阅读说明：本技术 一种优化环介导等温扩增反应的方法 (Method for optimizing loop-mediated isothermal amplification reaction ) 是由 王玉兰 滕新栋 付汝坤 杨慧丽 鲍春晓 于 2020-12-17 设计创作，主要内容包括：本发明属于生物技术领域,公开了一种优化环介导等温扩增反应的方法,所述优化环介导等温扩增反应的方法包括以下步骤：取石墨烯量子点、碳纳米管进行优化环介导等温扩增反应的扩增物的制备；依据扩增需求进行待扩增物的选择,通过待扩增物的信息进行扩增引物的设计,同时依照获取的待扩增物的信息进行待扩增物体积与扩增条件进行设定,后进行环介导等温扩增反应体系的建立；将优化环介导等温扩增反应的扩增物加入到建立的环介导等温扩增反应体系中,进行核酸的扩增,制备得到核酸反应体系。本发明能够实现对环介导等温扩增反应体系的优化,对环介导等温扩增的优化效果更好,同时本发明的操作简单,优化效果好,适用于分子检测等使用。(The invention belongs to the technical field of biology, and discloses a method for optimizing a loop-mediated isothermal amplification reaction, which comprises the following steps: preparing an amplification product of an optimized loop-mediated isothermal amplification reaction by taking the graphene quantum dots and the carbon nano tubes; selecting a substance to be amplified according to the amplification requirement, designing an amplification primer according to the information of the substance to be amplified, setting the volume of the substance to be amplified and the amplification condition according to the acquired information of the substance to be amplified, and then establishing a loop-mediated isothermal amplification reaction system; and adding the amplification product of the optimized loop-mediated isothermal amplification reaction into the established loop-mediated isothermal amplification reaction system to amplify the nucleic acid to prepare the nucleic acid reaction system. The invention can realize the optimization of the loop-mediated isothermal amplification reaction system, has better optimization effect on the loop-mediated isothermal amplification, has simple operation and good optimization effect, and is suitable for molecular detection and the like.)

1. A method for optimizing a loop-mediated isothermal amplification reaction, comprising the steps of:

respectively preparing graphene quantum dots and carbon nanotubes, weighing 8-12 parts of the graphene quantum dots and 2-3 parts of the carbon nanotubes in parts by mass as raw materials for preparing an amplification product for optimizing the loop-mediated isothermal amplification reaction, and purifying the graphene quantum dots;

the purifying treatment of the graphene quantum dots comprises the following steps:

(1.1) mixing the initial graphene quantum dot solution and a molecular sieve in a certain ratio at room temperature to obtain a reactant;

(1.2) centrifuging the reactant to obtain a graphene quantum dot-molecular sieve containing the graphene quantum dots;

(1.3) adding a desorption agent into the graphene quantum dot-molecular sieve, and desorbing the graphene quantum dot at the temperature of 70-100 ℃;

step two, purifying the carbon nano tube, and respectively primarily crushing the purified graphene quantum dot and the carbon nano tube powder; ball-milling the preliminarily crushed graphene quantum dots to obtain graphene quantum dot powder with the particle size of less than 800nm, ball-milling the crushed carbon nano tubes to obtain carbon nano tube powder with the particle size of less than 200 mu m, collecting the graphene quantum dot powder obtained after ball-milling treatment and mixing the graphene quantum dot powder with the carbon nano tube powder to obtain a mixture;

the purification treatment of the carbon nano tube comprises the following steps:

adding a parallel magnetic field along the growth direction of the carbon nano tube in a carbon nano tube growth area, enabling the magnetic field to interact with the magnetic nano particles so as to enable the magnetic nano particles to be subjected to magnetic field force in the growth direction, and enabling the magnetic nano catalyst particles to drive the carbon nano tube to grow directionally under the action of the magnetic field force to obtain a purified carbon nano tube;

dispersing the mixture obtained after treatment in deionized water, heating and separating out, and preparing an amplification product for optimizing the loop-mediated isothermal amplification reaction to obtain the amplification product for optimizing the loop-mediated isothermal amplification reaction;

the preparation of the amplificate for carrying out the optimized loop-mediated isothermal amplification reaction comprises the following steps:

(3.1) placing the mixture obtained after treatment in deionized water for ultrasonic dispersion;

(3.2) after the solid substances in the water are uniformly dispersed, heating the water solution;

(3.3) fully stirring the aqueous solution after heating, and standing for 1-2 h;

(3.4) rapidly cooling the water solution after standing to obtain an amplification product for optimizing the loop-mediated isothermal amplification reaction;

selecting a substance to be amplified according to the amplification requirement, consulting the characteristics of the substance to be amplified, designing an amplification primer according to the acquired information of the substance to be amplified, setting the volume of the substance to be amplified and the amplification condition according to the acquired information of the substance to be amplified, and then establishing a loop-mediated isothermal amplification reaction system;

and fifthly, adding the amplification product of the optimized loop-mediated isothermal amplification reaction into the established loop-mediated isothermal amplification reaction system, performing primary reaction of the loop-mediated isothermal amplification reaction system, adding the amplification product of the optimized loop-mediated isothermal amplification reaction into the loop-mediated isothermal amplification reaction system again after the amplification reaction starts, performing nucleic acid amplification, preparing a nucleic acid reaction system, and completing optimization of the loop-mediated isothermal amplification reaction.

2. The method for optimizing the loop-mediated isothermal amplification reaction according to claim 1, wherein in the first step, the preparation method of the graphene quantum dots comprises:

(1) weighing citric acid solid and grinding the citric acid solid to obtain citric acid powder;

(2) adding citric acid powder into a crucible for high-temperature heating;

(3) after heating is finished, taking out the solid in the crucible, adding excessive ultrapure water, and carrying out ultrasonic dissolution on the solid;

(4) and after the solid is fully dissolved, carrying out suction filtration on the mixed solution, removing the liquid to obtain the graphene quantum dots, and storing the graphene quantum dots at normal temperature in a dark place.

3. The method for optimizing the LAMP as claimed in claim 2, wherein the heating temperature of the high temperature heating in step (2) is 160-290 ℃ and the heating time is 35-50 min.

4. The method for optimizing LAMP as claimed in claim 1, wherein in the first step, the carbon nanotube is prepared by:

(1) selecting a substrate with two opposite surfaces;

(2) spin-coating a magnetic fluid on a substrate, and forming a catalyst layer on two opposite surfaces of the substrate;

(3) loading the substrate with catalyst layers formed on two opposite surfaces of the substrate into a chemical vapor deposition reaction chamber;

(4) and introducing carbon source gas into the reaction cavity to perform chemical vapor deposition to grow the carbon nano tube.

5. The method for optimizing the LAMP as claimed in claim 4, wherein in the step (2), the thickness of the catalyst layer is 300-800 nm.

6. The method for optimizing LAMP as claimed in claim 1, wherein in the step (3.1), the ultrasonic frequency is 50-55 kHz.

7. The method for optimizing LAMP as claimed in claim 1, wherein in the step (3.2), the heating temperature is set to 90-100 ℃ and the heating time is set to 8-12 min.

8. The method for optimizing LAMP as claimed in claim 1, wherein the step four includes designing the amplification primers based on the obtained information of the material to be amplified, including:

(1) designing a plurality of upstream primers and downstream primers according to the acquired information of the to-be-amplified object;

(2) excluding the designed primers from the unidirectional primers with sensitivity and specificity lower than a set threshold;

(3) pairing the remaining upstream primer and downstream primer after the elimination operation to generate a primer pair, and eliminating the unreasonable primer pair with lower sensitivity and specificity;

(4) and calculating the P values of the rest primer pairs, carrying out cluster analysis, and taking the primer pair with the minimum P value in each class as the designed amplification primer.

9. The method of optimizing a loop-mediated isothermal amplification reaction of claim 8, wherein the sensitivity threshold is 0.5-0.9 and the specificity threshold is 0.5-0.9.

10. The method for optimizing the loop-mediated isothermal amplification reaction according to claim 1, wherein in the fifth step, the total amount of the amplification product added in the optimized loop-mediated isothermal amplification reaction is 1/20 of the loop-mediated isothermal amplification reaction system.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a method for optimizing a loop-mediated isothermal amplification reaction.

Background

At present: loop-mediated isothermal amplification (Loop-mediated isothermal amplification, abbreviated as LAMP) is a technique for amplifying nucleic acid under isothermal conditions, and can be widely applied to the fields of food safety detection and the like. The technology has high sensitivity, 109-1010 times of amplicon can be generated after the amplification is carried out for 15min-1h under the isothermal condition, and if the experimental environment is polluted by aerosol, a false positive result is easily generated. In addition, because a plurality of primers are involved in the amplification reaction process, non-specific binding is easily generated among the primers to generate primer dimers, so that reaction substrates in a reaction system are consumed, the reaction efficiency and the detection sensitivity are reduced, and false positive results are easily caused to cause result misjudgment. Therefore, how to optimize the loop-mediated isothermal amplification reaction, so that the loop-mediated isothermal amplification reaction has higher detection sensitivity and inhibits nonspecific amplification is a technical problem which needs to be solved urgently in the technical field.

Graphene Quantum Dots (Graphene Quantum Dots) generally have a transverse dimension below 100nm and a longitudinal dimension below several nanometers, have a one-layer, two-layer or several-layer Graphene structure, have the characteristics of low biological toxicity, excellent water solubility, chemical inertness, stable photoluminescence, good surface modification and the like, and have important potential application in the fields of biology, medicine, materials, novel semiconductor devices and the like. However, in the prior art, the scheme for optimizing the graphene quantum dots for the loop-mediated isothermal amplification reaction has a poor optimization effect on the loop-mediated isothermal amplification and is complex to operate.

Through the above analysis, the problems and defects of the prior art are as follows: in the prior art, the scheme for optimizing the graphene quantum dots for the loop-mediated isothermal amplification reaction has poor optimization effect on the loop-mediated isothermal amplification and is complex to operate.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a method for optimizing a loop-mediated isothermal amplification reaction.

The invention is realized by a method for optimizing a loop-mediated isothermal amplification reaction, which comprises the following steps:

respectively preparing graphene quantum dots and carbon nanotubes, weighing 8-12 parts of the graphene quantum dots and 2-3 parts of the carbon nanotubes in parts by mass as raw materials for preparing an amplification product for optimizing the loop-mediated isothermal amplification reaction, and purifying the graphene quantum dots;

the purifying treatment of the graphene quantum dots comprises the following steps:

(1.1) mixing the initial graphene quantum dot solution and a molecular sieve in a certain ratio at room temperature to obtain a reactant;

(1.2) centrifuging the reactant to obtain a graphene quantum dot-molecular sieve containing the graphene quantum dots;

(1.3) adding a desorption agent into the graphene quantum dot-molecular sieve, and desorbing the graphene quantum dot at the temperature of 70-100 ℃;

step two, purifying the carbon nano tube, and respectively primarily crushing the purified graphene quantum dot and the carbon nano tube powder; ball-milling the preliminarily crushed graphene quantum dots to obtain graphene quantum dot powder with the particle size of less than 800nm, ball-milling the crushed carbon nano tubes to obtain carbon nano tube powder with the particle size of less than 200 mu m, collecting the graphene quantum dot powder obtained after ball-milling treatment and mixing the graphene quantum dot powder with the carbon nano tube powder to obtain a mixture;

the purification treatment of the carbon nano tube comprises the following steps:

adding a parallel magnetic field along the growth direction of the carbon nano tube in a carbon nano tube growth area, enabling the magnetic field to interact with the magnetic nano particles so as to enable the magnetic nano particles to be subjected to magnetic field force in the growth direction, and enabling the magnetic nano catalyst particles to drive the carbon nano tube to grow directionally under the action of the magnetic field force to obtain a purified carbon nano tube;

dispersing the mixture obtained after treatment in deionized water, heating and separating out, and preparing an amplification product for optimizing the loop-mediated isothermal amplification reaction to obtain the amplification product for optimizing the loop-mediated isothermal amplification reaction;

the preparation of the amplificate for carrying out the optimized loop-mediated isothermal amplification reaction comprises the following steps:

(3.1) placing the mixture obtained after treatment in deionized water for ultrasonic dispersion;

(3.2) after the solid substances in the water are uniformly dispersed, heating the water solution;

(3.3) fully stirring the aqueous solution after heating, and standing for 1-2 h;

(3.4) rapidly cooling the water solution after standing to obtain an amplification product for optimizing the loop-mediated isothermal amplification reaction;

selecting a substance to be amplified according to the amplification requirement, consulting the characteristics of the substance to be amplified, designing an amplification primer according to the acquired information of the substance to be amplified, setting the volume of the substance to be amplified and the amplification condition according to the acquired information of the substance to be amplified, and then establishing a loop-mediated isothermal amplification reaction system;

and fifthly, adding the amplification product of the optimized loop-mediated isothermal amplification reaction into the established loop-mediated isothermal amplification reaction system, performing primary reaction of the loop-mediated isothermal amplification reaction system, adding the amplification product of the optimized loop-mediated isothermal amplification reaction into the loop-mediated isothermal amplification reaction system again after the amplification reaction starts, performing nucleic acid amplification, preparing a nucleic acid reaction system, and completing optimization of the loop-mediated isothermal amplification reaction.

Further, in the first step, the preparation method of the graphene quantum dot comprises the following steps:

(1) weighing citric acid solid and grinding the citric acid solid to obtain citric acid powder;

(2) adding citric acid powder into a crucible for high-temperature heating;

(3) after heating is finished, taking out the solid in the crucible, adding excessive ultrapure water, and carrying out ultrasonic dissolution on the solid;

(4) and after the solid is fully dissolved, carrying out suction filtration on the mixed solution, removing the liquid to obtain the graphene quantum dots, and storing the graphene quantum dots at normal temperature in a dark place.

Further, in the step (2), the heating temperature of the high-temperature heating is 160-290 ℃, and the heating time is 35-50 min.

Further, in the first step, the preparation method of the carbon nanotube comprises:

(1) selecting a substrate with two opposite surfaces;

(2) spin-coating a magnetic fluid on a substrate, and forming a catalyst layer on two opposite surfaces of the substrate;

(3) loading the substrate with catalyst layers formed on two opposite surfaces of the substrate into a chemical vapor deposition reaction chamber;

(4) and introducing carbon source gas into the reaction cavity to perform chemical vapor deposition to grow the carbon nano tube.

Further, in the step (2), the thickness of the catalyst layer is 300-800 nm.

Further, in the step (3.1), the ultrasonic frequency is 50-55 kHz.

Further, in the step (3.2), the set heating temperature is 90-100 ℃, and the heating time is 8-12 min;

further, in the fourth step, the designing of the amplification primers according to the obtained information of the to-be-amplified product includes:

(1) designing a plurality of upstream primers and downstream primers according to the acquired information of the to-be-amplified object;

(2) excluding the designed primers from the unidirectional primers with sensitivity and specificity lower than a set threshold;

(3) pairing the remaining upstream primer and downstream primer after the elimination operation to generate a primer pair, and eliminating the unreasonable primer pair with lower sensitivity and specificity;

(4) and calculating the P values of the rest primer pairs, carrying out cluster analysis, and taking the primer pair with the minimum P value in each class as the designed amplification primer.

Further, the sensitivity threshold is 0.5-0.9 and the specificity threshold is 0.5-0.9.

Further, in the fifth step, the total amount of the amplification product added in the optimized loop-mediated isothermal amplification reaction is 1/20 of the loop-mediated isothermal amplification reaction system.

By combining all the technical schemes, the invention has the advantages and positive effects that: according to the invention, the graphene quantum dots and the carbon nano tubes are used for preparing the amplification product for optimizing the loop-mediated isothermal amplification reaction, and the preparation of the amplification product for optimizing the loop-mediated isothermal amplification reaction by the graphene quantum dots and the carbon nano tubes can realize the optimization of a loop-mediated isothermal amplification reaction system, so that the optimization effect of the loop-mediated isothermal amplification is better.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

FIG. 1 is a flow chart of a method for optimizing a loop-mediated isothermal amplification reaction according to an embodiment of the present invention.

Fig. 2 is a flowchart of a preparation method of graphene quantum dots provided in an embodiment of the present invention.

Fig. 3 is a flowchart of a method for manufacturing a carbon nanotube according to an embodiment of the present invention.

FIG. 4 is a flow chart of the preparation of an amplicon for performing an optimized loop-mediated isothermal amplification reaction according to an embodiment of the present invention.

FIG. 5 is a flow chart of the design of amplification primers by obtaining information of the substance to be amplified according to the embodiment of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In view of the problems of the prior art, the present invention provides a method for optimizing loop-mediated isothermal amplification reaction, which is described in detail below with reference to the accompanying drawings.

As shown in FIG. 1, the method for optimizing the loop-mediated isothermal amplification reaction provided by the embodiment of the present invention comprises the following steps:

s101, weighing 8-12 parts of graphene quantum dots and 2-3 parts of carbon nano tubes according to the mass parts as raw materials for preparing amplification products for carrying out the optimized loop-mediated isothermal amplification reaction, respectively purifying the graphene quantum dots and the carbon nano tubes, and respectively preliminarily crushing purified graphene quantum dots and carbon nano tube powder;

s102, performing ball milling on the preliminarily crushed graphene quantum dots to obtain graphene quantum dot powder with the particle size of less than 800nm, performing ball milling on the crushed carbon nano tubes to obtain carbon nano tube powder with the particle size of less than 200 mu m, collecting and mixing the graphene quantum dot powder and the carbon nano tube powder obtained after the ball milling treatment to obtain a mixture;

s103, dispersing the mixture obtained after treatment in deionized water, heating, precipitating, and preparing an amplification product for optimizing the loop-mediated isothermal amplification reaction to obtain the amplification product for optimizing the loop-mediated isothermal amplification reaction;

s104, selecting a to-be-amplified product according to amplification requirements, consulting the characteristics of the to-be-amplified product, designing an amplification primer according to the acquired information of the to-be-amplified product, setting the volume of the to-be-amplified product and amplification conditions according to the acquired information of the to-be-amplified product, and then establishing a loop-mediated isothermal amplification reaction system;

and S105, adding the amplification product for optimizing the loop-mediated isothermal amplification reaction into the established loop-mediated isothermal amplification reaction system, performing primary reaction of the loop-mediated isothermal amplification reaction system, adding the amplification product for optimizing the loop-mediated isothermal amplification reaction into the loop-mediated isothermal amplification reaction system again after the amplification reaction starts, performing nucleic acid amplification, preparing a nucleic acid reaction system, and completing optimization of the loop-mediated isothermal amplification reaction.

As shown in fig. 2, in step S101, a method for preparing a graphene quantum dot provided by the embodiment of the present invention includes:

s201, weighing citric acid solid and grinding the citric acid solid to obtain citric acid powder;

s202, adding citric acid powder into a crucible for high-temperature heating;

s203, after heating is finished, taking out the solid in the crucible, adding excessive ultrapure water, and carrying out ultrasonic dissolution on the solid;

and S204, after the solid is fully dissolved, carrying out suction filtration on the mixed solution, removing the liquid to obtain the graphene quantum dots, and storing the graphene quantum dots at normal temperature in a dark place.

In step S202, the heating temperature of the high-temperature heating provided by the embodiment of the invention is 160-290 ℃, and the heating time is 35-50 min.

The purification treatment of the graphene quantum dots provided by the embodiment of the invention comprises the following steps: mixing the initial graphene quantum dot solution and a molecular sieve according to a certain proportion at room temperature to obtain a reactant; centrifuging the reactant to obtain the graphene quantum dot-molecular sieve containing the graphene quantum dots; adding a desorption agent into the graphene quantum dot-molecular sieve, and desorbing the graphene quantum dot at the temperature of 70-100 ℃.

As shown in fig. 3, in step S101, the method for preparing a carbon nanotube according to the embodiment of the present invention includes:

s301, selecting a substrate with two opposite surfaces;

s302, spin-coating a magnetic fluid on a substrate, and forming a catalyst layer on two opposite surfaces of the substrate;

s303, loading the substrate with the catalyst layers formed on the two opposite surfaces of the substrate into a chemical vapor deposition reaction chamber;

s304, introducing carbon source gas into the reaction cavity, and growing the carbon nano tube by chemical vapor deposition.

In step S302, the thickness of the catalyst layer provided by the embodiment of the invention is 300-800 nm.

In step S101, the step of purifying the carbon nanotube provided in the embodiment of the present invention includes: and adding a parallel magnetic field along the growth direction of the carbon nano tube in the growth area of the carbon nano tube, so that the magnetic field and the magnetic nano particles interact with each other to further enable the magnetic nano particles to be subjected to the magnetic field force in the growth direction, and the magnetic nano catalyst particles drive the carbon nano tube to grow directionally under the action of the magnetic field force to obtain the purified carbon nano tube.

As shown in fig. 4, in step S102, the preparation of the amplification product for performing the optimized loop-mediated isothermal amplification reaction according to the embodiment of the present invention includes:

s401, placing the mixture obtained after treatment in deionized water, and setting ultrasonic frequency to be 50-55kHz for ultrasonic dispersion;

s402, after the solid substances in the water are uniformly dispersed, heating the water solution, wherein the heating temperature is set to be 90-100 ℃, and the heating time is set to be 8-12 min;

s403, fully stirring the aqueous solution after heating, and standing for 1-2 h;

s404, rapidly cooling the water solution after standing to obtain an amplification product for optimizing the loop-mediated isothermal amplification reaction.

As shown in fig. 5, in step S104, the designing of the amplification primers according to the information of the to-be-amplified product includes:

s501, designing a plurality of upstream primers and downstream primers according to the acquired information of the to-be-amplified object;

s502, excluding the unidirectional primers with sensitivity and specificity lower than a set threshold value from the designed primers;

s503, pairing the remaining upstream primer and downstream primer after the elimination operation to generate a primer pair, and eliminating the unreasonable primer pair with lower sensitivity and specificity;

s504, calculating the P values of the remaining primer pairs, carrying out cluster analysis, and taking the primer pair with the minimum P value in each class as the designed amplification primer.

The sensitivity threshold value provided by the embodiment of the invention is 0.5-0.9 and the specificity threshold value is 0.5-0.9.

In step S105, the total amount of the amplification product for optimizing the loop-mediated isothermal amplification reaction provided by the embodiment of the present invention is 1/20 of the loop-mediated isothermal amplification reaction system.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, and any modification, equivalent replacement, and improvement made by those skilled in the art within the technical scope of the present invention disclosed herein, which is within the spirit and principle of the present invention, should be covered by the present invention.