CN112680440A - Kit for rapidly extracting bacterial nucleic acid based on magnetic beads and extraction method - Google Patents
Kit for rapidly extracting bacterial nucleic acid based on magnetic beads and extraction method Download PDFInfo
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Abstract
The invention discloses a kit for rapidly extracting bacterial nucleic acid based on magnetic beads and an extraction method. The reagent of the kit comprises: lysis solution, magnetic bead solution, deproteinization rinsing solution, desalting rinsing solution and nucleic acid eluent. The method comprises the steps of adding magnetic bead solution and lysis solution into a sample for lysis, rinsing with rinsing solution, and eluting with nucleic acid eluent to obtain the bacterial nucleic acid. The kit for rapidly extracting the bacterial nucleic acid based on the magnetic beads is simple and convenient to extract, does not contain toxic reagents such as phenol, chloroform and the like, can realize manual operation, can integrate various automatic nucleic acid extractors, is simple and convenient in step, can automatically extract and obtain the nucleic acid only by adding a sample and without adding proteinase K, simplifies the experimental operation steps to the maximum extent and ensures the safety of experimenters. The requirements on nucleic acid extraction speed and flux and the requirements on nucleic acid extraction quality when a plurality of clinical samples are available in hospitals are met; meanwhile, the kit disclosed by the invention is low in toxicity, simple and convenient to operate, high in sample flux and better in nucleic acid extraction effect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a kit for rapidly extracting bacterial nucleic acid based on a paramagnetic particle method and a method for extracting bacterial nucleic acid.
Background
At present, a plurality of methods or kits for extracting bacterial genomes are available on the market, and the main methods comprise a traditional heating and boiling method, a Chelex-100 extraction method, an SDS-NaOH method, an SDS-enzyme cracking method, a CTAB method and a kit centrifugal column method.
However, these methods have problems including complicated steps, various reagents, toxic reagents such as phenol-containing chloroform, etc., and low extraction efficiency.
Disclosure of Invention
Therefore, the invention provides a kit for rapidly extracting bacterial nucleic acid based on magnetic beads, and aims to solve the problems that the steps for extracting bacterial nucleic acid are complex, the types of reagents are multiple, toxic reagents such as phenol, chloroform and the like are adopted, the extraction efficiency is low and the like in the prior art.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a kit for rapidly extracting bacterial nucleic acid based on magnetic beads, and the reagents of the kit comprise lysis solution, magnetic bead solution, deproteinization rinsing solution, desalting rinsing solution and nucleic acid eluent.
Further, the lysis solution comprises: 3-6mol/L guanidine salt, 5-20% of lauryl sodium sulfate, 20-200mmol/L ethylene diamine tetraacetic acid, 10-100mmol/L tris (hydroxymethyl) aminomethane, 100-1000mmol/L sodium chloride, 0.5-5% of sodium fatty alcohol polyoxyethylene ether sulfate and a surfactant.
Furthermore, the surfactant is one or more of TritonX-100, Tween-20, Tween-80, PEG4000 or NP-40.
Further, the deproteinization rinsing liquid contains 1-3mol/L guanidine salt, 100-1000mmol/L sodium chloride and 80% ethanol by volume ratio.
Further, the desalting rinse solution is ethanol with the volume ratio of 60-80%.
Further, the nucleic acid eluent contains 5-20mmol/L of tris (hydroxymethyl) aminomethane.
The second aspect of the present invention is a method for manually extracting bacterial nucleic acid using a magnetic bead-based kit for rapid extraction of bacterial nucleic acid, the method comprising:
step one, centrifuging a sample to obtain a suspended sample;
adding magnetic bead solution and lysis solution into the suspended sample, uniformly mixing for 30s in a vortex manner, adsorbing for 1min by using a magnetic frame, and discarding the supernatant;
adding 500 mu L deproteinized rinsing liquid, uniformly mixing for 30s in a vortex manner, adsorbing for 1min by a magnetic frame, and discarding the supernatant;
adding 800 mu L of desalting rinse solution, uniformly mixing for 30s in a vortex manner, adsorbing for 1min by a magnetic frame, and discarding the supernatant;
and step five, adding 100 mu L of nucleic acid eluent, heating in water bath at 65 ℃ for 10min, and eluting the nucleic acid to obtain the extracted bacterial nucleic acid.
In a third aspect of the present invention, there is provided a method for automatically extracting bacterial nucleic acid using a magnetic bead-based kit for rapid extraction of bacterial nucleic acid, the method comprising:
adding a reagent in a kit for rapidly extracting bacterial nucleic acid based on magnetic beads into a pre-sorting plate, adding 500 mu L of deproteinized rinsing liquid into columns 1 and 7, adding 300 mu L of lysate into columns 2 and 8, adding 800 mu L of desalted rinsing liquid into columns 3 and 9, and adding 100 mu L of nucleic acid eluent into columns 6 and 12;
step two, adding 200 mu L of sample solution into the columns 2 and 8 of the pre-separation plate;
and step three, putting the pre-partition plate into an automatic nucleic acid extractor, and operating a program to obtain the extracted bacterial nucleic acid.
Further, the sample solution is a clinical sample to be detected or a bacterial liquid.
The invention has the following advantages:
the kit for rapidly extracting the bacterial nucleic acid based on the magnetic beads is simple and convenient to extract, does not contain toxic reagents such as phenol, chloroform and the like, can realize manual operation, can integrate various automatic nucleic acid extractors, is simple and convenient in steps, can be used for extracting the nucleic acid on a computer only by adding a sample, simplifies the experimental operation steps to the maximum extent and ensures the safety of experimenters. The requirements on nucleic acid extraction speed and flux and the requirements on nucleic acid extraction quality when a plurality of clinical samples are available in hospitals are met; meanwhile, the kit disclosed by the invention is low in research and development toxicity, simple and convenient to operate, high in sample flux and good in nucleic acid extraction effect.
The method for rapidly extracting the bacterial genome by using the kit for rapidly extracting the bacterial nucleic acid based on the magnetic beads adopts a method of jointly cracking guanidine salt plasma and a special surfactant to promote the disintegration of nucleoproteins and inactivate nuclease in bacteria so that the released nucleic acid is not degraded, thereby achieving the purpose of completing the cracking combination in one step.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.
The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.
Fig. 1 is a diagram of agarose gel electrophoresis results of nucleic acid extraction from a kit for rapid extraction of bacterial nucleic acid based on magnetic beads and a kit for a competitive product according to an experimental example provided by the present invention, wherein 1 is the agarose gel electrophoresis result of nucleic acid extraction from the kit for a competitive product, and 2 is the agarose gel electrophoresis result of nucleic acid extraction from the kit for a competitive product according to the present invention.
Detailed Description
The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 method for extracting bacterial nucleic acid by hand using magnetic bead-based kit for rapid extraction of bacterial nucleic acid
1.1 this example utilizes magnetic bead based kit for rapid extraction of bacterial nucleic acid to extract nucleic acid by hand kit for nucleic acid extraction includes the following specific reagents:
1) lysis solution: 5mol/L guanidine hydrochloride, 10% sodium dodecyl sulfate, 50mmol/L ethylene diamine tetraacetic acid, 100mmol/L tris (hydroxymethyl) aminomethane, 200mmol/L sodium chloride, 2% sodium fatty alcohol-polyoxyethylene ether sulfate, 10% Triton X-100, and 5% PEG 4000;
2) deproteinizing rinse liquid: 2mol/L guanidine salt, 500mmol/L sodium chloride and 80% ethanol (V/V);
3) desalting and rinsing liquid: 80% ethanol (V/V);
4) nucleic acid eluent: 20mmol/L tris (hydroxymethyl) aminomethane.
1.2 a method for extracting bacterial nucleic acid by using a kit for rapidly extracting bacterial nucleic acid based on magnetic beads by a manual method, which comprises the following steps:
1) centrifuging 200 μ L-1mL of bacterial culture solution at 12000rpm/min for 2min, and removing supernatant; if the amount of the clinical sample is too small, the method can directly enter the next step without centrifugation, and 200-300 mu L of the suspension sample can be extracted.
2) Adding 20 mu L of magnetic bead solution and 300 mu L of lysis solution into the suspended sample, uniformly mixing by vortex for 30s, adsorbing by a magnetic frame for 1min, and discarding the supernatant;
3) continuously adding 500 μ L deproteinized rinsing solution, mixing uniformly for 30s by vortex, adsorbing for 1min by magnetic frame, and discarding the supernatant;
4) adding 800 μ L desalting rinse solution, mixing uniformly for 30s by vortex, adsorbing for 1min by magnetic frame, and removing supernatant;
5) finally, 100 mu L of nucleic acid eluent is added, and the nucleic acid is eluted by heating in water bath at 65 ℃ for 10 min;
6) the extracted nucleic acid extractive solution can be used immediately or stored at-20 deg.C.
Example 2 method for automatically extracting bacterial nucleic acid using magnetic bead-based kit for rapid extraction of bacterial nucleic acid
2.1 the reagent in the kit for rapidly extracting bacterial nucleic acid based on magnetic beads according to the embodiment adopts the reagent in the kit described in the embodiment 1;
automatic nucleic acid extractor: ji Van Purifier32, contains a pre-dividing plate.
2.2 a method for automatically extracting bacterial nucleic acid by using a magnetic bead-based kit for rapidly extracting bacterial nucleic acid, comprising the following steps:
1) adding the reagent in the kit into a pre-separation plate, adding 500 mu L deproteinized rinsing liquid into 1 and 7 columns, adding 300 mu L lysis solution into 2 and 8 columns, adding 800 mu L desalting rinsing liquid into 3 and 9 columns, and adding 100 mu L nucleic acid eluent into 6 and 12 columns;
2) adding 200 mu L of clinical sample solution to be detected, bacterial liquid or precipitate resuspension obtained by cracking 1mL of bacterial liquid by 200 mu L of cracking liquid into the columns 2 and 8 of pre-separation plates;
3) putting the pre-dividing plate into an automatic nucleic acid extractor, operating a program, and sucking out nucleic acid eluent; obtaining nucleic acid extracting solution;
4) the extracted nucleic acid extractive solution can be used immediately or stored at-20 deg.C.
Experimental example 1 method for manually extracting nucleic acid from Escherichia coli sample by using magnetic bead-based kit for rapidly extracting bacterial nucleic acid
1.1 the reagent kit for manually extracting the nucleic acid of the escherichia coli sample by using the magnetic bead-based reagent kit for rapidly extracting the bacterial nucleic acid in the experimental example comprises the following specific reagents:
1) lysis solution: 5mol/L guanidine hydrochloride, 10% sodium dodecyl sulfate, 50mmol/L ethylene diamine tetraacetic acid, 100mmol/L tris (hydroxymethyl) aminomethane, 200mmol/L sodium chloride, 2% sodium fatty alcohol-polyoxyethylene ether sulfate, 10% Triton X-100, and 5% PEG 4000;
2) deproteinizing rinse liquid: 2mol/L guanidine salt, 500mmol/L sodium chloride and 80% ethanol (V/V);
3) desalting and rinsing liquid: 80% ethanol (V/V);
4) nucleic acid eluent: 20mmol/L tris (hydroxymethyl) aminomethane.
1.2 method for manually extracting nucleic acid of escherichia coli sample by using magnetic bead-based kit for rapidly extracting bacterial nucleic acid
1) Culturing Escherichia coli for 16h, centrifuging 1mL of bacterial liquid at 12000rpm/min for 2min, and removing supernatant; adding 300 mu L of lysate and 20 mu L of 100mg/mL magnetic beads, uniformly mixing for 10min by vortex, placing the mixture on a magnetic frame for 30s, and discarding the supernatant; adding 500 mu L deproteinized rinsing liquid, mixing uniformly for 30s by vortex, placing on a magnetic frame for 30s, and removing the supernatant; adding 800 μ L desalting rinse solution, and mixing by vortex for 30 s; adding 100 μ L nucleic acid eluent, incubating at 65 deg.C for 10min to elute nucleic acid;
2) the method is carried out according to the instruction operation of a competition kit, and the competition kit is a MgPure virus RNA rapid purification kit (magnetic bead method) in Beiwoo medicine;
3) measuring the concentration of nucleic acid by the Qubit and the Nanodrop, measuring the purity of the nucleic acid by the Nanodrop, and running gel by 1% agarose electrophoresis; the results of the purity and yield of the nucleic acid extracted by the kit of the experimental example and the nucleic acid extracted by the kit of the competitive products are shown in table 1, and the results of the agarose gel electrophoresis of the nucleic acid extracted by the kit of the experimental example and the nucleic acid extracted by the kit of the competitive products are shown in table 1.
TABLE 1 nucleic acid purity and yield results
| Reagent kit | OD260/230 | OD260/280 | Yield of the product |
| Nucleic acid obtained from competitive products kit | 1.77 | 1.32 | 15mg |
| Experimental example 1 nucleic acid obtained from kit | 1.82 | 1.89 | 26mg |
Experimental example 2 method for manually extracting nucleic acid from alveolar lavage fluid sample by using magnetic bead-based kit for rapidly extracting bacterial nucleic acid
2.1 in the experimental example, the reagent in the kit for manually extracting the nucleic acid of the escherichia coli sample by using the kit for rapidly extracting the bacterial nucleic acid based on the magnetic beads in the experimental example 1 is adopted.
2.2 method for extracting nucleic acid of alveolar lavage fluid sample manually by using magnetic bead-based kit for rapid extraction of bacterial nucleic acid, comprising the following steps:
1) adding 200 μ L of alveolar lavage fluid, 300 μ L of lysate and 20 μ L of 100mg/mL magnetic bead solution into the same tube, mixing for 10min by vortex, placing on a magnetic frame for 30s, and discarding the supernatant; adding 500 mu L deproteinized rinsing liquid, mixing uniformly for 30s by vortex, placing on a magnetic frame for 30s, and removing the supernatant; adding 800 μ L desalting rinse solution, and mixing by vortex for 30 s; adding 100 μ L nucleic acid eluent, incubating at 65 deg.C for 10min, and eluting nucleic acid;
2) the method is carried out according to the instruction operation of a competition kit, and the competition kit is a MgPure virus RNA rapid purification kit (magnetic bead method) in Beiwoo medicine;
3) the nucleic acid yield results of the nucleic acid extracted by the method of the experimental example and the nucleic acid extracted by the competitive product kit are shown in table 2, and the results show that the yield of the nucleic acid of the alveolar lavage fluid extracted by the kit of the experimental example 2 is higher.
TABLE 2 nucleic acid concentration results Table
Experimental example 3 method for automatically extracting nucleic acid from pleural effusion and ascites by using magnetic bead-based kit for rapidly extracting bacterial nucleic acid
3.1 in the experimental example, reagents in the kit for manually extracting the nucleic acid of the escherichia coli sample are extracted by using the kit for quickly extracting the bacterial nucleic acid based on the magnetic beads in the experimental example 1;
automatic nucleic acid extractor: ji Van Purifier32, contains a pre-dividing plate.
3.2 method for extracting nucleic acid of pleural effusion and ascites automatically by using magnetic bead-based kit for rapid extraction of bacterial nucleic acid, comprising the following steps:
1) adding the reagents in the kit into a pre-split plate respectively, adding 500 mu L deproteinized rinsing liquid into 1 and 7 columns, adding 300 mu L lysis solution into 2 and 8 columns, adding 800 mu L desalting rinsing liquid into 3 and 9 columns, and adding 100 mu L nucleic acid eluent into 6 and 12 columns;
2) directly adding 200 mu L of clinical hydrothorax and ascites samples into the columns 2 and 8 of the pre-dividing plate;
3) putting the pre-dividing plate into an automatic nucleic acid extractor, operating a program, and sucking out nucleic acid eluent; the extracted nucleic acid can be used immediately or stored at-20 deg.C;
4) the method is carried out according to the instruction operation of a competition kit, and the competition kit is a MgPure virus RNA rapid purification kit (magnetic bead method) in Beiwoo medicine;
5) the nucleic acid concentration obtained by the experimental example and the nucleic acid concentration obtained by the competitive product kit are determined by the Qubit, the nucleic acid yield results of the nucleic acid extracted by the experimental example method and the nucleic acid extracted by the competitive product kit are shown in table 3, and the results show that the yield of the nucleic acid extracted from the hydrothorax and ascites is higher.
TABLE 3 nucleic acid concentration measurement results
Therefore, the method for extracting nucleic acid by using the kit for quickly extracting bacterial nucleic acid based on the magnetic beads is simple and convenient to operate, does not contain toxic reagents such as phenol, chloroform and the like, can realize manual operation, can integrate various automatic nucleic acid extractors, is simple and convenient in steps, can extract nucleic acid on a computer only by adding a sample, simplifies experimental operation steps to the maximum extent and ensures the safety of experimenters. Aiming at the requirements of a large number of clinical samples in a hospital on the extraction speed and flux of nucleic acid and the requirement on the extraction quality of nucleic acid, the research and development of the nucleic acid extraction method which has low toxicity, simple and convenient operation, high sample flux and good nucleic acid extraction effect is significant.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. A kit for rapidly extracting bacterial nucleic acid based on magnetic beads is characterized in that reagents of the kit comprise lysis solution, magnetic bead solution, deproteinization rinsing solution, desalting rinsing solution and nucleic acid eluent.
2. The kit for rapidly extracting bacterial nucleic acid based on magnetic beads according to claim 1, wherein the lysis solution comprises: 3-6mol/L guanidine salt, 5-20% of lauryl sodium sulfate, 20-200mmol/L ethylene diamine tetraacetic acid, 10-100mmol/L tris (hydroxymethyl) aminomethane, 100-1000mmol/L sodium chloride, 0.5-5% of sodium fatty alcohol polyoxyethylene ether sulfate and a surfactant.
3. The kit for rapidly extracting bacterial nucleic acid based on the magnetic beads as claimed in claim 2, wherein the surfactant is one or more of TritonX-100, Tween-20, Tween-80, PEG4000 or NP-40.
4. The kit for rapid extraction of bacterial nucleic acid based on magnetic beads as claimed in claim 1, wherein the deproteinization rinsing solution comprises 1-3mol/L guanidine salt, 100-1000mmol/L sodium chloride and 80% ethanol by volume.
5. The kit for rapidly extracting bacterial nucleic acid based on magnetic beads as claimed in claim 1, wherein the desalting rinse solution is ethanol with a volume ratio of 60-80%.
6. The kit for rapidly extracting bacterial nucleic acid based on magnetic beads as claimed in claim 1, wherein the nucleic acid eluent contains 5-20mmol/L tris (hydroxymethyl) aminomethane.
7. A method for manually extracting bacterial nucleic acid by using the magnetic bead-based kit for rapid extraction of bacterial nucleic acid according to claims 1 to 6, wherein the method comprises:
step one, centrifuging a sample to obtain a suspended sample;
adding magnetic bead solution and lysis solution into the suspended sample, uniformly mixing for 30s in a vortex manner, adsorbing for 1min by a magnetic frame, and discarding the supernatant;
adding 500 mu L deproteinized rinsing liquid, uniformly mixing for 30s in a vortex manner, adsorbing for 1min by a magnetic frame, and discarding the supernatant;
adding 800 mu L of desalting rinse solution, uniformly mixing for 30s in a vortex manner, adsorbing for 1min by a magnetic frame, and discarding the supernatant;
and step five, adding 100 mu L of nucleic acid eluent, heating in water bath at 65 ℃ for 10min, and eluting the nucleic acid to obtain the extracted bacterial nucleic acid.
8. A method for automatically extracting bacterial nucleic acid by using the magnetic bead-based kit for rapidly extracting bacterial nucleic acid according to claims 1 to 6, wherein the method comprises the following steps:
adding a reagent in a kit for rapidly extracting bacterial nucleic acid based on magnetic beads into a pre-sorting plate, adding 500 mu L of deproteinized rinsing liquid into columns 1 and 7, adding 300 mu L of lysate into columns 2 and 8, adding 800 mu L of desalted rinsing liquid into columns 3 and 9, and adding 100 mu L of nucleic acid eluent into columns 6 and 12;
step two, adding 200 mu L of sample solution into the columns 2 and 8 of the pre-separation plate;
and step three, putting the pre-partition plate into an automatic nucleic acid extractor, and operating a program to obtain the extracted bacterial nucleic acid.
9. The method for automatically extracting bacterial nucleic acid of the kit for rapidly extracting bacterial nucleic acid based on magnetic beads according to claim 8, wherein the sample solution is a clinical sample or a bacterial solution to be tested.
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Application publication date: 20210420 |
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