阅读说明：本技术 一种基于十六烷基三甲基溴化铵的磁珠核酸提取方法 (Magnetic bead nucleic acid extraction method based on cetyl trimethyl ammonium bromide ) 是由 张平 顾越星 于 2021-09-01 设计创作，主要内容包括：一种基于十六烷基三甲基溴化铵的磁珠核酸提取方法,采用金属浴、涡旋振荡器、低温离心机、移液器、真空抽吸器、磁力架和离心管作为提取的工具；采用四个步骤提取核酸,第一步是样品裂解；第二步磁珠结合核酸；第三步是磁珠清洗；第四步是核酸洗脱。本发明采用CTAB和SDS结合的方法能提高裂解效率,采用结合表面积大的磁珠法提高核酸结合量,进而提高了核酸产量。本发明不使用传统方法中的苯酚、氯仿等有毒试剂,对实验操作人员的伤害以及对环境的影响减少到最少,更加符合现代环保理念,更适合实现高通量自动化核酸提取。基于上述,本发明具有好的应用前景。(A magnetic bead nucleic acid extraction method based on cetyl trimethyl ammonium bromide adopts a metal bath, a vortex oscillator, a low-temperature centrifuge, a pipettor, a vacuum aspirator, a magnetic frame and a centrifuge tube as extraction tools; extracting nucleic acid by four steps, wherein the first step is sample cracking; second, magnetic beads bind nucleic acids; the third step is magnetic bead cleaning; the fourth step is nucleic acid elution. According to the invention, a CTAB and SDS combination method is adopted to improve the cracking efficiency, and a magnetic bead method with large combination surface area is adopted to improve the nucleic acid combination amount, so that the nucleic acid yield is improved. The invention does not use toxic reagents such as phenol, chloroform and the like in the traditional method, reduces the harm to experiment operators and the influence on the environment to the minimum, better accords with the modern environmental protection concept, and is more suitable for realizing high-flux automatic nucleic acid extraction. Based on the above, the invention has good application prospect.)

1. A magnetic bead nucleic acid extraction method based on cetyl trimethyl ammonium bromide adopts a metal bath, a vortex oscillator, a low-temperature centrifuge, a pipettor, a vacuum aspirator, a magnetic frame and a centrifuge tube as extraction tools; the method is characterized in that four steps are adopted for extracting nucleic acid, the first step is sample cracking, and the specific operation steps are as follows: adding an animal and plant powder sample or a homogenate sample into a 2ml centrifuge tube, adding CTAB lysate, uniformly mixing by using a vortex oscillator, incubating for a period of time at a certain temperature in a metal bath, adding 100ul SDS solution, continuously incubating for a period of time at a certain temperature, centrifuging for a period of time by using a low-temperature centrifuge under a certain temperature condition and 12000g, transferring 400ul of supernatant in the test tube by using a pipettor, adding the mixture sample after transferring the supernatant into a new 2ml centrifuge tube, adding 600ul of protein denaturant into the centrifuge tube containing the sample, and incubating for a period of time at a certain temperature after swirling for a plurality of seconds by using the vortex oscillator; and a second step of binding the magnetic beads with nucleic acids, which comprises the following specific operation steps: adding 1000ul of isopropanol into a centrifugal tube containing a sample, adding 10 ul of fully resuspended magnetic beads into the centrifugal tube, uniformly mixing by swirling for a plurality of seconds through a swirling oscillator, standing at room temperature for a period of time, uniformly mixing by swirling or reversing for a plurality of times in the middle, placing the centrifugal tube on a magnetic frame for a period of time, wherein the magnetic beads form compact precipitates, and the supernatant is clear; the third step is magnetic bead cleaning, and the specific operation steps are as follows: leaving the centrifuge tube on a magnetic frame, sucking the centrifuge tube by using a vacuum aspirator to suck the supernatant, adding the prepared 1ml of detergent into the centrifuge tube, rotating the centrifuge tube for a plurality of circles on the magnetic frame in situ, leaving the centrifuge tube on the magnetic frame for a period of time, then sucking the supernatant by using the vacuum aspirator, repeatedly cleaning 1ml of detergent to clean the magnetic beads once, sucking the supernatant by using the vacuum aspirator again, and drying at room temperature for a period of time; the fourth step is nucleic acid elution, and the specific operation steps are as follows: adding 75 mu l of eluent into a centrifugal tube, performing vortex oscillation on a vortex oscillator, uniformly mixing magnetic beads, incubating for a period of time at a certain temperature, performing instant centrifugation on the centrifugal tube, putting the centrifugal tube on a magnetic frame until the magnetic beads form compact precipitates, taking 70ul of supernatant, and putting a precipitation solution into a new 1.5ml centrifugal tube, wherein the eluent is the finished product of the extracted nucleic acid.

2. The method for extracting nucleic acid from magnetic beads based on cetyltrimethylammonium bromide according to claim 1, wherein in the first step of sample lysis, 1000ul of CTAB lysis solution is added, after vortex mixing by a vortex oscillator, incubation time is 30 minutes at 65 ℃ in a metal bath, adding SDS solution, incubation time is 15 minutes at 65 ℃, centrifugation time is 15 minutes at 4 ℃ at 12000g, and incubation time is 15 minutes at 65 ℃ after vortex of several seconds by a vortex oscillator.

3. The method for extracting nucleic acid from magnetic beads based on cetyl trimethyl ammonium bromide as claimed in claim 1, wherein the second step of mixing magnetic beads with nucleic acid is performed by vortex mixing for several seconds by vortex oscillator, the standing time at room temperature is 10 minutes, and the time for placing the centrifuge tube on the magnetic frame is 2 minutes.

4. The method for extracting nucleic acid from magnetic beads based on cetyltrimethylammonium bromide according to claim 1, wherein in the third step of bead washing, the time for leaving the centrifuge tube on the magnetic rack is 1 minute, and the time for drying at room temperature after sucking off the supernatant by using a vacuum aspirator is 5 minutes.

5. The method for extracting nucleic acid from magnetic beads based on cetyltrimethylammonium bromide according to claim 1, wherein in the fourth step of nucleic acid elution, the vortex oscillator vortexes and mixes the magnetic beads, and then the incubation time is 7 minutes at 65 ℃.

Technical Field

The invention relates to the technical field of nucleic acid extraction processes of animal and plant components, in particular to a magnetic bead nucleic acid extraction method based on cetyl trimethyl ammonium bromide.

Background

The CTAB (cetyl trimethyl ammonium bromide) method is a common animal and plant nucleic acid extraction method in a laboratory. CTAB is a cationic detergent which can dissolve animal and plant cell membranes, can form complexes with animal and plant nucleic acids, is soluble in high salt solution (phenol, chloroform, etc.), can precipitate from the solution when the salt concentration of the solution is reduced to a certain degree, can separate the CTAB-nucleic acid complexes from protein and polysaccharide substances by centrifugation of a centrifuge, and finally can prepare nucleic acids by ethanol or isopropanol precipitation (because CTAB can be removed by dissolving in ethanol or isopropanol, the animal and plant nucleic acids can be finally obtained).

However, the conventional CTAB method requires the use of a harmful organic solvent such as phenol or chloroform in combination to assist the removal of protein impurities and the like, due to technical limitations, thereby obtaining a nucleic acid solution with high purity. Although the CTAB method is characterized by high nucleic acid yield as compared with other mainstream nucleic acid extraction methods, it has disadvantages of many complicated centrifugation steps, and it involves the use of harmful organic solvents such as phenol and chloroform, which have great adverse effects on the health of workers and the environment, and thus has not been widely used in the market.

Disclosure of Invention

In order to overcome the defects that in the prior art, animal and plant nucleic acid is extracted based on a CTAB method experiment, because the centrifugal steps are more due to the limitation of the technology, and harmful organic solvents such as phenol, chloroform and the like are adopted, so that the health of operators and the environment are greatly adversely affected, the invention provides a method for extracting nucleic acid by using superparamagnetic silica nano magnetic beads and the like as extraction materials based on CTAB, wherein the magnetic beads can be specifically identified and efficiently combined with nucleic acid molecules on a micro interface, and the superparamagnetic property of the silica nano magnetic beads is utilized, so that the nucleic acid can be quickly separated from samples such as blood, animal tissues, food, pathogenic microorganisms and the like under the combined action of an external magnetic field and the adopted raw materials and preparation method, the yield is higher, toxic reagents such as phenol, chloroform and the like in the traditional method are not used, the harm to the experiment operators and the influence on the environment are minimized, and the method is more in line with the modern environmental protection concept, the magnetic bead nucleic acid extraction method based on the cetyl trimethyl ammonium bromide is more suitable for realizing high-throughput automatic nucleic acid extraction.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a magnetic bead nucleic acid extraction method based on cetyl trimethyl ammonium bromide adopts a metal bath, a vortex oscillator, a low-temperature centrifuge, a pipettor, a vacuum aspirator, a magnetic frame and a centrifuge tube as extraction tools; the method is characterized in that four steps are adopted for extracting nucleic acid, the first step is sample cracking, and the specific operation steps are as follows: adding an animal and plant powder sample or a homogenate sample into a 2ml centrifuge tube, adding CTAB lysate, uniformly mixing by using a vortex oscillator, incubating for a period of time at a certain temperature in a metal bath, adding 100ul SDS solution, continuously incubating for a period of time at a certain temperature, centrifuging for a period of time by using a low-temperature centrifuge under a certain temperature condition and 12000g, transferring 400ul of supernatant in the test tube by using a pipettor, adding the mixture sample after transferring the supernatant into a new 2ml centrifuge tube, adding 600ul of protein denaturant into the centrifuge tube containing the sample, and incubating for a period of time at a certain temperature after swirling for a plurality of seconds by using the vortex oscillator; and a second step of binding the magnetic beads with nucleic acids, which comprises the following specific operation steps: adding 1000ul of isopropanol into a centrifugal tube containing a sample, adding 10 ul of fully resuspended magnetic beads into the centrifugal tube, uniformly mixing by swirling for a plurality of seconds through a swirling oscillator, standing at room temperature for a period of time, uniformly mixing by swirling or reversing for a plurality of times in the middle, placing the centrifugal tube on a magnetic frame for a period of time, wherein the magnetic beads form compact precipitates, and the supernatant is clear; the third step is magnetic bead cleaning, and the specific operation steps are as follows: leaving the centrifuge tube on a magnetic frame, sucking the centrifuge tube by using a vacuum aspirator to suck the supernatant, adding the prepared 1ml of detergent into the centrifuge tube, rotating the centrifuge tube for a plurality of circles on the magnetic frame in situ, leaving the centrifuge tube on the magnetic frame for a period of time, then sucking the supernatant by using the vacuum aspirator, repeatedly cleaning 1ml of detergent to clean the magnetic beads once, sucking the supernatant by using the vacuum aspirator again, and drying at room temperature for a period of time; the fourth step is nucleic acid elution, and the specific operation steps are as follows: adding 75 mu l of eluent into a centrifugal tube, performing vortex oscillation on a vortex oscillator, uniformly mixing magnetic beads, incubating for a period of time at a certain temperature, performing instant centrifugation on the centrifugal tube, putting the centrifugal tube on a magnetic frame until the magnetic beads form compact precipitates, taking 70ul of supernatant, and putting a precipitation solution into a new 1.5ml centrifugal tube, wherein the eluent is the finished product of the extracted nucleic acid.

Further, in the first sample lysis, 1000ul of CTAB lysate is added, the CTAB lysate is uniformly vortexed by a vortex oscillator and then incubated in a metal bath at 65 ℃ for 30 minutes, the SDS solution is added, the incubation time is continued at 65 ℃ for 15 minutes, the centrifugation time is 15 minutes at 4 ℃ under 12000g, and the incubation time is 15 minutes at 65 ℃ after vortexing for several seconds by the vortex oscillator.

Further, in the second step of magnetic bead binding to nucleic acid, vortex mixing is carried out for several seconds by a vortex oscillator, the standing time at room temperature is 10 minutes, and the time for placing the centrifugal tube on a magnetic frame is 2 minutes.

Further, in the third step of magnetic bead washing, the time for leaving the centrifuge tube on the magnetic rack was 1 minute, and the time for drying at room temperature after aspirating the supernatant with a vacuum aspirator was 5 minutes.

Further, in the fourth step of nucleic acid elution, the vortex oscillator vortexes and mixes the magnetic beads, and then the incubation time is 7 minutes at the temperature of 65 ℃.

The invention has the beneficial effects that: according to the invention, a combination of CTAB and SDS is used as a cracking reagent to release high-yield nucleic acid in animal and plant samples, polysaccharide components inhibited by PCR (polymerase chain reaction) in the reaction are removed by CTAB, phenols inhibited by the PCR are removed by PVP (polyvinylpyrrolidone), residues, protein, grease, cholesterol and the like of cells are separated and removed by a low-temperature centrifugation method, isopropanol is used as a nucleic acid binding agent to help magnetic beads to bind the nucleic acid, a magnetic frame is used for separating supernatant and the magnetic beads, supernatant containing protein, inorganic salt and other organic solvents is sucked and removed, the magnetic beads are cleaned for 2 times by 80% ethanol to remove residual protein and salt ions, finally the nucleic acid bound by the magnetic beads is separated by eluent, and finally a nucleic acid finished product is obtained. According to the invention, a CTAB and SDS combination method is adopted to improve the cracking efficiency, and a magnetic bead method with large combination surface area is adopted to improve the nucleic acid combination amount, so that the nucleic acid yield is improved. The invention does not use toxic reagents such as phenol, chloroform and the like in the traditional method, reduces the harm to experiment operators and the influence on the environment to the minimum, better accords with the modern environmental protection concept, and is more suitable for realizing high-flux automatic nucleic acid extraction. Based on the above, the invention has good application prospect.

Drawings

FIG. 1 is a block diagram representation of the preparation process of the present invention.

Detailed Description

FIG. 1 shows a magnetic bead nucleic acid extraction method based on cetyl trimethyl ammonium bromide, which uses a metal bath, a vortex oscillator, a low-temperature centrifuge, a pipettor, a vacuum aspirator, a magnetic frame and a centrifuge tube as extraction tools; four steps are adopted to extract nucleic acid, the first step is sample cracking, and the specific operation steps are as follows: 200-300mg of animal and plant powder sample or homogenate sample is added into a 2ml centrifugal tube, adding lysis solution containing CTAB and PVP, CTAB will combine with part of polysaccharide in the sample, PVP will combine with part of phenols in the sample, mixing uniformly by vortex oscillator, incubating for a period of time at certain temperature in metal bath, then 100ul SDS (sodium dodecyl sulfate) solution is added to continue to incubate for a period of time at 65 ℃, the mixture is centrifuged for a period of time by a low-temperature centrifuge under the condition of 12000g at a certain temperature, then, 400ul of the supernatant in the test tube was transferred by a pipette, and then a sample of the mixture after transfer of the supernatant was added to a new 2ml centrifuge tube, adding 600ul of guanidinium isothiocyanate protein denaturant into a centrifugal tube containing a sample, performing vortex by using a vortex oscillator for a plurality of seconds, and incubating for a period of time at a certain temperature; and a second step of binding the magnetic beads with nucleic acids, which comprises the following specific operation steps: adding 1000ul of isopropanol into a centrifugal tube containing a sample, adding 10 ul of fully resuspended magnetic beads into the centrifugal tube, uniformly mixing by swirling for a plurality of seconds through a swirling oscillator, standing at room temperature for a period of time, uniformly mixing by swirling or reversing for a plurality of times in the middle, placing the centrifugal tube on a magnetic frame for a period of time, wherein the magnetic beads form compact precipitates, and the supernatant is clear; the third step is magnetic bead cleaning, and the specific operation steps are as follows: leaving the centrifuge tube on a magnetic frame, sucking the centrifuge tube by using a vacuum aspirator to suck the supernatant, adding 1ml of prepared detergent (80% ethanol) into the centrifuge tube, rotating the centrifuge tube for several circles on the magnetic frame in situ, leaving the centrifuge tube on the magnetic frame for a period of time, sucking the supernatant by using the vacuum aspirator, repeatedly washing 1ml of detergent (80% ethanol) to wash the magnetic beads once, sucking the supernatant by using the vacuum aspirator again, and drying the supernatant at room temperature for a period of time; the fourth step is nucleic acid elution, and the specific operation steps are as follows: adding 75 mu l of TE (Tris Tris-hydroxymethyl-aminomethane + EDTA-ethylene diamine tetraacetic acid) eluent into a centrifugal tube, performing vortex oscillation on a vortex oscillator to uniformly mix magnetic beads, incubating for a period of time at a certain temperature, performing instantaneous centrifugation on the centrifugal tube, placing the centrifugal tube on a magnetic frame until the magnetic beads form compact precipitates, taking 70ul of supernatant, and placing the precipitate into a new 1.5ml centrifugal tube, wherein the eluent is the finished product of the extracted nucleic acid.

As shown in FIG. 1, in the first step of sample lysis, 1000ul of CTAB lysate was added, after vortex mixing by a vortex oscillator, the incubation time was 30 minutes at 65 ℃ in a metal bath, the incubation time was 15 minutes at 65 ℃ after adding SDS solution, the centrifugation time was 15 minutes at 12000g at 4 ℃, and the incubation time was 15 minutes at 65 ℃ after vortex for several seconds by a vortex oscillator. And secondly, combining the magnetic beads with the nucleic acid, uniformly mixing by vortexing for a plurality of seconds through a vortexing oscillator, standing for 10 minutes at room temperature, and placing the centrifuge tube on a magnetic frame for 2 minutes. In the third step of magnetic bead washing, the time for leaving the centrifuge tube on the magnetic rack is 1 minute, and the time for drying at room temperature after sucking off the supernatant by using a vacuum aspirator is 5 minutes. In the fourth step of nucleic acid elution, the vortex oscillator is used for vortex oscillation and uniform mixing of the magnetic beads, and then the incubation time is 7 minutes at the temperature of 65 ℃.

As shown in FIG. 1, in the present invention, the first step of nucleic acid extraction is usually lysis to release nucleic acids. Lysis involved 2 levels: 1) cell membrane lysis to release nucleic acids; 2) the protein dissociates from the nucleic acid to release the nucleic acid. According to the invention, CTAB (cetyl trimethyl ammonium bromide) is used as a lysis reagent capable of dissolving cell membranes, and is usually used as a high-efficiency plant cell lysis reagent at 65 ℃, and the yield of the obtained nucleic acid is usually higher than that of other nucleic acid extraction reagents. The CTAB lysis reagent is also suitable for animal cells, and therefore can be used as a lysis reagent for simultaneously extracting nucleic acid from animal and plant tissues. The nucleic acid after cell lysis is free and also bound to the protein. The CTAB (cetyltrimethylammonium bromide) lysis reagent was worked at 65 ℃ for half an hour, and SDS (sodium dodecyl sulfate) was added to facilitate the separation of nucleic acids from proteins. SDS (sodium dodecyl sulfate) is an anionic detergent, which binds to amino acid residues 1:1 on proteins to negatively charge the proteins, thus leaving the secondary structure of the proteins fully open and all negatively charged, thus obtaining more free nucleic acids.

As shown in figure 1, in the invention, in the step of removing impurities, the impurities comprise protein, plant polysaccharide and polyphenol, animal fat and cholesterol, salt ions and extraction reagent components. The sample after the first step of lysis is put into a low-temperature centrifuge at 4 ℃ and centrifuged at 12000g for 15 minutes. The low temperature allows grease, cholesterol, etc. to precipitate in the sample solution, thus appearing on the surface layer where the sample floats after centrifugation. CTAB in lysates of high ionic strength, CTAB (cetyltrimethylammonium bromide) forms complexes with proteins and polysaccharides, and these CTAB complexes and cellular debris appear in the pellet fraction of the centrifugation. The intermediate clarified liquid fraction is removed to a new centrifuge tube and chaotropic salts (guanidinium hydrochloride, guanidinium isothiocyanate, etc.) are added. Unlike the conventional CTAB method, which requires harmful organic solvents such as phenol, chloroform, etc. to extract proteins, the method makes hydrophobic proteins more soluble in water by chaotropic salts that disrupt the stability of molecular forces (hydrogen bonds). Meanwhile, PVP (polyvinylpyrrolidone) in the lysis solution is a water-soluble polymer and can be combined with specific phenolic substances in plant tissues. Isopropanol is used to dissolve CTAB (cetyltrimethylammonium bromide) and the nucleic acid to be extracted is allowed to precipitate out in solution. To this end, the process dissolves as much of the contaminant components to be removed as possible in the supernatant solution which is subsequently discarded.

As shown in FIG. 1, in the present invention, magnetic beads are used as a carrier for nucleic acid binding, unlike conventional spin column nucleic acid extraction, the surface area of the magnetic beads for binding nucleic acid is much larger than that of a silica membrane in a spin column, and the amount of the magnetic beads can be increased to increase the probability of binding nucleic acid, and the yield of DNA binding by the magnetic bead method is several times that by the conventional spin column method. The precipitated nucleic acid is bound to the magnetic beads under the action of the binding agent isopropanol. Separating the magnetic beads and the supernatant by a magnetic frame, and sucking the supernatant containing CTAB, protein, plant polysaccharide, polyphenol and isopropanol. The magnetic beads are washed by 80% ethanol, and salt ions soluble in ethanol can be washed away.

In summary, as shown in fig. 1, according to the present invention, a combination of CTAB and SDS is used as a lysis reagent to release high-yield nucleic acid in animal and plant samples, CTAB is used to remove polysaccharide components inhibited by PCR (polymerase chain reaction) in the reaction, PVP (polyvinylpyrrolidone) is used to remove phenols inhibited by PCR, low-temperature centrifugation is used to separate and remove cell residues, proteins, oils, cholesterol, and the like, isopropanol is used as a nucleic acid binding agent to help magnetic beads to bind nucleic acid, a magnetic rack is used to separate supernatant and magnetic beads, supernatant containing proteins, inorganic salts, and other organic solvents is removed by suction, the magnetic beads are washed 2 times with 80% ethanol to remove residual proteins and salt ions, and finally, an eluent is used to separate nucleic acid bound to magnetic beads, thereby obtaining a nucleic acid product. According to the invention, a CTAB and SDS combination method is adopted to improve the cracking efficiency, and a magnetic bead method with large combination surface area is adopted to improve the nucleic acid combination amount, so that the nucleic acid yield is improved. The invention does not use toxic reagents such as phenol, chloroform and the like in the traditional method, reduces the harm to experiment operators and the influence on the environment to the minimum, better accords with the modern environmental protection concept, and is more suitable for realizing high-throughput automatic nucleic acid extraction. The background technology of the invention is as follows: the magnetic bead method nucleic acid extraction principle; the magnetic bead method is based on the same principle as the silica gel membrane centrifugal column, and the surface of the superparamagnetic nanoparticle is modified and surface-modified by using a nanotechnology to prepare the superparamagnetic silica nanobead. The magnetic beads can be specifically identified and efficiently combined with nucleic acid molecules on a microscopic interface. By utilizing the superparamagnetism of the silicon oxide nano microspheres, under the combined action of an external magnetic field and the raw materials and the preparation method adopted by the application, the sample nucleic acid such as blood, animal tissues, food, pathogenic microorganisms and the like can be separated, and the silicon oxide nano microspheres can be applied to various fields such as clinical disease diagnosis, blood transfusion safety, forensic medicine identification, environmental microorganism detection, food safety detection, molecular biology research and the like.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be apparent to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, but is capable of other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Furthermore, it should be understood that although the present description refers to embodiments, the embodiments do not include only one independent technical solution, and such description is only for clarity, and those skilled in the art should take the description as a whole, and the technical solutions in the embodiments may be appropriately combined to form other embodiments that can be understood by those skilled in the art.