CN111235145A - Nucleic acid purification reagent and purification method - Google Patents

Abstract

The invention discloses a nucleic acid purification reagent and a purification method, and belongs to the field of biology. The reagent comprises: magnetic beads, DNA binding solution, washing solution and eluent; the magnetic beads are silicon hydroxyl modified magnetic beads; the DNA binding solution is guanidine hydrochloride, sodium acetate and Triton X-100; the washing liquid comprises: reagent 1: guanidine hydrochloride, sodium acetate, Triton X-100; reagent 2: ethanol, sodium acetate, sodium citrate; the eluent is Tris-HCl, and in the implementation process, a DNA binding solution is added with a concentration ratio of 1: 1-5 of sodium cocoyl glutamate and sodium lauroyl glutamate can obviously improve the purification effect, the interaction of all reagents in the invention obviously improves the purification efficiency of nucleic acid and has high purity, so that the separation and purification yield can reach 98.76 percent, which is obviously higher than that of the prior art.

Description

Nucleic acid purification reagent and purification method

Technical Field

The invention belongs to the field of biology, and particularly relates to a nucleic acid purification reagent and a method for purifying nucleic acid by using the same.

Background

With the advancement of technology, nucleic acid purification has become an essential task in the preparation stage of molecular biology research. The first step in experimental procedures such as disease diagnosis, cloning, sequencing, amplification, hybridization, cDNA analysis is to purify the complete and high quality nucleic acid, otherwise the presence of large amounts of intracellular compositions (e.g., proteins, polysaccharides) or some components (organic solvents, metal ions) in the lysate may prevent the completion of these molecular biological downstream reactions.

Nucleic acids include both deoxyribonucleic acid and ribonucleic acid, and are present in cells in a state of being bound to proteins in many cases. The general principle for isolating and purifying nucleic acids is: (1) integrity, the complete primary structure makes the most essential requirement in order to guarantee the study of the structure and function of nucleic acids, since the genetic information is stored entirely within the primary structure, which also determines the form of its higher structure and the way it binds to other biological macromolecules; (2) purity, absence of enzyme inhibiting organic solvents and excessive concentrations of metal ions in nucleic acid samples, and contamination of other biological macromolecules such as proteins, polysaccharides and lipid molecules should be minimized.

The nucleic acid purification method mainly comprises two steps: firstly, cracking, namely breaking cells to release nucleic acid, wherein the cracking step is a key link of nucleic acid purification, and the cracking process mainly breaks the cells to release the nucleic acid; secondly, purification, i.e.separation of the released nucleic acids from other components in the cell, such as proteins, salts and other impurities. The existing widely used nucleic acid separation and purification methods comprise phenol-chloroform extraction method and centrifugal column purification, and the method has the defects of environmental pollution, complex operation, difficulty in realizing multiple fluxes and the like. Therefore, attempts have been made to purify the magnetic beads.

For example, chinese patent application 201810136469.6 discloses a nucleic acid purification reagent based on the magnetic bead method, wherein the nucleic acid purification reagent is an aqueous solution containing the following substances by mass: 1-5% carboxyl modified magnetic beads, 11.78-23.56% sodium chloride, 10-20% polyethylene glycol, 0.01-0.05% N-sarcosyl laurate, 0.16-0.79% Tris-HCl, 0.5-2% EDTA and 0.5-2% Tween 20. The nucleic acid purification and recovery kit comprises the nucleic acid purification reagent, a washing buffer solution and an elution buffer solution, the nucleic acid purification reagent and the nucleic acid recovery kit have high nucleic acid recovery efficiency, lower recovery loss on trace samples and better recovery efficiency on nucleic acid fragments of about 100bp or less, but the disposable kit in the application has larger usage amount, can increase the generation of laboratory waste and damage the environment, and the recovery purity still does not meet the requirement.

For another example, chinese patent application 201810632454.9 discloses a PCR purification reagent, i.e. a method for purifying PCR products using the reagent, wherein the PCR purification reagent comprises a DNA binding solution and an eluent, and the DNA binding solution has the following formula: 3mol/L sodium chloride and 30% ethanol by volume concentration; the formula of the eluent is as follows: 10mmol/L Tris, HCl, 100mmol/L NaCl, 1mmol/L EDTA, ethanol with volume concentration of 80%, and the pH value of Tris and HCl is 8.0. The PCR purification reagent can be successfully used for DNA purification without influencing the yield of DNA purification, and the PCR purification reagent and the regenerated silicon column are used together, so that the use quantity of disposable commercial kits can be reduced, the generation of laboratory garbage is reduced, the environment is protected, and social resources and use cost are saved, but the recovery rate and the purity of nucleic acid are not ideal.

Therefore, it is important to develop a purification reagent that can effectively improve the efficiency of separation and purification and improve the recovery rate and purity of nucleic acid.

Disclosure of Invention

In order to solve the defects in the prior art, the invention provides a nucleic acid purification reagent and a method for purifying nucleic acid by using the same.

The technical scheme for solving the technical problems is as follows:

in one aspect, the present invention discloses a nucleic acid purification reagent, comprising: magnetic beads, DNA binding solution, washing solution and eluent;

the concentration of the magnetic beads modified by silicon hydroxyl is 20-30mg/mL, and the particle size of the magnetic beads is 300-800 nm;

the DNA binding solution is 4-8M guanidine hydrochloride, 0.35-0.45M sodium acetate and 1-3% Triton X-100;

the washing liquid comprises: reagent 1: 4-8M guanidine hydrochloride, 0.25-0.35M sodium acetate, 1-3% Triton X-100;

reagent 2: ethanol with volume fraction of 70%, 0.1-0.2M sodium acetate, 10-20mM sodium citrate;

the eluent is 5-15mM Tris-HCl.

Preferably, the concentration of the magnetic beads modified by silicon hydroxyl is 22-28mg/mL, and the particle size of the magnetic beads is 400-600 nm;

the DNA binding solution is 5-7M guanidine hydrochloride, 0.40-0.45M sodium acetate and 1.5-2.5% Triton X-100;

the washing liquid comprises: reagent 1: 5-7M guanidine hydrochloride, 0.30-0.35M sodium acetate, 1.5-2.5% Triton X-100;

reagent 2: ethanol with volume fraction of 70%, 0.15-0.2M sodium acetate, and 12-18mM sodium citrate;

the eluent is 8-12mM Tris-HCl.

Preferably, the concentration of the magnetic beads modified by silicon hydroxyl is 25mg/mL, and the particle size of the magnetic beads is 500 nm;

the DNA binding solution is 6M guanidine hydrochloride, 0.40M sodium acetate and 2.0% Triton X-100;

the washing liquid comprises: reagent 1: 6M guanidine hydrochloride, 0.30M sodium acetate, 2.0% Triton X-100;

reagent 2: ethanol with the volume fraction of 70%, 0.15M sodium acetate and 15mM sodium citrate;

the eluent is 10mM Tris-HCl.

The pH value of the sodium acetate is 4.5-5, preferably 4.6-4.8, and more preferably 4.7.

The pH value of the sodium citrate is 4.5-5, preferably 4.6-4.8, and more preferably 4.6.

The pH value of the Tris-HCl is 8-9, and the preferable pH value is 8.5.

Preferably, the DNA binding solution further comprises a DNA having a concentration ratio of 1: 1-5 of sodium cocoyl glutamate and sodium lauroyl glutamate; preferably, the concentration ratio is 1: 2-4 of sodium cocoyl glutamate and sodium lauroyl glutamate; more preferably, the concentration ratio is 1: 3 sodium cocoyl glutamate and sodium lauroyl glutamate.

The concentration of the sodium cocoyl glutamate is 0.1-0.3M; the concentration of the sodium lauroyl glutamate is 0.3-0.6M.

In another aspect, the invention discloses a method for purifying nucleic acid, comprising the steps of:

(1) magnetic bead pretreatment: shaking up the silicon hydroxyl magnetic bead particles, sucking the silicon hydroxyl magnetic bead particles into a centrifugal tube, carrying out magnetic separation, and discarding the supernatant to obtain pretreated silicon hydroxyl magnetic bead particles;

(2) mixing: sucking DNA binding solution and a sample, adding the DNA binding solution and the sample into the pretreated silicon hydroxyl magnetic bead particles obtained in the step (1), standing after ultrasonic uniform mixing, performing magnetic separation, and discarding supernatant to obtain a substance A;

(3) adding the reagent 1 into the substance A obtained in the step (2), standing after ultrasonic mixing, performing magnetic separation, discarding the supernatant to obtain a substance B, and repeating the step once to obtain a substance C;

(4) adding the reagent 2 into the substance C obtained in the step (3), standing after ultrasonic mixing, performing magnetic separation, discarding the supernatant to obtain a substance D, and repeating the step once to obtain a substance E;

(5) slowly adding the eluent into the substance E obtained in the step (4), standing, keeping magnetic adsorption, and removing the supernatant to obtain a substance F;

(6) and (3) adding the eluent into the substance F obtained in the step (5), ultrasonically mixing uniformly, standing, ultrasonically mixing uniformly, carrying out magnetic adsorption, absorbing the supernatant, dissolving freeze-dried beads, and carrying out fluorescence PCR detection.

The volume ratio of the DNA binding solution to the sample in the step (2) is 70-90: 15-25;

preferably, the volume ratio of the DNA binding solution to the sample in the step (2) is 75-85: 18-22;

still preferably, the volume ratio of the DNA binding solution to the sample in the step (2) is 80: 20.

as a preferred technical scheme, the nucleic acid purification reagent comprises the following components: magnetic beads, DNA binding solution, washing solution and eluent;

the concentration of the magnetic beads modified by silicon hydroxyl is 25mg/mL, and the particle size of the magnetic beads is 500 nm;

the DNA binding solution is 6M guanidine hydrochloride, 0.1M sodium cocoyl glutamate, 0.3M sodium lauroyl glutamate, 0.40M sodium acetate, pH 4.7 and 2.0 percent Triton X-100;

the washing liquid comprises: reagent 1: 6M guanidine hydrochloride, 0.30M sodium acetate pH 4.7, 2% Triton X-100;

reagent 2: 70% by volume ethanol, 0.15M sodium acetate pH 4.7, 15mM sodium citrate pH 4.6;

the eluent is 10mM Tris-HCl with the pH value of 8.5.

A method for purifying nucleic acid by using the purification reagent comprises the following steps:

(1) magnetic bead pretreatment: shaking up 10 mu L of silicon hydroxyl magnetic bead particles, sucking the particles into a centrifuge tube, carrying out magnetic separation, and discarding the supernatant to obtain pretreated silicon hydroxyl magnetic bead particles;

(2) mixing: sucking 80 mu L of DNA binding solution and 20 mu L of sample, adding the DNA binding solution and the 20 mu L of sample into the pretreated silicon hydroxyl magnetic bead particles obtained in the step (1), standing for 90s after ultrasonic uniform mixing, carrying out magnetic separation, and removing supernatant to obtain a substance A;

(3) adding 100 mu L of reagent 1 into the substance A obtained in the step (2), standing for 90s after ultrasonic mixing, carrying out magnetic separation, removing supernatant to obtain a substance B, and repeating the step once to obtain a substance C;

(4) adding 100 mu L of reagent 2 into the substance C obtained in the step (3), standing for 90s after ultrasonic mixing, carrying out magnetic separation, removing supernatant to obtain a substance D, and repeating the step once to obtain a substance E;

(5) slowly adding 100 mu L of eluent into the substance E obtained in the step (4), standing for 10-20s, keeping magnetic adsorption, and removing supernatant to obtain a substance F;

(6) and (3) adding 100 mu L of eluent into the substance F obtained in the step (5), ultrasonically mixing the eluent uniformly, standing the mixture for 3min at the temperature of 55 ℃, ultrasonically mixing the mixture uniformly, carrying out magnetic adsorption, and absorbing the supernatant to dissolve freeze-dried beads for carrying out fluorescence PCR detection.

The invention also provides a method for full-automatic purification on a microfluidic chip by using the purification reagent, which comprises the following steps:

(1) putting 10 mu L of magnetic bead micro powder into a purification bin, and putting one PCR bead into a PCR bin;

(2) sucking the uniformly mixed DNA binding solution and a sample, uniformly mixing, pumping into a purification bin, ultrasonically mixing, standing, ultrasonically mixing, magnetically adsorbing, and sucking the liquid in the purification bin;

(3) sucking the uniformly mixed reagent 1, pumping into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(4) sucking the uniformly mixed reagent 2 into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(5) slowly pumping the eluent into a purification bin, standing, sucking the liquid in the purification bin, heating the area around the purification bin, introducing the eluent into the purification bin through a heating area, ultrasonically mixing, standing, repeating for 3 times, and performing magnetic adsorption;

(6) pushing the liquid in the purification bin into the PCR bin to fill the PCR bin with the liquid, and sealing;

(7) and carrying out temperature control circulation and fluorescence acquisition according to a set program.

The standing time in the step (2) is 40-50s, preferably 45 s.

The first standing time in the step (5) is 15 to 25s, preferably 20 s.

The heating temperature in the step (5) is 55 to 60 ℃, preferably 55 to 56 ℃.

The second standing time in the step (5) is 55-65s, preferably 60 s.

Compared with the prior art, the invention has the following excellent effects:

(1) the magnetic bead particles used in the invention are silicon hydroxyl magnetic beads, so that nucleic acid can be better separated and purified, the interaction of the magnetic beads and other purification reagents of the invention obviously improves the purification efficiency of the nucleic acid and has high purity, and the separation and purification yield can reach 98.76%, which is obviously higher than that of the prior art.

(2) In the implementation process of the invention, the DNA binding solution is added with the components with the concentration ratio of 1: 1-5 of sodium cocoyl glutamate and sodium lauroyl glutamate, obviously improves the purification effect.

(3) The invention strictly controls the components of the magnetic bead binding solution and the concentration of each component in the implementation process, and respectively uses two washing solutions to carry out layered washing and purification on nucleic acid, thereby improving the purification efficiency.

Drawings

FIG. 1 is a graph of fluorescence amplification of purified products;

FIG. 2 is a UV spectrophotometer quantitative test chart of the purified product;

reference numerals: a-control sample fluorescence amplification curve, B-example 3 purified product fluorescence amplification curve.

Detailed Description

The principles and features of this invention are described below in conjunction with specific embodiments, which are set forth merely for purposes of explanation of their legal names and not intended to limit the scope of the invention.

Example 1

A nucleic acid purification reagent:

silicon hydroxyl magnetic beads with the concentration of 20mg/mL and the particle size of 300 nm;

DNA binding solution: 4M guanidine hydrochloride, 0.35M sodium acetate pH 4.5, 1% Triton X-100;

washing liquid: reagent 1: 4M guanidine hydrochloride, 0.25M sodium acetate pH 4.5, 1% Triton X-100;

reagent 2: ethanol with volume fraction of 70%, 0.1M sodium acetate pH 4.5, 10mM sodium citrate pH 4.5;

eluent: 5mM Tris-HCl pH 8.

The purification method comprises the following steps:

(1) magnetic bead pretreatment: shaking up 5 mu L of silicon hydroxyl magnetic bead particles, sucking the particles into a centrifuge tube, carrying out magnetic separation, and discarding the supernatant to obtain pretreated silicon hydroxyl magnetic bead particles;

(2) mixing: adding 70 mu L of DNA binding solution and 15 mu L of sample into the pretreated silicon hydroxyl magnetic bead particles obtained in the step (1), standing for 90s after ultrasonic uniform mixing, carrying out magnetic separation, and discarding the supernatant to obtain a substance A;

(3) adding 90 mu L of reagent 1 into the substance A obtained in the step (2), standing for 90s after ultrasonic mixing, carrying out magnetic separation, removing supernatant to obtain a substance B, and repeating the step once to obtain a substance C;

(4) adding 90 mu L of reagent 2 into the substance C obtained in the step (3), standing for 90s after ultrasonic mixing, carrying out magnetic separation, removing supernatant to obtain a substance D, and repeating the step once to obtain a substance E;

(5) slowly adding 100 mu L of eluent into the substance E obtained in the step (4), standing for 10s, keeping magnetic adsorption, and removing supernatant to obtain a substance F;

(6) and (3) adding 100 mu L of eluent into the substance F obtained in the step (5), ultrasonically mixing the eluent uniformly, standing the mixture for 3min at the temperature of 55 ℃, ultrasonically mixing the mixture uniformly, carrying out magnetic adsorption, and absorbing the supernatant to dissolve freeze-dried beads for carrying out fluorescence PCR detection.

Example 2

A nucleic acid purification reagent:

silicon hydroxyl magnetic beads with the concentration of 30mg/mL and the particle size of 800 nm;

DNA binding solution: 8M guanidine hydrochloride, 0.2M sodium cocoyl glutamate, 0.4M sodium lauroyl glutamate, 0.45M sodium acetate pH 5, 3% Triton X-100;

washing liquid: reagent 1: 8M guanidine hydrochloride, 0.35M sodium acetate pH 5, 3% Triton X-100;

reagent 2: ethanol with volume fraction of 70%, 0.2M sodium acetate pH 5, 20mM sodium citrate pH 5;

eluent: 15mM Tris-HCl pH 9.

The purification method comprises the following steps:

(1) magnetic bead pretreatment: shaking up 15 mu L of silicon hydroxyl magnetic bead particles, sucking the particles into a centrifuge tube, carrying out magnetic separation, and discarding the supernatant to obtain pretreated silicon hydroxyl magnetic bead particles;

(2) mixing: adding 90 mu L of DNA binding solution and 25 mu L of sample into the pretreated silicon hydroxyl magnetic bead particles obtained in the step (1), standing for 90s after ultrasonic uniform mixing, carrying out magnetic separation, and discarding the supernatant to obtain a substance A;

(3) adding 110 mu L of reagent 1 into the substance A obtained in the step (2), standing for 90s after ultrasonic mixing, carrying out magnetic separation, removing supernatant to obtain a substance B, and repeating the step once to obtain a substance C;

(4) adding 110 mu L of reagent 2 into the substance C obtained in the step (3), standing for 90s after ultrasonic mixing, carrying out magnetic separation, removing supernatant to obtain a substance D, and repeating the step once to obtain a substance E;

(5) slowly adding 100 mu L of eluent into the substance E obtained in the step (4), standing for 10s, keeping magnetic adsorption, and removing supernatant to obtain a substance F;

(6) and (3) adding 100 mu L of eluent into the substance F obtained in the step (5), ultrasonically mixing the eluent uniformly, standing the mixture for 3min at the temperature of 55 ℃, ultrasonically mixing the mixture uniformly, carrying out magnetic adsorption, and absorbing the supernatant to dissolve freeze-dried beads for carrying out fluorescence PCR detection.

Example 3

A nucleic acid purification reagent:

silicon hydroxyl magnetic beads with the concentration of 25mg/mL and the particle size of 500 nm;

DNA binding solution: 6M guanidine hydrochloride, 0.2M sodium cocoyl glutamate, 0.6M sodium lauroyl glutamate, 0.40M sodium acetate pH 4.7, 2% Triton X-100;

washing liquid: reagent 1: 6M guanidine hydrochloride, 0.30M sodium acetate pH 4.7, 2% Triton X-100;

reagent 2: ethanol with volume fraction of 70%, 0.15M sodium acetate pH 4.7, 15mM sodium citrate pH 4.6;

eluent: 10mM Tris-HCl pH 8.5.

The purification method comprises the following steps:

(1) magnetic bead pretreatment: shaking up 10 mu L of silicon hydroxyl magnetic bead particles, sucking the particles into a centrifuge tube, carrying out magnetic separation, and discarding the supernatant to obtain pretreated silicon hydroxyl magnetic bead particles;

(2) mixing: sucking 80 mu L of DNA binding solution and 20 mu L of sample, adding the DNA binding solution and the 20 mu L of sample into the pretreated silicon hydroxyl magnetic bead particles obtained in the step (1), standing for 90s after ultrasonic uniform mixing, carrying out magnetic separation, and removing supernatant to obtain a substance A;

(3) adding 100 mu L of reagent 1 into the substance A obtained in the step (2), standing for 90s after ultrasonic mixing, carrying out magnetic separation, removing supernatant to obtain a substance B, and repeating the step once to obtain a substance C;

(4) adding 100 mu L of reagent 2 into the substance C obtained in the step (3), standing for 90s after ultrasonic mixing, carrying out magnetic separation, removing supernatant to obtain a substance D, and repeating the step once to obtain a substance E;

(5) slowly adding 100 mu L of eluent into the substance E obtained in the step (4), standing for 10-20s, keeping magnetic adsorption, and removing supernatant to obtain a substance F;

(6) and (3) adding 100 mu L of eluent into the substance F obtained in the step (5), ultrasonically mixing the eluent uniformly, standing the mixture for 3min at the temperature of 55 ℃, ultrasonically mixing the mixture uniformly, carrying out magnetic adsorption, and absorbing the supernatant to dissolve freeze-dried beads for carrying out fluorescence PCR detection.

Example 4

A nucleic acid purification reagent:

silicon hydroxyl magnetic beads with the concentration of 20mg/mL and the particle size of 300 nm;

DNA binding solution: 4M guanidine hydrochloride, 0.1M sodium cocoyl glutamate, 0.3M sodium lauroyl glutamate, 0.35M sodium acetate pH 4.5, 1% Triton X-100;

washing liquid: reagent 1: 4M guanidine hydrochloride, 0.25M sodium acetate pH 4.5, 1% Triton X-100;

reagent 2: ethanol with volume fraction of 70%, 0.1M sodium acetate pH 4.5, 10mM sodium citrate pH 4.5;

eluent: 5mM Tris-HCl pH 8.

The purification method comprises the following steps:

(1) placing the magnetic bead micro powder in a purification bin, and placing one PCR bead in a PCR bin;

(2) sucking the uniformly mixed DNA binding solution and a sample, uniformly mixing, pumping into a purification bin, ultrasonically mixing uniformly, standing for 40s, ultrasonically mixing uniformly, standing for 15s, performing magnetic adsorption, and sucking the liquid in the purification bin;

(3) sucking the uniformly mixed reagent 1, pumping into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(4) sucking the uniformly mixed reagent 2 into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(5) slowly pumping the eluent into a purification bin, standing for 15s, then sucking the liquid in the purification bin, heating the area around the purification bin to 55 ℃, introducing the eluent into the purification bin through the heated area, ultrasonically mixing and standing for 55s, repeating for 3 times, and then carrying out magnetic adsorption;

(6) pushing the liquid in the purification bin into the PCR bin to fill the PCR bin with the liquid, and sealing;

(7) and carrying out temperature control circulation and fluorescence acquisition according to a set program.

Example 5

A nucleic acid purification reagent:

silicon hydroxyl magnetic beads with the concentration of 30mg/mL and the particle size of 800 nm;

DNA binding solution: 8M guanidine hydrochloride, 0.2M sodium cocoyl glutamate, 0.4M sodium lauroyl glutamate, 0.45M sodium acetate pH 5, 3% Triton X-100;

washing liquid: reagent 1: 8M guanidine hydrochloride, 0.35M sodium acetate pH 5, 3% Triton X-100;

reagent 2: ethanol with volume fraction of 70%, 0.2M sodium acetate pH 5, 20mM sodium citrate pH 5;

eluent: 15mM Tris-HCl pH 9.

The purification method comprises the following steps:

(1) placing the magnetic bead micro powder in a purification bin, and placing one PCR bead in a PCR bin;

(2) sucking the uniformly mixed DNA binding solution and a sample, uniformly mixing, pumping into a purification bin, ultrasonically mixing uniformly, standing for 50s, ultrasonically mixing uniformly, standing for 15s, performing magnetic adsorption, and sucking the liquid in the purification bin;

(3) sucking the uniformly mixed reagent 1, pumping into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(4) sucking the uniformly mixed reagent 2 into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(5) slowly pumping the eluent into a purification bin, standing for 25s, then sucking the liquid in the purification bin, heating the area around the purification bin to 60 ℃, introducing the eluent into the purification bin through the heated area, ultrasonically mixing and standing for 65s, repeating for 3 times, and then carrying out magnetic adsorption;

(6) pushing the liquid in the purification bin into the PCR bin to fill the PCR bin with the liquid, and sealing;

(7) and carrying out temperature control circulation and fluorescence acquisition according to a set program.

Example 6

A nucleic acid purification reagent:

silicon hydroxyl magnetic beads with the concentration of 25mg/mL and the particle size of 500 nm;

DNA binding solution: 6M guanidine hydrochloride, 0.2M sodium cocoyl glutamate, 0.6M sodium lauroyl glutamate, 0.40M sodium acetate pH 4.7, 2% Triton X-100;

washing liquid: reagent 1: 6M guanidine hydrochloride, 0.30M sodium acetate pH 4.7, 2% Triton X-100;

reagent 2: ethanol with volume fraction of 70%, 0.15M sodium acetate pH 4.7, 15mM sodium citrate pH 4.6;

eluent: 10mM Tris-HCl pH 8.5.

The purification method comprises the following steps:

(1) placing the magnetic bead micro powder in a purification bin, and placing PCR beads in a PCR bin;

(2) sucking the uniformly mixed DNA binding solution and a sample, uniformly mixing, pumping into a purification bin, ultrasonically mixing uniformly, standing for 45s, ultrasonically mixing uniformly, standing for 15s, performing magnetic adsorption, and sucking the liquid in the purification bin;

(3) sucking the uniformly mixed reagent 1, pumping into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(4) sucking the uniformly mixed reagent 2 into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(5) slowly pumping the eluent into a purification bin, standing for 20s, then sucking the liquid in the purification bin, heating the area around the purification bin to 55 ℃, introducing the eluent into the purification bin through the heated area, ultrasonically mixing and standing for 60s, repeating for 3 times, and then carrying out magnetic adsorption;

(6) pushing the liquid in the purification bin into the PCR bin to fill the PCR bin with the liquid, and sealing;

(7) and carrying out temperature control circulation and fluorescence acquisition according to a set program.

Comparative example 1

The difference from example 3 is that: the same procedure as in example 3 was repeated except that only reagent 2 was used as the washing solution, that is, reagent 2 was used in the purification method (3).

Comparative example 2

The difference from example 3 is that:

DNA binding solution: 6M guanidine hydrochloride, 0.2M sodium cocoyl glutamate, 0.40M sodium acetate pH 4.7, 2% Triton X-100;

the other operations and steps are the same as in example 3.

Comparative example 3

The difference from example 3 is that:

DNA binding solution: 6M guanidine hydrochloride, 0.8M sodium cocoyl glutamate, 0.1M sodium lauroyl glutamate, 0.40M sodium acetate pH 4.7, 2% Triton X-100;

namely, the concentration ratio of sodium cocoyl glutamate to sodium lauroyl glutamate is 8: 1; the other operations and steps are the same as in example 3.

Comparative example 4

The difference from example 3 is that the magnetic beads used were carboxyl group-modified magnetic bead particles, and the other operations were the same as in example 3.

Comparative example 5

The difference from example 3 is that the volume ratio of the DNA binding solution to the sample was 50: 30, namely 100 mu L of DNA binding solution and 60 mu L of sample; the other operations and steps are the same as in example 3.

Test example 1 detection of efficiency of separation and purification of nucleic acid at a minute concentration

The detection method comprises the following steps: adding a purification reagent into the nucleic acid with the concentration of 20 ng/mu L for purification, measuring the concentration of the nucleic acid in the purified product, and calculating to obtain the purification yield, wherein specific detection data are shown in the following tables 1 and 2; and simultaneously, carrying out fluorescent amplification and quantification on the purified product by using a fluorescent quantitative PCR instrument.

TABLE 1

TABLE 2

As is apparent from the test data in Table 1 and Table 2 above, the nucleic acid purified by the purification reagents provided in examples 1 to 6 of the present invention has a separation and purification yield of about 96%, which is as high as 98.76%, the purification efficiency of comparative example 1 is decreased by washing with a washing solution, the purification rate of nucleic acid is also affected to some extent by changing the type or concentration ratio of the DNA binding solution reagent in comparative examples 2 and 3, and the purification yield of nucleic acid is decreased, and the yield is decreased by changing the type of magnetic beads in comparative example 4, and the yield is most affected by changing the volume ratio of the DNA binding solution to the sample in comparative example 6, which indicates that the purification yield of nucleic acid can be increased only by purifying nucleic acid with the purification reagents prepared according to the present invention.

Test example 2 detection of separation and purification efficiency of nucleic acids having different lengths

The detection method comprises the following steps: adding a purification reagent into the nucleic acid with the fragments of 120bp and 460bp for purification, measuring the nucleic acid concentration in the product before and after purification, and calculating to obtain the purification yield, wherein specific detection data are shown in the following tables 3 and 4; and simultaneously, carrying out fluorescent amplification and quantification on the purified product by using a fluorescent quantitative PCR instrument.

TABLE 3

	Fragments (bp)	Purification efficiency%	Fragments (bp)	Purification efficiency%
					Example 1	120	95.58	460	94.83
Example 2	120	97.15	460	95.64
					Example 3	120	98.24	460	98.32
Example 4	120	96.46	460	96.15
					Example 5	120	96.84	460	96.52
Examples6	120	97.35	460	97.03

TABLE 4

According to the detection data in the above table 3 and table 4, it is obvious that when the nucleic acid is purified by using the purification reagents provided in the embodiments 1 to 6 of the present invention, the separation and purification yield is about 95%, and the highest separation and purification yield can reach 98.32%, the purification yield is reduced when the comparative example 1 is washed by using a washing solution, the purification yield is reduced when the comparative examples 2 and 3 change the type or concentration ratio of the DNA binding solution reagent also affects the purification rate of the nucleic acid to a certain extent, so that the purification efficiency of the nucleic acid is reduced, the purification efficiency is reduced when the comparative example 4 changes the type of the magnetic beads, and the purification efficiency is maximally affected by the comparative example 6 change the volume ratio of the DNA binding solution and the sample, so that the purification yield of the nucleic acid can be higher only when the purification reagent prepared by the present invention is used for nucleic acid purification.

The invention carries out fluorescence amplification detection and ultraviolet spectrophotometer quantitative detection on the nucleic acid purified in the embodiment 3, and as can be seen from the attached drawings 1 and 2, the trend of the fluorescence amplification curve of the purified nucleic acid is the same as that of the fluorescence curve of a control group (the control group is high-purity nucleic acid), which indicates that the purification effect of the purified nucleic acid is good and the purity of the obtained nucleic acid is high; after the amplification of the fluorescent quantitative PCR instrument, the baseline of the fluorescent curve is stable, the exponential amplification interval is longer, the comparative analysis is facilitated, the amplification effect is good, the product has no inhibition effect on the PCR process, the overlap ratio of the fluorescent curves of a plurality of samples is higher, the difference among the samples is smaller, and the purification system is more stable.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. A nucleic acid purification reagent, characterized in that: the reagent comprises: magnetic beads, DNA binding solution, washing solution and eluent;

the concentration of the magnetic beads modified by silicon hydroxyl is 20-30mg/mL, and the particle size of the magnetic beads is 300-800 nm;

the DNA binding solution is 4-8M guanidine hydrochloride, 0.35-0.45M sodium acetate and 1-3% Triton X-100;

the washing liquid comprises: reagent 1: 4-8M guanidine hydrochloride, 0.25-0.35M sodium acetate, 1-3% Triton X-100;

reagent 2: ethanol with volume fraction of 70%, 0.1-0.2M sodium acetate, 10-20mM sodium citrate;

the eluent is 5-15mM Tris-HCl.

2. The purification reagent according to claim 1, wherein: magnetic beads, DNA binding solution, washing solution and eluent;

the concentration of the magnetic beads modified by silicon hydroxyl is 25mg/mL, and the particle size of the magnetic beads is 500 nm;

the DNA binding solution is 6M guanidine hydrochloride, 0.40M sodium acetate and 2.0% Triton X-100;

the washing liquid comprises: reagent 1: 6M guanidine hydrochloride, 0.30M sodium acetate, 2.0% Triton X-10;

reagent 2: ethanol with the volume fraction of 70%, 0.15M sodium acetate and 15mM sodium citrate;

the eluent is 10mM Tris-HCl.

3. The purification reagent according to claim 2, characterized in that: magnetic beads, DNA binding solution, washing solution and eluent;

the concentration of the magnetic beads modified by silicon hydroxyl is 25mg/mL, and the particle size of the magnetic beads is 500 nm;

the DNA binding solution is 6M guanidine hydrochloride, 0.40M sodium acetate pH 4.7, 2.0% Triton X-100;

the washing liquid comprises: reagent 1: 6M guanidine hydrochloride, 0.30M sodium acetate pH 4.7, 2.0% Triton X-100;

reagent 2: 70% by volume ethanol, 0.15M sodium acetate pH 4.7, 15mM sodium citrate pH 4.6;

the washing solution is 10mM Tris-HCl with the pH value of 8.5.

4. The purification reagent of claim 3, wherein: the DNA binding solution also contains a DNA with a concentration ratio of 1: 1-5 of sodium cocoyl glutamate and sodium lauroyl glutamate.

5. The purification reagent of claim 4, wherein: the concentration of the sodium cocoyl glutamate is 0.1-0.3M; the concentration of the sodium lauroyl glutamate is 0.3-0.6M.

6. The method for purifying nucleic acid using the purification reagent according to any one of claims 1 to 5, wherein: the method comprises the following steps:

(1) magnetic bead pretreatment: shaking up the silicon hydroxyl magnetic bead particles, sucking the silicon hydroxyl magnetic bead particles into a centrifugal tube, carrying out magnetic separation, and discarding the supernatant to obtain pretreated silicon hydroxyl magnetic bead particles;

(2) mixing: sucking DNA binding solution and a sample, adding the DNA binding solution and the sample into the pretreated silicon hydroxyl magnetic bead particles obtained in the step (1), standing after ultrasonic uniform mixing, performing magnetic separation, and discarding supernatant to obtain a substance A;

(3) adding the reagent 1 into the substance A obtained in the step (2), standing after ultrasonic mixing, performing magnetic separation, discarding the supernatant to obtain a substance B, and repeating the step once to obtain a substance C;

(4) adding the reagent 2 into the substance C obtained in the step (3), standing after ultrasonic mixing, performing magnetic separation, discarding the supernatant to obtain a substance D, and repeating the step once to obtain a substance E;

(5) slowly adding the eluent into the substance E obtained in the step (4), standing, keeping magnetic adsorption, and removing the supernatant to obtain a substance F;

(6) and (3) adding the eluent into the substance F obtained in the step (5), ultrasonically mixing uniformly, standing, ultrasonically mixing uniformly, carrying out magnetic adsorption, absorbing the supernatant, dissolving freeze-dried beads, and carrying out fluorescence PCR detection.

7. The method of claim 6, wherein: the volume ratio of the DNA binding solution to the sample in the step (2) is 70-90: 15-25.

8. The method of claim 7, wherein: the volume ratio of the DNA binding solution to the sample in the step (2) is 80: 20.

9. the method for full-automatic purification of a purification reagent according to any one of claims 1 to 5 on a microfluidic chip, wherein: the method comprises the following steps:

(1) placing the magnetic bead micro powder in a purification bin, and placing PCR beads in a PCR bin;

(2) sucking the uniformly mixed DNA binding solution and a sample, uniformly mixing, pumping into a purification bin, ultrasonically mixing, standing, ultrasonically mixing, magnetically adsorbing, and sucking the liquid in the purification bin;

(3) sucking the uniformly mixed reagent 1, pumping into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(4) sucking the uniformly mixed reagent 2 into a purification bin, ultrasonically mixing uniformly, performing magnetic adsorption, sucking the liquid in the purification bin to be dry, and repeating the step once;

(5) slowly pumping the eluent into a purification bin, standing, sucking the liquid in the purification bin, heating the area around the purification bin, introducing the eluent into the purification bin through a heating area, ultrasonically mixing, standing, repeating for 3 times, and performing magnetic adsorption;

(6) pushing the liquid in the purification bin into the PCR bin to fill the PCR bin with the liquid, and sealing;

(7) and carrying out temperature control circulation and fluorescence acquisition according to a set program.

10. The method of claim 9, wherein: the heating temperature in the step (5) is 55-60 ℃.

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