CN118256485A

CN118256485A - Kit for rapidly extracting nucleic acid and application

Info

Publication number: CN118256485A
Application number: CN202211686708.8A
Authority: CN
Inventors: 赵艳峰; 陈林松; 李玉萍; 何文新
Original assignee: Shanghai Pulmonary Hospital
Current assignee: Shanghai Pulmonary Hospital
Filing date: 2022-12-27
Publication date: 2024-06-28

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a kit for rapidly extracting nucleic acid and application thereof. The kit disclosed by the invention comprises SDS, EDTA and Triton X-100, and the kit is used for extracting nucleic acid, so that the extraction cost is low, the operation is simple, the time consumption is short, the PCR amplification is not influenced by an extraction product, and the extraction efficiency is extremely high because the step of nucleic acid loss cannot be introduced in the operation process.

Description

Kit for rapidly extracting nucleic acid and application

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a kit for rapidly extracting nucleic acid and application thereof.

Background

Along with the continuous progress of molecular diagnosis technology, lung cancer driving genes are continuously discovered, and clinical researchers can detect specific gene mutation generated by driving lung cancer from a molecular level so as to guide patients to take medicines in a targeted way and realize accurate treatment of lung cancer. The accurate diagnosis and treatment mode of lung cancer can not only greatly improve the survival time of patients, but also enable more patients with advanced lung cancer to be treated with dignity and have quality cancer survival. The accurate diagnosis and treatment of lung cancer are based on molecular diagnosis, and nucleic acid extraction is a precondition technology for molecular diagnosis.

The methods currently used for nucleic acid extraction mainly include conventional extraction methods, spin column methods and magnetic bead methods.

Traditional extraction methods include boiling cleavage, phenol chloroform extraction, anionic detergent method and guanidine isothiocyanate/phenol method (Trizol method). Boiling lysis is commonly used for manual extraction of DNA. The chromosome DNA is much larger than the plasmid DNA molecule, the chromosome DNA is a linear molecule, and the plasmid DNA is a covalent closed loop molecule; when the DNA solution is heated, the linear chromosome DNA is easy to denature, and the covalently closed plasmid DNA restores its natural conformation when cooled; the variable chromosomal DNA fragments combine with denatured proteins and cell debris to form a precipitate, while the renatured supercoiled plasmid DNA molecules are present in the liquid phase in a solubilized state, so that the two can be separated by centrifugation. The boiling cleavage method is used for extracting DNA, has less yield and more impurities, and is mainly suitable for some rough experiments, wherein the DNA can be broken.

Phenol chloroform extraction is a classical method for extracting nucleic acids, especially DNA, mainly by using two different organic solvents to alternately extract and remove proteins, while phenol inhibits the degradation of DNase and RNase, protein molecules are dissolved in the phenol phase, and nucleic acids are dissolved in the aqueous phase. After centrifugation and delamination, the aqueous layer is taken out, the operation is repeated for a plurality of times, and then the aqueous phase containing the nucleic acid is combined, and DNA or RNA is precipitated by ethanol by utilizing the property that the nucleic acid is insoluble in alcohol. The phenol chloroform extraction has the greatest advantages of low cost and lower requirements on experimental conditions, and the extracted DNA is kept in a natural state. The obtained DNA has high purity, large fragment and good effect, and has the defects of complicated operation and toxic organic solvent.

The anionic detergent method is to denature proteins by using detergents such as SDS or sodium xylylate, and break electrostatic attraction or coordination bonds between nucleic acids and proteins, thereby releasing the nucleic acids. The SDS method is simple and mild in operation, but the obtained product contains more saccharide impurities.

The guanidine isothiocyanate/phenol method (Trizol method) is a classical method for extracting RNA, and in homogenized or solubilized samples, the Trizol reagent can maintain the integrity of RNA while destroying cells and solubilized cell components. After centrifugation with chloroform, the lysate was separated into aqueous and organic phases. RNA is present in the aqueous phase. After aqueous phase transfer, RNA was recovered by isopropanol precipitation. After removal of the aqueous phase, the DNA can be precipitated from the intermediate phase with ethanol and the protein can be obtained from the organic phase by precipitation with isopropanol. The Trizol method is suitable for RNA extraction experiments of common plant tissues, animal tissues, fungi, bacteria and the like.

The centrifugal column method for extracting nucleic acid is a simpler method for separating and purifying trace nucleic acid, and the basic principle is that the cell is broken by using the lysate, so that the nucleic acid in the cell is released. The released nucleic acid is specifically adsorbed on a specific silicon carrier, and the carrier has strong affinity and adsorption force only for nucleic acid, and is basically not adsorbed on other biochemical components such as protein, polysaccharide and lipid, so that the nucleic acid is thrown out of the column during centrifugation. Eluting the nucleic acid adsorbed on the specific carrier with eluent, and separating to obtain purified nucleic acid. The centrifugal column method nucleic acid extraction kit has low price, relatively simple operation and wider application in the market. However, the method has the disadvantages of large sample demand, large loss, incapacitation of rare samples, inconvenience in high throughput, automation operation and the like.

The magnetic bead method utilizes the principle that active groups of magnetic particles can be combined with and dissociated from nucleic acid under certain conditions, cells are firstly cracked by using cell lysate, the magnetic particles with the active groups can specifically adsorb nucleic acid molecules which are dissociated from the cells, other interferents in a sample are well removed, the magnetic particles and the liquid are separated under the action of a magnetic field, finally, the particles are recovered, and pure DNA or RNA can be obtained by eluting with eluent, so that a nucleic acid template with higher quality is obtained. The magnetic bead method does not need centrifugation, does not need to add various reagents, is simple to operate and meets the requirement of automatic extraction of nucleic acid. But has higher cost, and is difficult to popularize for scientific research end use.

The methods have the advantages of complex operation, high cost and great nucleic acid loss caused by multiple operations, and the maximum extraction efficiency can only reach 40-50%.

Therefore, there is a strong need for a nucleic acid extraction method for molecular diagnostics that is efficient and rapid and does not affect the efficiency of PCR amplification.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a rapid extraction kit suitable for animal nucleic acid and application thereof. The extraction kit and the extraction method have the advantages of low extraction cost, simple operation, short time consumption, no influence on PCR amplification of the extracted product, no introduction of the step of nucleic acid loss in the operation process, and high extraction efficiency.

In order to achieve the above and other related objects, the present invention adopts the following technical scheme:

In a first aspect of the invention, a nucleic acid extraction kit is provided comprising SDS, EDTA and Triton X-100.

Specifically, the chemical structural formula of SDS is(Formula I). The molecular formula of SDS is CH ₃(CH₂)₁₁OSO₃ Na and the molecular weight is 288.38. The CAS registry number for SDS is 151-21-3. The English name of the SDS is: sodium dodecyl sulfate. The Chinese name of the SDS is: sodium dodecyl sulfate.

The chemical structural formula of the EDTA isThe molecular formula of the EDTA is (HO ₂CCH₂)₂NCH₂CH₂N(CH₂CO₂H)₂, molecular weight is 292.24, CAS registration number of the EDTA is 60-00-4, english name of the EDTA is Ethylenedinitrilotetraacetic acid, chinese name of the EDTA is ethylenediamine tetraacetic acid.

The chemical structural formula of the Triton X-100 isThe molecular formula of Triton X-100 is t-Oct-C ₆H₄-(OCH₂CH₂)_x OH, x=9-10, and the molecular weight is 647. The CAS registry number for Triton X-100 is 9036-19-5. The Chinese name of Triton X-100 is: polyethylene glycol octyl phenyl ether (triton X-100).

The kit of the invention employs a combination of SDS, EDTA and Triton X-100 to extract nucleic acids, the combination of which and the final concentration used are critical for the kit of the invention.

In the kit of the invention, the SDS can fully lyse cells under the proper temperature condition to separate chromosomes and denature proteins, and simultaneously, the SDS is combined with the proteins and the polysaccharides to form a complex, so that the nucleic acid is released.

Further, the final concentration of SDS was used at 0.1% to 1%. The unit of the final concentration is mass percent.

Further, the final concentration of SDS used is 0.1% to 0.25%. The unit of the final concentration is mass percent.

Further, the final concentration of SDS was used at 0.25% to 1%. The unit of the final concentration is mass percent.

In the kit, EDTA can be combined with auxiliary factors of intracellular enzyme activity, inhibit the activities of nuclease and protease, and protect nucleic acid from degradation or denaturation. The cofactor for the enzymatic activity may be divalent metal ions within the cell, such as Mg ²⁺、Ca²⁺ and Fe ²⁺, etc.

Further, the final concentration of EDTA used is 0.05% to 0.8%. The unit of the final concentration is mass percent.

Further, the final concentration of EDTA used is 0.05% to 0.2%. The unit of the final concentration is mass percent.

Further, the final concentration of EDTA used is 0.2% to 0.8%. The unit of the final concentration is mass percent.

In the kit of the invention, the Triton X-100 can dissolve lipid on cell membranes and cell nuclear membranes to assist in releasing nucleic acid.

Further, the final concentration of Triton X-100 used is 0.01% -0.2%. The unit of the final concentration is mass percent.

Further, the final concentration of Triton X-100 used is 0.01% -0.05%. The unit of the final concentration is mass percent.

Further, the final concentration of Triton X-100 is 0.05% -0.2%; the unit of the final concentration is mass percent.

Further, the kit may also include other auxiliary reagents. For example, DEPC water.

In a second aspect of the invention there is provided the use of the kit in the extraction of nucleic acids.

The extraction object of the kit is nucleic acid in a sample to be detected, wherein the nucleic acid comprises DNA and/or RNA.

Further, the nucleic acid is selected from animal nucleic acids.

Further, the animal is a mammal. The mammal is preferably a rodent, artiodactyla, perissodactyla, lagomorpha, primate, etc. The rodent is selected from a rat or a mouse. The primate is selected from the group consisting of a monkey, ape, and a human.

Further, the sample to be tested is selected from: blood samples, sputum samples, alveolar lavage samples, in vitro cultured cell lines, animal tissue samples, paraffin samples.

Further, the blood sample may be a peripheral blood sample.

Further, the in vitro cultured cell line is selected from tumor cell lines. For example, lung cancer cell lines.

Further, the animal tissue sample is a mouse tissue sample. Such as lung tissue.

In a third aspect of the present invention, there is provided a method for extracting nucleic acid, comprising the steps of: nucleic acid extraction is performed using the kit of the first aspect of the invention.

Further, the method comprises the steps of:

(1) Adding a sample to be tested into a test container, and adding SDS, EDTA and Triton X-100;

(2) Fully and uniformly mixing a sample to be tested with the added reagent;

(3) Collecting the sample adhered to the pipe wall and the added reagent to the bottom of the pipe;

(4) Fully lysing the cells, isolating chromosomes, inactivating nucleases, denaturing proteins, and releasing nucleic acids;

(5) Precipitating proteins, polysaccharides and other impurities, releasing the nucleic acid into the supernatant;

(6) Taking the supernatant.

Further, in the step (1), the test vessel is selected from EP tubes.

Further, in the step (1), the sample to be tested is added to the test container, and the nucleic acid protecting agent is added first.

The nucleic acid protecting agent can protect extracted nucleic acid from degradation and from contamination by source RNA and DNA.

The nucleic acid protecting agent may be DEPC water.

Further, in the step (2), a vortex oscillation mode can be adopted.

Further, in the step (3), a transient centrifugation mode can be adopted.

Further, in the step (4), a heating mode may be adopted.

Further, in the step (5), a centrifugal mode can be adopted.

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

The nucleic acid lifting kit of the invention adopts specific composition components and proportions, so that the nucleic acid in a detected sample is thoroughly released under the unique final concentration condition when the kit is used, and the residual trace SDS, EDTA and Triton X-100 can form precipitation with organic matters in the solution, and the protein is denatured at high temperature, so that only trace inorganic salt ions and water-soluble nucleic acid remain in the solution after centrifugation, and the nucleic acid released by the cleavage method can be directly used for nucleic acid detection without magnetic bead or column purification. However, more alcohol is needed to be added for cleaning in the traditional nucleic acid extraction process, and the technical scheme of the invention does not need to carry out the step of alcohol cleaning, so that the unpurified nucleic acid in the technical scheme of the invention has no influence on normal PCR amplification. That is, the amplification is not affected by higher concentration of salt ions or organic molecules in the PCR amplification process, but the nucleic acid loss is low due to fewer operation steps, so that the recovery rate of the nucleic acid is greatly improved, and the recovery rate of the nucleic acid in the technical scheme of the invention can be up to 80%, but the recovery rate of the nucleic acid in the prior art is only about 40%. Therefore, the technical scheme of the invention has the advantages of low cost (each part is not more than 0.5 yuan, only one twentieth of the prior art), simple operation, no influence on PCR amplification due to the extraction of the product, and 80 percent of extraction efficiency because no step of nucleic acid loss is introduced in the operation process.

Drawings

Fig. 1: results of DNA measurement in blood.

Fig. 2: results of RNA determination in blood.

Fig. 3: DNA determination results of sputum samples.

Fig. 4: DNA assay results of alveolar lavage fluid samples.

Fig. 5: RNA assay results of in vitro cultured cells.

Fig. 6: DNA determination results of mouse tissue samples.

Fig. 7: results of DNA determination of paraffin samples.

Detailed Description

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention. The test methods in the following examples, in which specific conditions are not noted, are generally conducted under conventional conditions or under conditions recommended by the respective manufacturers.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art, see in particular Sambrook et al MOLECULAR CLONING：A LABORATORY MANUAL,Second edition,Cold Spring HarborLaboratory Press,1989and Third edition,2001;Ausubel et al ,CURRENT PROTOCOLS INMOLECULAR BIOLOGY,John Wiley&Sons,New York,1987and periodic updates;theseries METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATINSTRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODSIN ENZYMOLOGY,Vol.304,Chromatin(P.M.Wassarman and A.P.Wolffe,eds.),AcademicPress,San Diego,1999; and METHODS IN MOLECULAR BIOLOGY, vol.119, chromatinProtocols (P.B. Becker, ed.) Humana Press, totowa,1999, et al.

Example 1 construction of the kit for rapid nucleic acid extraction of the present invention

The rapid nucleic acid extraction kit of the invention comprises SDS, EDTA and Triton X-100. After the sample to be tested is added, the final concentration (mass percent) of SDS is 0.1-1%. The final concentration (mass percent) of EDTA is 0.05-0.8%. The final concentration (mass percent) of Triton X-100 is 0.01% -0.2%.

Example 2 application of the kit of the invention in DNA extraction of blood samples

2.1 Purpose of test

The application and the efficiency of the kit in DNA extraction of blood samples are verified. The kits used in this example include SDS, EDTA and Triton X-100, as shown in Table 1 below.

Table 1 kit composition

2.2 Test methods

(1) Test sample: the peripheral blood sample is derived from the remaining blood after the clinical laboratory test procedure is completed. The instrumentation involved in this test includes: PCR instrument (ABI 7300), EP tube, centrifuge, metal bath (for 90 ℃ C. Heating).

(2) Experimental group: the present example 2 includes 3 experimental groups, specifically, the 1 experimental group uses the 1 kit in table 1, the 2 experimental group uses the 2 kit, and the 3 experimental group uses the 3 kit. For test group 1, 100. Mu.l of whole blood sample was added to a 1.5ml of nuclease-free EP tube, 100. Mu.l of DEPC water was added, and the reagents of kit 1 were added by calculation so that the final concentrations of SDS, EDTA and Triton X-100 were used at 0.25%,0.2% and 0.05% of the reagents of kit 1, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For test group 2, an additional 100. Mu.l of whole blood sample was taken and added to the 1.5ml of the non-ribozyme EP tube, and the procedure was the same as that of test group 1 of this example except that the reagents of kit 2 were made to use SDS, EDTA and Triton X-100 at final concentrations of 0.1%,0.8% and 0.2%. For test group 3, an additional 100. Mu.l of whole blood sample was taken and added to the 1.5ml of the non-ribozyme EP tube, and the procedure was the same as that of test group 1 of this example except that the final concentrations of reagent for test kit 3 were 1%,0.05% and 0.01% SDS, EDTA and Triton X-100.

(3) Control group: this example 2 included 3 control groups, specifically, control group No. 1 used the comparative kits in table 1; the control group No. 2 uses a commercial magnetic bead method kit, and the control group No. 3 uses a commercial column kit. For control group 1, 100. Mu.l of whole blood sample of the same sample of the experimental group of this example was taken in parallel and added to a 1.5ml of nuclease-free EP tube, 100. Mu.l of DEPC water was added, and the reagents added to the comparative kit of Table 1 were calculated so that the final concentrations of SDS, EDTA and Triton X-100 were used at 2%,1% and 0.5% of the final concentrations in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For both control No. 2 and control No. 3, peripheral blood DNA was extracted strictly according to the instructions of the corresponding kit, taking about 40 minutes.

(4) Nucleic acid determination: detecting the concentration of nucleic acid DNA obtained in the experimental group and the control group, respectively taking 2ng as a template, and adopting BerleThe EGFR gene of interest was quantified on the ABI 7300 by Green Supermix. The reaction conditions are as follows: 30sec at 95 ℃;95℃for 5sec,60℃for 30sec,40 cycles; 30sec at 72 ℃.

The results of the detection are shown in Table 2 and FIG. 1 below.

TABLE 2 determination of DNA in blood

As can be seen from table 2 and fig. 1, the highest fluorescence intensity analysis results for EGFR detection CT values and amplification curves show that: the concentration (mass percent) of SDS (0.1% -1%), EDTA (0.05% -0.8%) and Triton X-100 (0.01% -0.2%) of human blood sample DNA extracted within the range of the kit does not have adverse effect on PCR amplification, the CT value of target gene detection is smaller than that outside the concentration range of the kit, and the detection CT value (CT value average 3-4) corresponding to the sample extracted by the centrifugation column method and the magnetic bead method, which shows that the extraction method not only does not influence PCR amplification, but also has the extraction efficiency far higher than that of the control group column method and the magnetic bead method (the extraction efficiency is about 8-16 times higher and the extraction time is about 4 times shorter).

Example 3 application of the kit of the invention in the extraction of RNA from blood samples

3.1 Purpose of test

The application and the efficiency of the kit in blood sample RNA extraction are verified. The kit used in this example includes SDS, EDTA and Triton X-100, as shown in Table 1 of example 2.

3.2 Test methods

(1) Test sample: the peripheral blood sample was derived from the remaining blood after completion of the clinical laboratory test, and was tested using the same batch of whole blood as in example 1. The instrumentation involved in this test includes: PCR instrument (ABI 7300), EP tube, centrifuge, metal bath (for 90 ℃ C. Heating).

(2) Experimental group: the present example 3 includes 3 experimental groups, specifically, the 1 experimental group uses the 1 kit in the table 1 of the example 2, the 2 experimental group uses the 2 kit, and the 3 experimental group uses the 3 kit. For test group 1, 100. Mu.l of whole blood sample was added to a 1.5ml of nuclease-free EP tube, 100. Mu.l of DEPC water was added, and the reagents of kit 1 were added by calculation so that the final concentrations of SDS, EDTA and Triton X-100 were used at 0.25%,0.2% and 0.05% of the reagents of kit 1, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For test group 2, an additional 100. Mu.l of whole blood sample was taken and added to the 1.5ml of the non-ribozyme EP tube, and the procedure was the same as that of test group 1 of this example except that the reagents of kit 2 were made to use SDS, EDTA and Triton X-100 at final concentrations of 0.1%,0.8% and 0.2%. For test group 3, an additional 100. Mu.l of whole blood sample was taken and added to the 1.5ml of the non-ribozyme EP tube, and the procedure was the same as that of test group 1 of this example except that the final concentrations of reagent for test kit 3 were 1%,0.05% and 0.01% SDS, EDTA and Triton X-100.

(3) Control group: this example 3 included 3 control groups, specifically, control group No. 1 used the comparative kits of example 2, table 1; the control group No. 2 uses a commercial magnetic bead method kit, and the control group No. 3 uses a commercial column kit. For control group 1, 100. Mu.l of whole blood sample of the same sample of the experimental group of this example was taken in parallel and added to an EP tube of 1.5ml of the nuclease-free, 100. Mu.l of DEPC water was added, and the reagents added to the comparative kit of Table 1 were calculated so that the final concentrations of use of the comparative kit were 2%,1% and 0.5% of SDS, EDTA and Triton X-100, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For both control No. 2 and control No. 3, peripheral blood RNA extraction was performed strictly according to the instructions of the corresponding kit, taking about 40 minutes.

(4) Nucleic acid determination: nucleic acid RNAs obtained in the experimental group and the control group were quantified by NanoDrop, 500ngRNA was taken, and RNA was reverse transcribed into cDNA using the iScript ^TM cDNA synthesis kit, and then the synthesized cDNA was used as a template. Respectively taking 2ng as templates, and using BereThe EGFR gene of interest was quantified on the ABI 7300 by Green Supermix. The reaction conditions are as follows: 30sec at 95 ℃;95℃for 5sec,60℃for 30sec,40 cycles; 30sec at 72 ℃.

The detection results are shown in Table 3 and FIG. 2 below.

TABLE 3 determination of RNA in blood

From table 3 and fig. 2, the highest fluorescence intensity analysis results for EGFR detection CT values and amplification curves show that: the concentration (mass percent) of SDS (0.1% -1%), EDTA (0.05% -0.8%) and Triton X-100 (0.01% -0.2%) of human blood sample RNA extracted within the range of the kit does not have adverse effect on PCR amplification, the CT value of target gene detection is smaller than that outside the concentration range of the kit, and the detection CT value (CT value average 3-4) corresponding to the sample extracted by the centrifugation column method and the magnetic bead method, which shows that the extraction method not only does not influence PCR amplification, but also has the extraction efficiency far higher than that of the control group column method and the magnetic bead method (the extraction efficiency is about 8-16 times higher and the extraction time is about 4 times shorter).

Example 4 application of the kit of the invention in the extraction of DNA from sputum samples

4.1 Purpose of test

The application and the efficiency of the kit in the DNA extraction of sputum samples are verified. The kit used in this example includes SDS, EDTA and Triton X-100, as shown in Table 1 of example 2.

4.2 Test methods

(1) Test sample: the sputum to be expected during the practice of sputum production during hospitalization of a lung transplant patient was used as a sample for comparative analysis. The instrumentation involved in this test includes: PCR instrument (ABI 7300), EP tube, centrifuge, metal bath (for 90 ℃ C. Heating).

(2) Experimental group: the present example 4 includes 3 experimental groups, specifically, the 1 experimental group uses the 1 kit in the table 1 of the example 2, the 2 experimental group uses the 2 kit, and the 3 experimental group uses the 3 kit. For test group 1, 1ml was placed in a 1.5ml EP tube without ribozyme after sputum liquefaction, 12000Xg was centrifuged for 2 minutes to discard the supernatant and precipitate, 100. Mu.l DEPC water was added to the precipitate, and the reagents of kit 1 were added by calculation so that the final concentrations of 0.25%,0.2% and 0.05% SDS, EDTA and Triton X-100 were used as the reagents of kit 1, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For test group 2, 1ml of the sputum-liquefied treatment was placed in an EP tube of 1.5ml without ribozyme, and the procedure was the same as that of test group 1 of this example except that SDS, EDTA and Triton X-100 were used in the final concentrations of 0.1%,0.8% and 0.2% of the reagents of the kit No. 2. For test group 3, 1ml was placed in an EP tube of 1.5ml without ribozyme after the sputum liquefaction treatment, and the procedure was the same as that of test group 1 of this example except that SDS, EDTA and Triton X-100 were used in the final concentrations of 1%,0.05% and 0.01% for the reagents of the kit No. 3.

(3) Control group: this example 3 included 3 control groups, specifically, control group No. 1 used the comparative kits of example 2, table 1; the control group No. 2 uses a commercial magnetic bead method kit, and the control group No. 3 uses a commercial column kit. For control group 1, 1ml of sputum of the same sample of the experimental group of this example was liquefied and placed in a 1.5ml of non-ribozyme EP tube, 12000Xg was centrifuged for 2 minutes, and the supernatant was discarded, 100. Mu.l of DEPC water was added to the pellet, and the reagents of the comparative kit of Table 1 were calculated so that the final concentrations of the comparative kit were 2%,1% and 0.5% SDS, EDTA and Triton X-100, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For both control No. 2 and control No. 3, the extraction of DNA from sputum samples took about 40 minutes, strictly following the instructions of the corresponding kit.

(4) Nucleic acid determination: measuring nucleic acid concentration obtained in experimental group and control group, respectively taking 2ng as template, and using BereThe EGFR gene of interest was quantified on the ABI 7300 by Green Supermix. The reaction conditions are as follows: 30sec at 95 ℃;95℃for 5sec,60℃for 30sec,40 cycles; 30sec at 72 ℃.

4.3 Test results

The detection results are shown in the following table 4 and fig. 3.

TABLE 4 DNA determination results of sputum samples

As can be seen from the results of Table 4 and FIG. 3, the DNA of the sputum sample extracted by the extraction method of the kit of the present invention did not affect PCR amplification, but the extraction efficiency was much higher than that by the column-passing method and the magnetic bead method (the extraction efficiency was about 8-16 times higher, and the extraction time was about 4 times shorter.

Example 5 use of the kit of the invention in DNA extraction in alveolar lavage fluid

5.1 Purpose of test

The application and the efficiency of the kit in DNA extraction of alveolar lavage fluid samples are verified. The kit used in this example includes SDS, EDTA and Triton X-100, as shown in Table 1 of example 2.

5.2 Test methods

(1) Test sample: the alveolar lavage fluid left when the lung transplant patient performs alveolar lavage is used as a sample for comparison analysis. The instrumentation involved in this test includes: PCR instrument (ABI 7300), EP tube, centrifuge, metal bath (for 90 ℃ C. Heating).

(2) Experimental group: the present example 4 includes 3 experimental groups, specifically, the 1 experimental group uses the 1 kit in the table 1 of the example 2, the 2 experimental group uses the 2 kit, and the 3 experimental group uses the 3 kit. For test group 1, 2ml of alveolar lavage fluid sample was centrifuged at 12000Xg for 2 min, the supernatant was discarded, 100. Mu.l of DEPC water was added to the pellet, and the reagents of kit 1 were added by calculation so that the final concentrations of SDS, EDTA and Triton X-100 were used at 0.25%,0.2% and 0.05% of the reagent of kit 1, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For experimental group 2, 2ml of alveolar lavage fluid was taken, and the procedure was the same as that of experimental group 1 of this example except that the final concentrations of reagents of kit 2 were 0.1%,0.8% and 0.2% SDS, EDTA and Triton X-100. For test group 3, 2ml of alveolar lavage fluid was taken, and the procedure was the same as that of test group 1 of this example except that the final concentrations of reagent used in the kit 3 were 1%,0.05% and 0.01% SDS, EDTA and Triton X-100.

(3) Control group: this example 4 included 3 control groups, specifically, control group No. 1 used the comparative kits of example 2, table 1; the control group No. 2 uses a commercial magnetic bead method kit, and the control group No. 3 uses a commercial column kit. For control group 1, 2ml of alveolar lavage fluid samples of the same sample of the experimental group of this example were taken in parallel, centrifuged at 12000Xg for 2 minutes, and the supernatant was discarded, 100. Mu.l of DEPC water was added to the pellet, and the reagents added to the comparative kit of Table 1 were calculated so that the final concentrations of use of the comparative kit were 2%,1% and 0.5% SDS, EDTA and Triton X-100, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For both control No. 2 and control No. 3, the extraction of alveolar lavage DNA took about 40 minutes, strictly following the instructions of the corresponding kit.

5.3 Test results

The detection results are shown in the following table 5 and fig. 4.

TABLE 5 DNA determination results of alveolar lavage samples

As can be seen from Table 5 and FIG. 4, the extraction of DNA from alveolar lavage fluid samples by the kit extraction method of the present invention did not affect PCR amplification, but rather the extraction efficiency was much higher than that by the column-passing method and the magnetic bead method (the extraction efficiency was about 8-16 times higher, and the extraction time was about 4 times shorter).

Example 6 application of the kit of the invention in the extraction of RNA from in vitro cultured cell lines

6.1 Purpose of test

The application and the efficiency of the kit in the extraction of RNA of in vitro cultured cell line samples are verified. The kit used in this example includes SDS, EDTA and Triton X-100, as shown in Table 1 of example 2.

6.2, Test method

(1) Test sample: lung cancer cell lines a549 and H1299 were each inoculated into 6-well plates at a density of 2 x 10 ⁶, cultured for 24 hours, dispersed by trypsin digestion and collected into different EP tubes, and examined on the same batch of cultured cell lines as samples. The instrumentation involved in this test includes: PCR instrument (ABI 7300), EP tube, centrifuge, metal bath (for 90 ℃ C. Heating).

(2) Experimental group: the present example 4 includes 3 experimental groups, specifically, the 1 experimental group uses the 1 kit in the table 1 of the example 2, the 2 experimental group uses the 2 kit, and the 3 experimental group uses the 3 kit. For experiment group 1, each of the collected A549 and H1299 tubes was taken, 100. Mu.l of DEPC water was added, and the reagent of kit 1 was added by calculation so that the final concentrations of the reagent of kit 1 were 0.25%,0.2% and 0.05% SDS, EDTA and Triton X-100, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For experimental group No. 2, the same operations as those of experimental group No. 1 of this example were conducted except that one tube of each of collected a549 and H1299 was used so that the final concentrations of the reagent of kit No. 2 were 0.1%,0.8% and 0.2% SDS, EDTA and Triton X-100. For test group 3, the same operations as test group 1 of this example were conducted except that one tube of each of collected A549 and H1299 was used so that the final concentrations of the reagent used in the kit 3 were 1%,0.05% and 0.01% SDS, EDTA and Triton X-100.

(3) Control group: this example 4 included 4 control groups, specifically, control group No. 1 used the comparative kits of example 2, table 1; the control group No. 2 uses a commercial magnetic bead method kit, the control group No. 3 uses a commercial column kit, and the control group No. 4 uses a commercial Trizol. For control group 1, A549 and H1299 cultured in the same 6-well plate of the experimental group of this example were taken in parallel, 100. Mu.l of DEPC water was added to each tube, and the reagents for the comparative kits in Table 1 were added by calculation so that the final concentrations of SDS, EDTA and Triton X-100 were 2%,1% and 0.5% in terms of mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For the control No. 2, the control No. 3 and the control No. 4, the extraction of the lung cancer cell lines a549 and H1299RNA took about 40 minutes, strictly according to the instructions of the corresponding kit.

(4) Nucleic acid determination: RNA extracted from the experimental group and the control group was quantified with NanoDrop, 500ng of RNA was taken, and RNA was reverse transcribed into cDNA using the iScript ^TM cDNA synthesis kit, and then the synthesized cDNA was used as a template. Respectively taking 2ng as templates, and using BereThe EGFR gene of interest was quantified on the ABI 7300 by Green Supermix. The reaction conditions are as follows: 30sec at 95 ℃;95℃for 5sec,60℃for 30sec,40 cycles; 30sec at 72 ℃.

6.3 Test results

The detection results are shown in the following table 6 and fig. 5.

TABLE 6 RNA determination results of in vitro cultured cell line samples

As can be seen from Table 6 and FIG. 5, the extraction of RNA from the cell line by the extraction method of the kit of the present invention did not affect PCR amplification, but rather the extraction efficiency was much higher than that by the column-passing method and the magnetic bead method (the extraction efficiency was about 8-16 times higher, and the extraction time was about 4 times shorter).

Example 7 application of the kit of the invention in the extraction of DNA from tissue samples of mice

7.1 Purpose of test

The application and the efficiency of the kit in the DNA extraction of the mouse tissue samples are verified. The kit used in this example includes SDS, EDTA and Triton X-100, as shown in Table 1 of example 2.

7.2 Test methods

(1) Test sample: the same batch of lung tissue samples of mice are taken as samples for detection. The instrumentation involved in this test includes: PCR instrument (ABI 7300), EP tube, centrifuge, metal bath (for 90 ℃ C. Heating).

(2) Experimental group: the present example 4 includes 3 experimental groups, specifically, the 1 experimental group uses the 1 kit in the table 1 of the example 2, the 2 experimental group uses the 2 kit, and the 3 experimental group uses the 3 kit. For experiment group 1, a fresh sample of mouse lung was taken and homogenized twice with a tissue homogenizer 50HZ for 45 seconds, one of which was placed in a 1.5ml non-ribozyme EP tube and 100 μl of DEPC water was added, followed by calculation of the reagent added to kit 1 such that the final concentrations of 0.25%,0.2% and 0.05% SDS, EDTA and Triton X-100 were used for the kit 1, where the final concentrations were used in mass percent. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For experiment group 2, another homogenized sample of mouse lung was taken, and the procedure was the same as in experiment group 1 of this example, except that the final concentrations of reagents of kit 2 were 0.1%,0.8% and 0.2% SDS, EDTA and Triton X-100. For experiment group 3, another homogenized sample of mouse lung was taken, and the procedure was the same as in experiment group 1 of this example, except that the final concentrations of reagent used in kit 3 were 1%,0.05% and 0.01% SDS, EDTA and Triton X-100.

(3) Control group: this example 4 included 3 control groups, specifically, control group No. 1 used the comparative kits of example 2, table 1; the control group No. 2 uses a commercial magnetic bead method kit, and the control group No. 3 uses a commercial column kit. For control group 1, homogenized mouse lung samples of the same sample of the experimental group of this example were taken in parallel, placed in a 1.5ml of non-ribozyme EP tube, 100. Mu.l of DEPC water was added, and the reagents for the comparative kit of Table 1 were added by calculation so that the final concentrations of the comparative kit were 2%,1% and 0.5% SDS, EDTA and Triton X-100, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For both control No. 2 and control No. 3, the extraction of the mouse lung sample DNA took about 40 minutes, strictly following the instructions of the corresponding kit.

7.3 Test results

The detection results are shown in the following table 7 and fig. 6.

TABLE 7 DNA determination results of mouse tissue samples

As can be seen from the results of Table 7 and FIG. 6, the extraction of DNA from the tissue sample of the mouse by the extraction method of the kit of the present invention did not affect PCR amplification, but the extraction efficiency was much higher than that by the column-passing and magnetic bead method (the extraction efficiency was about 8-16 times higher, and the extraction time was about 4 times shorter).

Example 8 application of the kit of the invention in the extraction of Paraffin sample DNA

7.1 Purpose of test

The application and the efficiency of the kit in the extraction of the paraffin sample DNA are verified. The kit used in this example includes SDS, EDTA and Triton X-100, as shown in Table 1 of example 2.

7.2 Test methods

(1) Test sample: paraffin embedded samples of non-small cell lung cancer patients after surgery were taken and sectioned at a thickness of 5 μm and used for DNA extraction in experimental and control groups, respectively. The instrumentation involved in this test includes: PCR instrument (ABI 7300), EP tube, centrifuge, metal bath (for 90 ℃ C. Heating).

(2) Experimental group: the present example 4 includes 3 experimental groups, specifically, the 1 experimental group uses the 1 kit in the table 1 of the example 2, the 2 experimental group uses the 2 kit, and the 3 experimental group uses the 3 kit. For experiment group 1, paraffin section samples of 5 non-small cell lung cancer patients were taken, scraped into 1.5ml of non-ribozyme EP tube, dewaxed with PBS 3 times, placed in an oven at 56℃for 10 minutes to evaporate the dewaxed organic solvent completely, 100. Mu.l of DEPC water was added, and the reagents of kit 1 were calculated to give final concentrations of 0.25%,0.2% and 0.05% SDS, EDTA and Triton X-100, where the final concentrations were used in mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For test group 2, paraffin section samples of another 5 non-small cell lung cancer patients were taken, and the same procedure as in test group 1 of this example was conducted except that the final concentrations of the reagents of the kit 2 were 0.1%,0.8% and 0.2% SDS, EDTA and Triton X-100. For experiment group 3, paraffin section samples of another 5 non-small cell lung cancer patients were taken, and the same procedure as in experiment group 1 of this example was conducted except that the final concentrations of the reagents of the kit 3 were 1%,0.05% and 0.01% SDS, EDTA and Triton X-100.

(3) Control group: this example 4 included 3 control groups, specifically, control group No. 1 used the comparative kits of example 2, table 1; the control group No. 2 uses a commercial magnetic bead method kit, and the control group No. 3 uses a commercial column kit. For control group 1,5 of the above paraffin section samples of the same sample of the experimental group of this example were taken in parallel, scraped into an EP tube of 1.5ml of a non-ribozyme, dewaxed and washed 3 times with PBS, the organic solvent was completely volatilized in the same manner as described above, 100. Mu.l of DEPC water was added, and the reagents of the comparative kit of Table 1 were calculated so that the final concentrations of 2%,1% and 0.5% of SDS, EDTA and Triton X-100 were used in the final concentrations of mass%. Vortex shaking for 30 seconds, and instantaneous centrifugation; heating at 90 ℃ for 3 minutes; centrifuging at 8000rpm for 3 minutes; taking the supernatant (for later use). For both control No. 2 and control No. 3, the extraction of non-small cell lung cancer paraffin embedded sample DNA took about 40 minutes, strictly according to the instructions of the corresponding kit.

8.3 Test results

The detection results are shown in the following table 8 and fig. 7.

TABLE 8 DNA determination results of Paraffin samples

As can be seen from the results of the above Table 8 and FIG. 7, the DNA of the paraffin tissue sample of non-small cell lung cancer extracted by the extraction method of the kit of the present invention does not affect PCR amplification, but has much higher extraction efficiency than the column-passing method and the magnetic bead method (the extraction efficiency is about 8-16 times higher, and the extraction time is about 4 times shorter).

While the invention has been described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and additions may be made without departing from the scope of the invention. Equivalent embodiments of the present invention will be apparent to those skilled in the art having the benefit of the teachings disclosed herein, when considered in the light of the foregoing disclosure, and without departing from the spirit and scope of the invention; meanwhile, any equivalent changes, modifications and evolution of the above embodiments according to the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A nucleic acid extraction kit comprising SDS, EDTA and Triton X-100.

2. The kit of claim 1, further comprising any one of: (1) the final concentration of SDS is 0.1% -1%; (2) the final concentration of SDS is 0.1% -0.25%; (3) the final concentration of SDS is 0.25% -1%; the unit of the final concentration is mass percent.

3. The kit of claim 1, further comprising any one of: (1) the final concentration of EDTA used is 0.05% to 0.8%; (2) the final concentration of EDTA is 0.05% -0.2%; (3) the final use concentration of EDTA is 0.2% -0.8%; the unit of the final concentration is mass percent.

4. The kit of claim 1, further comprising any one of: (1) The final concentration of Triton X-100 is 0.01% -0.2%; (2) The final concentration of Triton X-100 is 0.01% -0.05%; (3) The final concentration of Triton X-100 is 0.05% -0.2%; the unit of the final concentration is mass percent.

5. The kit of claim 1, further comprising a combination of any one or more of: (1) The SDS can sufficiently lyse cells under the proper temperature condition to separate chromosomes and denature proteins, and simultaneously, the SDS is combined with the proteins and the polysaccharide to form a complex, so that the nucleic acid is released; (2) The EDTA can be combined with auxiliary factors of intracellular enzyme activity, inhibit the activities of nuclease and protease, and protect nucleic acid from degradation or denaturation; (3) The Triton X-100 can dissolve lipids on cell membranes and nuclear membranes to assist in the release of nucleic acids.

6. Use of a kit according to any one of claims 1-5 for extracting nucleic acids.

7. The use according to claim 6, wherein the nucleic acid comprises DNA and/or RNA.

8. The use according to claim 6, wherein the nucleic acid is from a mammal.

9. A method for extracting nucleic acid, comprising the steps of: extraction using the kit of any one of claims 1-5.

10. The method according to claim 9, comprising the steps of: (1) Adding a sample to be tested into a test container, and adding SDS, EDTA and Triton X-100; (2) fully and uniformly mixing the sample to be tested with the added reagent; (3) Collecting the sample adhered to the pipe wall and the added reagent to the bottom of the pipe; (4) Fully lysing the cells, isolating chromosomes, inactivating nucleases, denaturing proteins, and releasing nucleic acids; (5) Precipitating proteins, polysaccharides and other impurities, releasing the nucleic acid into the supernatant; (6) taking the supernatant.