CN118440934A

CN118440934A - DNA extraction kit and extraction method

Info

Publication number: CN118440934A
Application number: CN202410530574.3A
Authority: CN
Inventors: 何东华; 唐续; 李雪玲; 刘怡; 聂晶
Original assignee: Guangzhou Yarui Biotechnology Co ltd
Current assignee: Guangzhou Yarui Biotechnology Co ltd
Priority date: 2024-04-29
Filing date: 2024-04-29
Publication date: 2024-08-06

Abstract

The invention discloses a DNA extraction kit and an extraction method, belonging to the field of DNA detection; the technical key points are as follows: the composition comprises the following components: lysate, magnetic bead suspension, resuspension, cleaning solution, eluent. The resuspension comprises: guanidine hydrochloride, ethanol and a solvent; the cleaning liquid comprises: sodium chloride, tris-HCl, ethanol, a pH regulator and a solvent; the eluent comprises: tris-HCl, pH regulator, solvent. The invention aims to provide a DNA extraction kit and an extraction method, which optimize reagent components, can complete experiments at normal temperature, achieve the aim of efficiently recovering free DNA of different segments in plasma, save reagent cost and operation time, and can better meet the requirement of downstream detection of extracted nucleic acid.

Description

DNA extraction kit and extraction method

Technical Field

The invention relates to the field of DNA detection, in particular to a DNA extraction kit and an extraction method.

Background

The free DNA (cfDNA) in the plasma sample is a complex mixture containing disintegrated white blood cells, necrotic tumor cells and pathogen DNA, and the healthy human plasma contains only a small amount of free DNA, and the fragment size is mainly below 200 bp. At present, free DNA has been used as a novel biomarker in the fields of clinical prenatal diagnosis of pregnant women, tumor detection, detection after organ transplantation, detection of viral infection and the like. With the continuous appearance of new research results, the potential value of cfDNA in the aspects of early diagnosis, prognosis evaluation, efficacy monitoring and the like of diseases is receiving more and more attention.

The detection of cfDNA in plasma or serum can be considered as a "liquid biopsy", which is useful for performing various relevant diagnostics and can avoid invasive tissue biopsies. Blood is taken as a detection sample, so that repeated collection of the detection sample becomes possible, and the method is favorable for tracking and monitoring the development of diseases and the treatment process of tumors. However, the content of free DNA in plasma is generally low, fragments are small, and extraction is difficult, which greatly influences the gene detection of free nucleic acid and the development of various researches. Therefore, how to extract free DNA is a research and development hotspot.

The free DNA extraction method comprises the following three technical routes:

First, TRizol method (e.g., CN110951724A, CN109295047A, scheme). The TRizol method has the disadvantage that it is very toxic to operators, and therefore this technical route is now increasingly abandoned.

Second, the column method (e.g., CN106754892A, CN114525274A, etc.). The method has the defects of more samples, more consumables, high requirements on centrifugal equipment and limited sample treatment.

Third, the magnetic bead method (e.g., CN110607297A, CN116590279A, CN114107288A, etc.). The magnetic bead method has the advantages that: simple operation, short time, safety and innocuity, and is suitable for high-flux treatment of more samples. However, the nucleic acid extraction yield by the magnetic bead method is not higher than that by the spin column method.

Therefore, the existing magnetic bead extraction method is improved, a set of simpler, faster and more affordable nucleic acid extraction and purification method is developed, and the plasma free nucleic acid extraction method which has no influence on the subsequent nucleic acid detection experiment becomes a technical problem to be solved at present.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art, in particular to an extraction kit which has high safety, strong operation convenience and more pertinency and high efficiency in recovering plasma free DNA by optimizing reagent components and a magnetic bead method of the magnetic bead method and improving the nucleic acid extraction yield of the magnetic bead method in order to solve the problems of the safety of the conventional TRizol method and the operation convenience and flux deficiency of a centrifugal column method.

Another object of the present invention is to provide an extraction method.

The technical scheme of the invention is as follows:

A DNA extraction kit belongs to a plasma free DNA extraction kit, and consists of the following components:

a, a lysate; the lysate comprises: guanidine isothiocyanate, ammonium sulfate, sodium taurodeoxycholate, sodium dodecyl sulfate, nonionic surfactant and solvent;

b, magnetic bead suspension; the magnetic bead suspension comprises: superparamagnetic microspheres, pH modifiers, solvents;

c, resuspension; the heavy suspension comprises: guanidine hydrochloride, ethanol and a solvent;

d, cleaning liquid; the cleaning liquid comprises: sodium chloride, tris-HCl, ethanol, a pH regulator and a solvent;

e, eluting the eluent; the eluent comprises: tris-HCl, pH regulator and solvent.

Preferably, the lysate comprises: 2-4M guanidine isothiocyanate, 0.5-1mol of ammonium sulfate, 10-100mM sodium taurodeoxycholate, 0.1% -2% sodium dodecyl sulfate and 1-12% nonionic surfactant, wherein the solvent is enzyme-free sterile water.

Preferably, the nonionic surfactant of the lysate is:

NP40 with the volume percentage content of 6-12%;

Or alternatively, the first and second heat exchangers may be,

Triton X-100 with the volume percentage content of 3-10%;

Or alternatively, the first and second heat exchangers may be,

Igecal CA-630% by volume;

or tween 20 with the volume percentage content of 1-3 percent.

Preferably, the magnetic bead suspension comprises: the volume fraction of the superparamagnetic microspheres is 70-80%, the particle size of the superparamagnetic microspheres is 10-60 nm, the pH regulator is concentrated hydrochloric acid, and the solvent is enzyme-free sterile water;

wherein the pH of the magnetic bead suspension is 4.0-6.0, and the magnetic bead suspension cannot be frozen.

Preferably, the resuspension components include: 2-4M guanidine hydrochloride, 70-80% ethanol, and the solvent is sterile water without enzyme.

Preferably, the cleaning solution comprises the following components: 5-30mM/L sodium chloride, 10-50mM Tris-HCl, 75-85% ethanol, concentrated hydrochloric acid as pH regulator, sterile water as solvent, and pH of the cleaning solution of 6.5-8.0.

Preferably, the eluent comprises: 10-30mM Tris-HCl, wherein the pH regulator is concentrated hydrochloric acid, and the solvent is enzyme-free sterile water; the pH of the eluent is 7.5-9.5.

A method for extracting free DNA using the above extraction kit, comprising the steps of:

s1, adding 2.5-5ml of lysate and 25-50ul of magnetic bead suspension into a centrifuge tube filled with 2-4 ml of sample, mixing the mixture uniformly in a reverse way, and incubating the mixture at room temperature for 15min;

s2, placing the centrifuge tube on a magnetic rack, adsorbing magnetic beads, and sucking out and discarding the solution;

S3, adding 300-500ul heavy suspension into the centrifuge tube, vibrating and uniformly mixing, placing the mixture on a magnetic rack to adsorb magnetic beads, and sucking out and discarding the solution;

s4, adding 300-500ul of cleaning solution into the centrifuge tube, vibrating and uniformly mixing, placing the mixture on a magnetic rack to adsorb magnetic beads, and sucking out and discarding the solution;

s5, uncovering the centrifuge tube and placing for 5 minutes to volatilize the ethanol completely;

S6, adding 25-100ul of eluent, shaking and mixing uniformly, standing for 5 minutes, placing on a magnetic rack to adsorb magnetic beads, transferring the clarified solution to a new centrifuge tube, and obtaining the free DNA of the extracted sample.

The application has the beneficial effects that:

first, a first application point of the present application is that: the free plasma extraction kit provided by the application has the advantages that the operation of the cracking process is simple, heating is not needed, the nucleic acid and the nucleic acid binding protein can be rapidly and thoroughly separated and released from the nucleic acid in the plasma sample at room temperature, sodium taurodeoxycholate in the cracking liquid belongs to a powerful anion remover, and the sodium dodecyl sulfate in the cracking liquid is matched, so that the protein can be denatured to a greater extent in the cracking process, and the protein has higher solubility so as to be better separated from the nucleic acid.

Second, a second invention point of the present invention is that: the ammonium sulfate component added into the lysis solution can play a role in protecting nucleic acid in the lysis process, has a certain inhibition effect on nuclease, can inhibit the degradation effect of the nuclease on the nucleic acid, and further protects the integrity of the nucleic acid fragments after the sample is lysed; the magnetic beads selected by the magnetic bead suspension liquid have super-strong paramagnetism, the particle size is 10-60 nm, the particle size is small, the particle size distribution range is narrow, the magnetic response is strong enough, the magnetic beads can be quickly aggregated under the action of an external magnetic field, can be uniformly dispersed after leaving the magnetic field, do not generate aggregation phenomenon, do not generate sedimentation due to the fact that the particle size is too large, and can be specifically combined with nucleic acid in solution under the action of high salt (guanidine hydrochloride, guanidine isothiocyanate and the like) and the external magnetic field and under the low pH condition through hydrophobic action, hydrogen bonding action, electrostatic action and the like, so that the nucleic acid is not combined with other impurities, the nucleic acid can be quickly separated from a biological sample, and impurities in the sample can be removed in the process of heavy suspension and washing liquid, so that the quality of the nucleic acid can be guaranteed, and the requirement of downstream detection can be better met.

Drawings

The invention is described in further detail below in connection with the embodiments in the drawings, but is not to be construed as limiting the invention in any way.

FIG. 1.A is an amplification curve of the first three groups of simulated plasma samples of example 1.

FIG. 1.B is an amplification curve of the four groups after a simulated plasma sample of example 1.

FIG. 2 is an amplification curve of human plasma samples of example 2.

FIG. 3 is an amplification curve of the lysates of example 3 after nucleic acid extraction from human plasma samples at varying concentrations of surfactant.

FIG. 4 is an amplification curve of the kit of example 4 of the present invention after nucleic acid extraction from human plasma samples with different particle size magnetic bead suspensions.

Detailed Description

The objects, technical solutions and advantages of the present invention will become more apparent by the following detailed description of the present invention with reference to the accompanying drawings. It should be understood that the description is only illustrative and is not intended to limit the scope of the invention. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the present invention.

< Protocol for the kit for extraction of free plasma DNA of the present application >

The application provides a kit for extracting free plasma DNA, which comprises:

(1) And (3) a lysate. The components of the lysate are as follows: 2-4M guanidine isothiocyanate, 0.5-1mol of ammonium sulfate, 10-100mM sodium taurodeoxycholate, 0.1% -2% sodium dodecyl sulfate and 1-12% nonionic surfactant, wherein the solvent is enzyme-free sterile water. The nonionic surfactant of the lysate is any one of NP40 with the volume percentage content of 6-12%, triton X-100 with the volume percentage content of 3-10%, igecal CA-630 with the volume percentage content of 2-5% and Tween 20 with the volume percentage content of 1-3%.

(2) A magnetic bead suspension. The magnetic bead suspension comprises the following components: magnetic balls with the volume fraction of 70-80%, the particle size of the magnetic balls is 10-60 nm, concentrated hydrochloric acid is used as a pH regulator, and the solvent is 50% of enzyme-free sterile water. The pH of the magnetic bead suspension is 4.0-6.0, and the magnetic bead suspension is stored at 4-8 ℃ and cannot be frozen.

(3) And (5) re-suspending. The components of the heavy suspension are as follows: 2-4M guanidine hydrochloride, 70-80% ethanol, and the solvent is sterile water without enzyme.

(4) And (5) cleaning the liquid. The cleaning liquid comprises the following components: 15-30mM/L sodium chloride, 10-50mM Tris-HCl, 75-85% ethanol, concentrated hydrochloric acid as pH regulator, sterile water as solvent, and pH of the cleaning solution of 6.5-8.0.

(5) And (3) eluting the eluent. The eluent comprises the following components: 10-30mM Tris-HCl, wherein the pH regulator is concentrated hydrochloric acid, and the solvent is enzyme-free sterile water; the pH of the eluent is 7.5-9.5.

< Description of test verification >

The kit and the commercial column extraction plasma extraction kit are respectively used for extracting nucleic acid from different samples, the same volume of eluent is respectively used for eluting the nucleic acid during the elution, the obtained nucleic acid is respectively and simultaneously subjected to real-time fluorescent quantitative PCR detection, the CT values of the genomic interleukin 11 (IL 11) DNA fragment and the plasmid RND3 are measured,

Wherein the base sequence of the qPCR primer combined with the genome IL11 DNA fragment is as follows:

tagaaagatatcctgacattggccaggcatggtggctcacgcctgtaatcctggcactttgggaggacgaagcgagtggatcactgaag tccaagagttcgagaccggcctgcgagaca.

the base sequence of the primer combined with the plasmid RND3 is as follows:

acacacaaagaatagagttgagcctgtgggacacttcgggttctccttactatgacaatgtccgccccctctcttaccctgattcggatgct gtgctgatttgctttgacatcagtagaccagaga.

example 1 ]

<1.1: Preparation of test article ]

Simulation of plasma samples:

(1) Preparing 100ml of BSA solution with the concentration of 50 g/L;

(2) Weighing 5g of BSA dry powder, adding the BSA dry powder into a solvent 1 XTE until the BSA is completely dissolved and then fixing the volume to 100ml;

(3) 2ml of a BSA solution with a concentration of 50g/L was taken, and genomic DNA nucleic acid and RND3 plasmid were added thereto in a ladder amount, respectively, and mixed uniformly to simulate the free plasma state as a simulated plasma sample.

<1.2: DNA nucleic acid extraction method-

The free plasma DNA extraction reagent of the invention extracts nucleic acid from a simulated plasma sample according to the following steps:

s100, adding 2.5ml of lysate and 25ul of magnetic bead suspension into a centrifuge tube filled with 2ml of prepared samples, reversing and mixing uniformly, and incubating for 15min at room temperature;

placing the centrifuge tube on a magnetic rack, adsorbing magnetic beads, and sucking out and discarding the solution;

adding 300ul of heavy suspension into a centrifuge tube, vibrating and uniformly mixing, placing the mixture on a magnetic rack to adsorb magnetic beads, and sucking out and discarding the solution;

adding 300ul of cleaning solution into the centrifuge tube, vibrating and uniformly mixing, placing the mixture on a magnetic rack to adsorb magnetic beads, and sucking out and discarding the solution;

The centrifuge tube is uncapped and placed for 5 minutes, so that the ethanol is completely volatilized;

Adding 50ul of eluent, shaking and mixing uniformly, standing for 5 minutes at room temperature, placing on a magnetic rack to adsorb magnetic beads, transferring the clarified solution to a new centrifuge tube, and obtaining the free DNA of the extracted sample.

<1.3: Detection results ]

The nucleic acid extracted by the kit of the invention is eluted by the same volume of eluent as the nucleic acid extracted by the commercial column extraction plasma extraction kit, and the DNA concentration and purity of the nucleic acid obtained by simulating the extraction of a plasma sample are measured by using Nanodrop, and the result is shown in Table 1.

The nucleic acid extracted by the kit and the nucleic acid extracted by the commercial column extraction plasma extraction kit are respectively and evenly mixed with 2ul of PCR reaction liquid and 18ul of PCR reaction liquid for real-time fluorescence quantitative PCR amplification, and the extraction effects of the two reagents on different fragments of the nucleic acid in simulated plasma are compared, and the results are shown in Table 1.2.

Table 1.1: the concentration and purity of the simulated plasma samples of example 1

Table 1.2: ct value of the simulated plasma sample of example 1

Example 2 ]

<2.1: Preparation of test article ]

Plasma samples: adding RND3 plasmid with concentration of 1.69×10X10-copy/ul into human plasma sample, mixing, and dividing into two groups.

<2.2: DNA nucleic acid extraction method-

The free plasma DNA extraction reagent of the invention extracts nucleic acid from human plasma samples according to the following steps:

(1) Adding 2.5ml of lysate and 25ul of magnetic bead suspension into a centrifuge tube filled with 2ml of prepared sample, mixing the mixture upside down, incubating the mixture at room temperature for 1h, and mixing the mixture upside down every 5min;

(2) Placing the centrifuge tube on a magnetic rack to enable the magnetic beads to be adsorbed, and sucking out and discarding the solution;

(3) Adding 300ul of heavy suspension into a centrifuge tube, vibrating and uniformly mixing, placing the mixture on a magnetic rack to adsorb magnetic beads, and sucking out and discarding the solution;

(4) Adding 300ul of cleaning solution into the centrifuge tube, vibrating and uniformly mixing, placing the mixture on a magnetic rack to adsorb magnetic beads, and sucking out and discarding the solution;

(5) The centrifuge tube is uncapped and placed for 5 minutes, so that the ethanol is completely volatilized;

(6) Adding 50ul of eluent, shaking and mixing uniformly, standing for 5 minutes, placing on a magnetic rack to adsorb magnetic beads, transferring the clarified solution to a new centrifuge tube, and obtaining the free DNA of the extracted sample.

<2.3: Detection results ]

The nucleic acid extracted by the kit has the same elution volume as the nucleic acid extracted by the commercial column extraction plasma extraction kit, and the DNA purity of the nucleic acid extracted by the human plasma sample is measured by adopting Nanodrop, and the result is shown in Table 2.1.

The nucleic acid extracted by the kit and the nucleic acid extracted by the commercial column extraction plasma extraction kit are respectively and uniformly mixed with 2ul and 18ul of PCR reaction liquid to carry out real-time fluorescence quantitative PCR amplification, and the extraction effects of the two reagents on different segments of free nucleic acid in human plasma are compared, and the results are shown in Table 2.2.

Table 2.2: ct value of human plasma sample test of example 2

Example 3 ]

<3.1: Preparation of test article ]

Plasma samples: adding RND3 plasmid with concentration of 1.69 x 10-4 copies/ul into human plasma sample, mixing, and extracting, testing and comparing with different surfactant concentrations by using the lysate of the present invention to extract the prepared human plasma sample.

<3.2: DNA nucleic acid extraction method-

<3.3: Detection results ]

The concentration and purity of DNA of human plasma sample nucleic acid extracted from different surfactant concentrations of the lysate are determined by using Nanodrop, and the results are shown in Table 3.1.

Table 3.1: example 3 nucleic acid concentration and purity

The nucleic acid extracted by the kit lysate with different surfactant concentrations is respectively and evenly mixed with the PCR reaction liquid with 2ul and 18ul, and real-time fluorescence quantitative PCR amplification is carried out to compare the extraction effects of the different surfactant concentrations, and the results are shown in Table 3.2.

Table 3.2: CT value for nucleic acid test of example 3

Example 4 ]

<4.1: Preparation of test article ]

Plasma samples: adding RND3 plasmid with concentration of 1.69 x 10-4 copies/ul into human plasma sample, mixing, the magnetic bead suspension liquid matched with the kit of the invention is used for extracting the prepared human plasma sample compared with the magnetic beads matched with the kit of the invention with other particle sizes.

<4.2: DNA nucleic acid extraction method-

<4.3: Detection results ]

The magnetic bead suspension liquid matched with the kit provided by the invention is compared with magnetic beads with other particle sizes matched with the kit provided by the invention to perform extraction effect comparison, the prepared nucleic acid extracted from the human plasma sample is subjected to DNA concentration and purity determination by using Nanodrop, and the result is shown in Table 4.1.

TABLE 4.1 comparison of nucleic acid concentration and purity extracted from magnetic beads having different particle sizes

The kit of the invention is matched with the magnetic bead suspension of the invention and the magnetic beads with other particle sizes, 2ul of nucleic acid extracted from the prepared human plasma sample is respectively and evenly mixed with 18ul of PCR reaction liquid, and real-time fluorescence quantitative PCR amplification is carried out to compare the magnetic bead suspension of the invention with the magnetic bead extraction effects with other particle sizes, and the results are shown in Table 4.2.

TABLE 4.2 CT values of nucleic acids extracted from magnetic beads having different particle diameters

< Conclusion analysis >

According to the results of tables 1.1 and 1.2, the DNA yield and purity of the kit of the present invention were superior to commercial column extraction plasma extraction kits in simulated plasma samples.

According to the results of tables 2.1 and 2.2, in a real human plasma sample, the extraction effect of the kit on plasmid RND3 is obviously improved compared with that of a comparative commercial column extraction plasma extraction kit, which proves that the kit has better recovery effect on small fragment nucleic acid such as plasmid in the human plasma sample and has strong application to the extraction of the nucleic acid of the human free plasma sample.

According to the results of tables 3.1 and 3.2, the extraction effect of the non-ionic surfactant Igecal CA-630 concentration of the kit lysate of the present invention on human plasma samples was better at 5% in real human plasma samples, which demonstrated that the non-ionic surfactant Igecal CA-630 at 5% concentration was more complete in nucleic acid retention.

According to the results of tables 4.1 and 4.2, the magnetic beads with the particle diameters of 10-60nm matched with the kit of the invention have stronger capability of acquiring nucleic acid compared with the magnetic beads with other particle diameters in a real human plasma sample. The invention has the advantages of optimizing reagent components, saving reagent cost and operation time, reducing the requirement on experimental equipment in the operation process, and ensuring the quality and recovery yield of free DNA in blood plasma as shown in the examples 1-4.

The above examples are provided for convenience of description of the present invention and are not to be construed as limiting the invention in any way, and any person skilled in the art will make partial changes or modifications to the invention by using the disclosed technical content without departing from the technical features of the invention.

Claims

1. A DNA extraction kit, which belongs to a plasma free DNA extraction kit, and is characterized by comprising the following components:

2. The DNA extraction kit of claim 1, wherein said lysate comprises: 2-4M guanidine isothiocyanate, 0.5-1mol of ammonium sulfate, 10-100mM sodium taurodeoxycholate, 0.1% -2% sodium dodecyl sulfate and 1-12% nonionic surfactant, wherein the solvent is enzyme-free sterile water.

3. The DNA extraction kit of claim 2, wherein the non-ionic surfactant of the lysate is:

NP40 with the volume percentage content of 6-12%;

Or alternatively, the first and second heat exchangers may be,

Triton X-100 with the volume percentage content of 3-10%;

Or alternatively, the first and second heat exchangers may be,

Igecal CA-630% by volume;

or tween 20 with the volume percentage content of 1-3 percent.

4. A DNA extraction kit according to claim 1,2 or 3, wherein the magnetic bead suspension comprises: the volume fraction of the superparamagnetic microspheres is 70-80%, the particle size of the superparamagnetic microspheres is 10-60 nm, the pH regulator is concentrated hydrochloric acid, and the solvent is enzyme-free sterile water;

5. A DNA extraction kit according to claim 1,2 or 3, wherein the resuspension components comprise: 2-4M guanidine hydrochloride, 70-80% ethanol, and the solvent is sterile water without enzyme.

6. A DNA extraction kit according to claim 1,2 or 3, wherein the wash solution comprises: 5-30mM/L sodium chloride, 10-50mM Tris-HCl, 75-85% ethanol, concentrated hydrochloric acid as pH regulator, sterile water as solvent, and pH of the cleaning solution of 6.5-8.0.

7. A DNA extraction kit according to claim 1,2 or 3, wherein the eluent comprises: 10-30mM Tris-HCl, wherein the pH regulator is concentrated hydrochloric acid, and the solvent is enzyme-free sterile water; the pH of the eluent is 7.5-9.5.

8. A method of extracting free DNA using the extraction kit of claim 1, comprising the steps of: