanol, wherein the volume ratio of the polyether alcohol to the total volume of the lysate is 0.1-30%.
As a further improvement of the invention, the magnetic bead binding solution comprises magnetic beads, sodium chloride and tris (hydroxymethyl) aminomethane and is prepared from sterilized water; the pH value of the magnetic bead binding solution is 6.8-7.2.
As a further improvement of the invention, 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 bead is modified with hydroxyl or carboxyl.
As a further improvement of the invention, the washing solution I is a mixed aqueous solution containing 2-6M guanidine hydrochloride, 0.01-100 mM sodium chloride and 30-50% ethanol.
As a further improvement of the invention, the pH value of the DNase I digestive juice is 6.8-7.2; the DNase I digestive juice is an aqueous solution containing 1-10 wt% of DNase I, 30-50 wt% of glycerol, 1-5 mM of calcium chloride and 10-100 mM of tris (hydroxymethyl) aminomethane.
In a further improvement of the present invention, the concentration of sodium chloride in the magnetic bead binding solution is 0.01 to 1M, and the concentration of tris (hydroxymethyl) aminomethane is 0.02 to 1M.
As a further improvement of the invention, the washing solution II is an isopropanol aqueous solution or an ethanol aqueous solution with the volume fraction of 70-85%; the eluent is one of distilled water, deionized water and ultrapure water.
A method for extracting complete bacterial total RNA adopts the kit for extracting complete bacterial total RNA by the magnetic bead method, and comprises the following steps:
s1, taking 1-4 mL of bacteria culture solution to a centrifuge tube, carrying out centrifugal treatment for 1-5 minutes, removing supernatant, adding 100-200 mu L of physiological saline solution, and carrying out vortex treatment to obtain a uniformly mixed bacterial solution;
s2, respectively adding 300-600 mu L of lysis solution and 10-100 mu L of magnetic bead binding solution into the centrifugal tube filled with the uniformly mixed bacterial solution obtained in the step S1, standing for 5-15 minutes after vortex, then performing magnetic separation by using a magnetic frame, and sucking away the liquid;
s3, adding 200-700 mu L of washing liquid II into the centrifuge tube treated in the step S2, performing vortex and standing, performing magnetic separation by using a magnetic frame, removing liquid, and opening a cover to stand at room temperature for 3-5 minutes;
s4, adding 80-200 mu L of DNase I digestive juice into the centrifuge tube treated in the step S3, and standing for 5-10 minutes at room temperature after vortexing; continuously adding 200-700 mu L of washing solution I into the centrifugal tube, standing after vortex, then carrying out magnetic separation by using a magnetic frame, and absorbing and discarding the liquid;
s5, adding 200-700 mu L of washing liquid II into the centrifuge tube treated in the step S4, performing vortex and standing, performing magnetic separation by using a magnetic frame, removing liquid, and opening a cover to stand at room temperature for 3-5 minutes;
s6, adding 40-160 mu L of eluent into the centrifuge tube processed in the step S5, standing for 1-3 minutes after vortex, then carrying out magnetic separation by using a magnetic frame, and absorbing supernatant liquid into a new centrifuge tube to obtain the complete bacterial total RNA.
As a further improvement of the invention, in step S2, in the standing process, the centrifuge tube is reversed 5 to 6 times every 5 minutes to mix evenly; in the steps S1 to S6, the time of the vortex is 10 to 15 seconds; in steps S3 to S5, after the washing solution I or the washing solution II is added, the time for standing after vortex is 10 to 30 seconds.
As a further improvement of the invention, in step S1, the rotation speed of the centrifugal treatment is 10000-12000 rpm; in step S2, the magnetic bead binding solution is manually turned over and uniformly mixed for 10 to 20 times before use; in steps S3 to S5, the centrifuge tube is held on the magnetic rack when the liquid is discarded.
The invention has the beneficial effects that:
1. the invention provides a kit for extracting complete bacteria total RNA by a paramagnetic particle method and an extraction method thereof, wherein the kit comprises an extraction reagent group for extracting the bacteria total RNA; the extraction reagent group comprises magnetic bead binding solution, lysis solution, washing solution I, washing solution II, eluent and DNase I digestive juice; the lysis solution comprises sodium citrate, sodium lauroyl sarcosinate, sodium chloride, sodium metabisulfite, guanidine hydrochloride, polyether alcohol and ethanol. The total RNA extracted by the kit is complete in fragment, high in concentration and not easy to degrade after agarose gel electrophoresis; the overall performance is superior to that of the same type of kits in the market, and the kit has better application prospect and market value.
2. According to the invention, the raw materials of the lysis solution are reasonably compounded to obtain the optimal proportion and extraction condition of the lysis solution compounding system, and the raw materials play a synergistic effect in the RNA cracking and combining process, so that the lysis solution system has excellent binding force and stable binding property to the total RNA of bacteria. When the bacteria are cracked by the lysate, the components such as protein, RNase and RNA in the bacteria are all released, at the moment, the RNase has strong degradability on the RNA, and meanwhile, the protein and the RNA are mutually adsorbed together due to electrostatic interaction, so that certain RNA loss is caused; however, after sodium salt sodium citrate, sodium lauroyl sarcosinate, sodium chloride, polyether alcohol, reducing agent sodium metabisulfite, guanidine hydrochloride and hydroxyl or carboxyl magnetic beads with proper concentration are added, the sodium ions increase the RNA adsorption amount of the magnetic beads, and simultaneously, the citrate can effectively protect the group activity on the surfaces of the magnetic beads and enhance the RNA binding capacity of the magnetic beads; the high-concentration guanidine hydrochloride can not only crack bacteria, but also denature protein and inhibit the degradation of RNase to RNA; meanwhile, the polyether alcohol can keep the activity of the RNA to a certain extent, and is more beneficial to the generation of a salt bridge between the RNA and the magnetic beads; sodium pyrosulfite can prevent RNA from being oxidized and denatured, and ensures effective combination of magnetic beads and RNA; the lauroyl sodium sarcosinate can keep the stability of the whole system so as to prevent the degradation of the extraction effect caused by the system change of the cracking liquid; the integrity of the finally extracted total RNA can be ensured by the treatment of the lysate of the system.
3. The extraction method of the complete bacteria total RNA has excellent extraction effect on gram-negative bacteria and gram-positive bacteria, and in the extraction process, agents with pungent odor such as mercaptoethanol or dithiothreitol do not need to be additionally added, lysozyme does not need to be used for pretreatment, and the cost is saved. Meanwhile, the used reagent can be stored at normal temperature for a long time, has small environmental pollution and can be used for replacing most of like products on the market.
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FIG. 1 shows the results of electrophoresis of E.coli and Staphylococcus aureus extracted from the kits of example 1 and comparative examples 1-2, respectively, according to the present invention; wherein, 1 and 2 are the invention; 3. 4 is OMEGA; 5. 6 is Tiangen; the odd number is colibacillus and the even number is staphylococcus aureus.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
It should be noted that, in order to avoid obscuring the present invention with unnecessary details, only the structures and/or processing steps closely related to the aspects of the present invention are shown in the drawings, and other details not closely related to the present invention are omitted.
In addition, it is also to be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
A kit for extracting complete bacteria total RNA by a paramagnetic particle method comprises an extraction reagent group for extracting bacteria total RNA; the extraction reagent group comprises magnetic bead binding solution, lysis solution, washing solution I, washing solution II, eluent and DNase I digestive juice; the lysate comprises 1-100 mM sodium citrate, 1-100 mM sodium lauroyl sarcosinate, 0.1-10M sodium chloride, 0.01-1M sodium metabisulfite, 1.5-6M guanidine hydrochloride, 0.1-30% of polyether alcohol and 30-70% of ethanol, wherein the total volume ratio of the lysate to the polyether alcohol is 0.1-30%.
Particularly, after the bacteria are lysed by the lysis solution, components such as protein, RNase and RNA in the bacteria are all released, and at the moment, the RNase has strong degradability on the RNA, and meanwhile, the protein and the RNA are adsorbed together due to electrostatic interaction, so that certain RNA loss is caused. However, after sodium citrate, sodium lauroyl sarcosinate, sodium chloride, polyether alcohol, a reducing agent sodium metabisulfite, guanidine hydrochloride and magnetic beads with hydroxyl or carboxyl groups on the surfaces are added, sodium ions increase the RNA adsorption amount of the magnetic beads, and meanwhile, the citrate can effectively protect the group activity on the surfaces of the magnetic beads and enhance the RNA binding capacity of the magnetic beads; the high-concentration guanidine hydrochloride can not only crack bacteria, but also denature protein and inhibit the degradation of RNase to RNA; meanwhile, the polyether alcohol can keep the activity of the RNA to a certain degree, and salt bridges between the RNA and magnetic beads can be generated; sodium metabisulfite can prevent RNA from being oxidized and denatured, and effective combination of magnetic beads and RNA is ensured; the lauroyl sodium sarcosinate can keep the stability of the whole system so as to prevent the degradation of the extraction effect caused by the system change of the cracking liquid; the extracted RNA fragments were left intact.
Because in the process of RNA extraction, the RNA is very easy to denature and degrade when in cleavage combination, and the addition of the polyether alcohol can protect the terminal group of the RNA from denaturation; and the RNA is of a single-stranded structure and is easy to denature under the influence of environmental factors, so the requirement on oxidation resistance is higher, and the addition of sodium metabisulfite can protect the RNA from oxidative denaturation, so that the action of matching polyether alcohol to prevent the denaturation and degradation of the total RNA in the extraction process is realized. The sodium metabisulfite aqueous solution is generally acidic, and can influence the stability of the reagent if being directly applied to the aspect of RNA extraction, so the invention avoids the influence of the sodium metabisulfite on the reagent by regulating and controlling the proportion of a lysate system; the raw materials of the lysis solution are reasonably compounded to obtain the optimal proportion and extraction conditions of a lysis solution compounding system, and the raw materials play a synergistic effect, so that the lysis solution system has excellent binding force and stable binding property to the total RNA of bacteria.
Specifically, the magnetic bead binding solution comprises magnetic beads, sodium chloride and tris (hydroxymethyl) aminomethane and is prepared from sterilized 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 mu m, and the content is 10-200 mg/mL; the surface of the magnetic bead is modified with hydroxyl or carboxyl. 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. RNA can react with groups on the surface of the magnetic beads in the extraction process and is adsorbed on the surface of the magnetic beads; the particle size, content and surface groups of the magnetic beads are accordingly limited.
The washing solution I is a mixed aqueous solution containing 2-6M guanidine hydrochloride, 0.01-100 mM sodium chloride and 30-50% ethanol. In the process of total RNA cracking combination, the cracking system has a good protection effect on RNA, and the obtained RNA amount is high, so that the washing solution I only needs to play a conventional washing effect, the use of reagents is reduced, and the cost is saved.
Specifically, the pH value of the DNase I digestive juice is 6.8-7.2; the DNase I digestive juice is an aqueous solution containing 1-10 wt% of DNase I, 30-50 wt% of glycerol, 1-5 mM of calcium chloride and 10-100 mM of tris (hydroxymethyl) aminomethane. The washing liquid II is an isopropanol water solution or an ethanol water solution with the volume fraction of 70-85%; the eluent is one of distilled water, deionized water and ultrapure water.
A method for extracting total RNA of intact bacteria adopts a kit for extracting the total RNA of the intact bacteria by a magnetic bead method, and comprises the following steps:
s1, taking 1-4 mL of bacteria culture solution to a centrifuge tube for centrifugal treatment for 1-5 minutes at the rotation speed of 10000-12000 r/min; then removing supernatant liquid, adding 100-200 mu L of physiological saline solution, and carrying out vortex for 10-15 seconds to obtain uniformly mixed bacterial liquid;
s2, respectively adding 300-600 mu L of lysis solution and 10-100 mu L of magnetic bead binding solution into the centrifugal tube filled with the uniformly mixed bacterial solution obtained in the step S1, swirling for 10-15 seconds, standing for 5-15 minutes, and reversing the centrifugal tube 5-6 times every 5 minutes to uniformly mix; then, carrying out magnetic separation by using a magnetic frame, and absorbing and discarding the liquid;
s3, adding 200-700 mu L of washing liquid II into the centrifuge tube treated in the step S2, whirling for 10-15 seconds, standing for 10-30 seconds, then performing magnetic separation by using a magnetic frame, sucking away liquid, uncovering, and standing for 3-5 minutes at room temperature;
s4, adding 80-200 mu L of DNase I digestive juice into the centrifuge tube treated in the step S3, and standing for 5-10 minutes at room temperature after vortexing; continuously adding 200-700 mu L of washing liquid I into the centrifuge tube, whirling for 10-15 seconds, standing for 10-30 seconds, then performing magnetic separation by using a magnetic frame, and absorbing and discarding liquid;
s5, adding 200-700 mu L of washing liquid II into the centrifuge tube treated in the step S4, whirling for 10-15 seconds, standing for 10-30 seconds, then performing magnetic separation by using a magnetic frame, sucking away liquid, uncovering, and standing for 3-5 minutes at room temperature;
s6, adding 40-160 mu L of eluent into the centrifuge tube processed in the step S5, vortexing for 10-15 seconds, standing for 1-3 minutes, then performing magnetic separation by using a magnetic frame, and absorbing supernatant liquid into a new centrifuge tube to obtain the complete bacteria total RNA.
Specifically, in step S1, the mixed bacteria solution is turbid; in the steps S3 to S5, the centrifugal tube is kept on the magnetic frame when the liquid is sucked and discarded; in step S2, the magnetic bead-binding solution should be mixed by hand in reverse for 10 to 20 times before use.
The extraction method has excellent extraction effect on gram-negative bacteria and gram-positive bacteria, and does not need to additionally add reagents with pungent odor such as mercaptoethanol or dithiothreitol and the like in the extraction process, and does not need to use lysozyme for pretreatment. Meanwhile, the used reagent can be stored at normal temperature for a long time, has little pollution to the environment and can be used for replacing most similar products on the market.
Example 1
The embodiment provides a kit for extracting complete bacterial total RNA by a paramagnetic particle method and an extraction method thereof, wherein the kit comprises an extraction reagent group for extracting the bacterial total RNA; the extraction reagent group consists of magnetic bead binding solution, lysis solution, washing solution I, washing solution II, eluent and DNase I digestive juice. Wherein the magnetic bead binding solution consists of 80mg/mL magnetic beads, 0.5M sodium chloride and 0.2M tris (hydroxymethyl) aminomethane; the lysate included 20mM sodium citrate, 40mM sodium lauroyl sarcosinate, 0.2M sodium chloride, 5% polyetherol, 0.1M sodium metabisulfite, 2M guanidine hydrochloride and 45% ethanol; washing solution I is a mixed water solution containing 3M guanidine hydrochloride, 50mM sodium chloride and 50% ethanol; the washing liquid II is ethanol water solution with volume fraction of 75%; the eluent is distilled water; the DNase I digestion solution comprises an aqueous solution containing DNase I with a mass concentration of 5wt%, glycerol with a mass concentration of 40wt%, calcium chloride with a concentration of 2.5mM, and tris (hydroxymethyl) aminomethane with a concentration of 50 mM.
The method for extracting the Escherichia coli/staphylococcus aureus RNA by using the kit for extracting the complete bacteria total RNA by the paramagnetic particle method comprises the following steps:
s1, taking 1-4 mL of fresh overnight-cultured bacteria culture solution into a centrifuge tube, centrifuging for 1-5 minutes at 10000-12000 rpm, discarding supernatant, adding 100-200 mu L of physiological saline solution, swirling for 10-15 seconds, and uniformly mixing the bacteria solution;
s2, respectively adding 300-600 mu L of lysis solution and 10-100 mu L of magnetic bead binding solution into the bacterial solution obtained in the step S1, carrying out vortex for 10-15 seconds, standing for 5-15 minutes, reversing and uniformly mixing for 5-6 times every 5 minutes, then carrying out magnetic separation by using a magnetic frame, and absorbing and discarding the liquid;
s3, adding 200-700 mu L of washing liquid II into the centrifugal tube in the step S2, carrying out vortex for 10-15 seconds, then standing for 10-30 seconds, then carrying out magnetic separation by using a magnetic frame, removing the liquid, and standing for 3-5 minutes at room temperature after uncovering;
s4, adding 80-200 mu L of DNase I digestive juice into the centrifuge tube obtained in the step S3, vortexing for 10-15 seconds, standing for 5-10 minutes at room temperature, then adding 200-700 mu L of washing liquid I into the centrifuge tube, vortexing for 10-15 seconds, standing for 10-30 seconds, then performing magnetic separation by using a magnetic frame, and absorbing and discarding liquid;
s5, adding 200-700 mu L of washing liquid II into the centrifugal tube in the step S4, carrying out vortex for 10-15 seconds, then standing for 10-30 seconds, then carrying out magnetic separation by using a magnetic frame, removing the liquid, and standing for 3-5 minutes at room temperature after uncovering;
s6, adding 40-160 mu L of eluent into the centrifuge tube in the step S5, swirling for 10-15 seconds, standing for 1-3 minutes, then carrying out magnetic separation by using a magnetic frame, and absorbing supernatant liquid into a new centrifuge tube to obtain the total RNA of the bacteria.
Comparative examples 1 to 2
Comparative example 1 extraction of total RNA from Escherichia coli/Staphylococcus aureus was performed using a commercially available magnetic bead method total RNA extraction kit (M6930) from OMEGA.
Comparative example 2 extraction of total RNA from Escherichia coli/Staphylococcus aureus was carried out using a commercially available kit for extracting total RNA from cultured bacteria (DP 430) from Tiangen Biochemical technology.
Referring to fig. 1, fig. 1 shows the electrophoresis results of the kit for example 1 and comparative examples 1-2, which respectively extracts escherichia coli and staphylococcus aureus; wherein, 1, 2 are examples 1,3, 4 are comparative examples 1,5, 6 are comparative example 2; the odd number is colibacillus and the even number is staphylococcus aureus. As can be seen from FIG. 1, the total RNA fragments of Escherichia coli and Staphylococcus aureus extracted by the kit of example 1 of the present invention are complete and clear; the M6930 kit of comparative example 1 has a better effect of extracting total RNA of Escherichia coli, but is still inferior to that of example 1, and has a poor effect of extracting total RNA of Staphylococcus aureus. The DP430 kit of comparative example 2 was less effective in extracting total RNA from both e.
Examples 2 to 5 and comparative examples 3 to 6
Examples 2 to 5 and comparative examples 3 to 6 provide a kit for extracting total RNA from intact bacteria by a magnetic bead method and an extraction method thereof, which are different from example 1 in that the content of polyether alcohol and the concentration of sodium metabisulfite in a lysate are shown in the following table, and the rest are substantially the same as example 1, and are not repeated herein.
TABLE 1 parameters of polyether alcohol and sodium metabisulfite in the kits of examples 2-5 and comparative examples 3-6
| 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 |
Total RNA of Escherichia coli and Staphylococcus aureus (bacteria amount 5X 10E 5) extracted in examples 1 to 5 and comparative examples 3 to 6 was subjected to quality measurement (purity range 1.9-2.2), and the results are shown in the following table.
TABLE 2 results of quality test of total RNA extracted in examples 2 to 5 and comparative examples 3 to 6
As can be seen from Table 2, when the polyether alcohol content is in the range of 0.1% -30% and the sodium metabisulfite concentration is in the range of 0.01-1M, the concentration and purity of the extracted Escherichia coli and Staphylococcus aureus are high, which indicates that the total bacterial RNA extracted by using the lysate system has excellent binding force and stable binding property. As can be seen from comparative examples 3 to 4, when the lysate does not contain polyether alcohol, the terminal group of RNA is easily denatured and degraded, resulting in a decrease in purity; when sodium metabisulfite is not included, RNA is susceptible to oxidation by environmental factors, resulting in a decrease in nucleic acid concentration. As can be seen from comparative examples 5 to 6, when the polyether alcohol and sodium metabisulfite contents are both too high, the nucleic acid content and purity are low, since the aqueous sodium metabisulfite solution is generally acidic and, if added too much, affects the stability of the reagent; similarly, the excessive amount of polyether alcohol can destroy the complex system of the raw materials of the lysate, and adversely affect the extraction process of the total RNA of bacteria.
In summary, the invention provides a kit for extracting total RNA from bacteria by a paramagnetic particle method and an extraction method thereof, wherein the kit comprises an extraction reagent group for extracting total RNA from bacteria; the extraction reagent group comprises magnetic bead binding solution, lysis solution, washing solution I, washing solution II, eluent and DNase I digestive juice; the lysate comprises 1-100 mM sodium citrate, 1-100 mM sodium lauroyl sarcosinate, 0.1-10M sodium chloride, 0.01-1M sodium metabisulfite, 1.5-6M guanidine hydrochloride, 0.1-30% of polyether alcohol and 30-70% of ethanol, wherein the total volume ratio of the lysate to the polyether alcohol is 0.1-30%. When bacteria are cracked by a lysate, sodium salt sodium citrate, sodium lauroyl sarcosinate, sodium chloride, polyether alcohol, a reducing agent sodium metabisulfite, guanidine hydrochloride and magnetic beads containing hydroxyl or carboxyl on the surface are added at proper concentration; the sodium ions increase the RNA adsorption capacity of the magnetic beads, and the citrate can effectively protect the radical activity on the surfaces of the magnetic beads and enhance the RNA binding capacity of the magnetic beads; the high-concentration guanidine hydrochloride can not only crack bacteria, but also denature protein and inhibit the degradation of RNase to RNA; meanwhile, the polyether alcohol can keep the activity of the RNA to a certain extent, and is more beneficial to the generation of a salt bridge between the RNA and the magnetic beads; sodium pyrosulfite can prevent RNA from being oxidized and denatured, and ensures effective combination of magnetic beads and RNA; the lauroyl sodium sarcosinate can keep the stability of the whole system so as to prevent the degradation of the extraction effect caused by the system change of the cracking liquid; the extracted RNA fragments were left intact. According to the invention, the raw materials of the lysis solution are reasonably compounded to obtain the optimal proportion and extraction condition of the lysis solution compounding system, and the raw materials play a synergistic effect in the RNA cracking and combining process, so that the lysis solution system has excellent binding force and stable binding property to the total RNA of bacteria. The total RNA extracted by the kit is complete in fragment, high in concentration and not easy to degrade after agarose gel electrophoresis; the overall performance is superior to that of the same type of kits in the market, and the kit has better application prospect and market value.
Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the spirit and scope of the present invention.
Claims (10)
1. A kit for extracting complete bacteria total RNA by a magnetic bead method is characterized by comprising an extraction reagent group for extracting bacteria total RNA; the extraction reagent group comprises magnetic bead binding solution, lysis solution, washing solution I, washing solution II, eluent and DNase I digestive juice; the lysate comprises 1-100 mM of sodium citrate, 1-100 mM of sodium lauroyl sarcosinate, 0.1-10M of sodium chloride, 0.01-1M of sodium metabisulfite, 1.5-6M of guanidine hydrochloride, 0.1-30% of polyether alcohol and 30-70% of ethanol, wherein the volume ratio of the polyether alcohol to the total volume of the lysate is 0.1-30%.
2. The kit for extracting the total RNA of the intact bacteria by the magnetic bead method according to claim 1, wherein the magnetic bead binding solution comprises magnetic beads, sodium chloride and tris (hydroxymethyl) aminomethane and is prepared from sterilized water; the pH value of the magnetic bead binding solution is 6.8-7.2.
3. The kit for extracting the total RNA of the intact bacteria by the magnetic bead method according to claim 2, wherein the magnetic bead has a particle size range of 0.3-2 μm and a content of 10-200 mg/mL; the surface of the magnetic bead is modified with hydroxyl or carboxyl.
4. The kit for extracting total RNA of intact bacteria by the magnetic bead method according to claim 1, wherein the washing solution I is a mixed aqueous solution containing 2-6M guanidine hydrochloride, 0.01-100 mM sodium chloride and 30-50% ethanol.
5. The kit for extracting the complete bacterial total RNA according to the claim 1, wherein the pH value of the DNase I digestive juice is 6.8-7.2; the DNase I digestive juice is an aqueous solution containing 1-10 wt% of DNase I, 30-50 wt% of glycerol, 1-5 mM of calcium chloride and 10-100 mM of tris (hydroxymethyl) aminomethane.
6. The kit for extracting total RNA from intact bacteria by the magnetic bead method according to claim 2, wherein the concentration of sodium chloride in the binding solution for magnetic beads is 0.01-1M, and the concentration of tris (hydroxymethyl) aminomethane is 0.02-1M.
7. The kit for extracting the total RNA of the intact bacteria by the magnetic bead method according to claim 1, wherein the washing solution II is 70-85% by volume of an isopropanol aqueous solution or an ethanol aqueous solution; the eluent is one of distilled water, deionized water and ultrapure water.
8. A method for extracting total RNA of intact bacteria, which is characterized in that the kit for extracting total RNA of intact bacteria by the magnetic bead method of any one of claims 1 to 7 comprises the following steps:
s1, taking 1-4 mL of bacteria culture solution to a centrifuge tube, carrying out centrifugal treatment for 1-5 minutes, removing supernatant, adding 100-200 mu L of physiological saline solution, and carrying out vortex treatment to obtain a uniformly mixed bacterial solution;
s2, respectively adding 300-600 mu L of lysis solution and 10-100 mu L of magnetic bead binding solution into the centrifugal tube filled with the uniformly mixed bacterial solution obtained in the step S1, standing for 5-15 minutes after vortex, then performing magnetic separation by using a magnetic frame, and sucking away the liquid;
s3, adding 200-700 mu L of washing liquid II into the centrifuge tube treated in the step S2, whirling, standing, performing magnetic separation by using a magnetic frame, removing liquid, opening a cover, and standing at room temperature for 3-5 minutes;
s4, adding 80-200 mu L of DNase I digestive juice into the centrifuge tube treated in the step S3, and standing for 5-10 minutes at room temperature after vortexing; continuously adding 200-700 mu L of washing solution I into the centrifugal tube, standing after vortex, then carrying out magnetic separation by using a magnetic frame, and absorbing and discarding the liquid;
s5, adding 200-700 mu L of washing liquid II into the centrifuge tube treated in the step S4, performing vortex and standing, performing magnetic separation by using a magnetic frame, removing liquid, and opening a cover to stand at room temperature for 3-5 minutes;
s6, adding 40-160 mu L of eluent into the centrifuge tube treated in the step S5, whirling, standing for 1-3 minutes, performing magnetic separation by using a magnetic frame, and absorbing supernatant liquid into a new centrifuge tube to obtain the complete bacterial total RNA.
9. The method for extracting the kit for extracting the complete bacterial total RNA by the paramagnetic particle method according to claim 8, wherein in the step S2, the centrifuge tube is reversed 5 to 6 times every 5 minutes in the standing process to uniformly mix the bacteria; in the steps S1 to S6, the time of the vortex is 10 to 15 seconds; in steps S3 to S5, after the washing solution I or the washing solution II is added, the mixture is vortexed and then kept standing for 10 to 30 seconds.
10. The method for extracting the kit for extracting the total RNA of the intact bacteria by the magnetic bead method according to claim 8, wherein in the step S1, the rotation speed of the centrifugal treatment is 10000-12000 r/min; in step S2, the magnetic bead binding solution is manually turned over and uniformly mixed for 10 to 20 times before use; in steps S3 to S5, the centrifuge tube is held on the magnetic rack when the liquid is discarded.
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| CN202211026045.7A CN115305246A (en) | 2022-08-25 | 2022-08-25 | Kit for extracting complete bacteria total RNA by paramagnetic particle method and extraction method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202211026045.7A CN115305246A (en) | 2022-08-25 | 2022-08-25 | Kit for extracting complete bacteria total RNA by paramagnetic particle method and extraction method thereof |
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| CN202211026045.7A Pending CN115305246A (en) | 2022-08-25 | 2022-08-25 | Kit for extracting complete bacteria total RNA by paramagnetic particle method and extraction method thereof |
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