CN109810974B - Reagent composition for extracting HIV-1 nucleic acid and method thereof - Google Patents
Reagent composition for extracting HIV-1 nucleic acid and method thereof Download PDFInfo
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Images
Abstract
The invention relates to the field of nucleic acid extraction, in particular to a reagent combination product for extracting human immunodeficiency virus HIV-1 nucleic acid and a method thereof. The reagent combination product comprises lysis solution: 2-6mol/L of guanidinium isothiocyanate, 3-20mmol/L of chelating agent, 1-10% of anionic surfactant, 100-4 mol/L of nonionic surfactant, 1-4mol/L of isopropanol and 0.1-5% of CTAB; washing liquid: 15-45mmol/L of anhydrous citric acid, 10-40mmol/L of sodium citrate dihydrate, 5-3ml/L of Proclin9500.5, and the pH value of the mixture is 4.7-5.3; eluent: 10-40mmol/L of trihydroxymethyl aminomethane, 1-15mmol/L of trihydroxymethyl aminomethane hydrochloride, 1.5-3 ml/L of Proclin 9500, and 0.3-2 mu g/mu L of Carrier RNA. The components are cooperated, so that the extraction sensitivity, the nucleic acid quality and the extraction efficiency are improved, and the extraction stability is good.
Description
Technical Field
The invention relates to the field of nucleic acid extraction, in particular to a reagent combination product for extracting human immunodeficiency virus HIV-1 nucleic acid and a method thereof.
Background
Since the first reported example of AIDS in the United states in 1981, the AIDS causes over 2800 million deaths, and the virus has high lethality and infectivity, and no effective treatment exists so far, and is listed as one of the major high-risk prevention and control viruses in the world.
AIDS (Human Immunodeficiency Virus, HIV) is a reverse transcription RNA Virus, and the main pathogenic principle of AIDS is that various viruses cannot be killed in time due to the breakdown of immune mechanisms of Human bodies, and the various viruses are normally propagated in the Human bodies to generate clinical syndromes such as tumors and the like, so the lethality is extremely high. AIDS is divided into HIV-1 type and HIV-2 type according to its genotype, and HIV-1 has stronger infectivity and pathogenicity compared with HIV-2. The gene structure of HIV is basically the same as that of other retroviruses, and the HIV is an RNA double molecule, wherein a single-stranded RNA molecule of the HIV consists of 9749 nucleotides, and has 3 groups of 9 genes. The first group is the genes common to retroviruses, i.e., gag, pol, and env genes, which are structural genes of the virus and encode viral proteins. The HIV-2 virus discovered worldwide is mainly epidemic in Nonzea, most of the HIV-2 virus is concentrated in West Africa regions, the HIV epidemic in China is HIV-1 type strains, because the AIDS is retrovirus, and the genes are bound to have diversity, the HIV-1 strains epidemic in different regions in China are not completely the same and have a plurality of subtypes, and the HIV-1 mainly has strong mutation capability due to the high mutability of the genes env, gag and pol in the M group of the HIV-1 in China. Therefore, in order to accurately detect AIDS, a high-degree conserved region must be found during sequence selection, and a plurality of sections are selected as much as possible for design.
The detection method of AIDS mainly comprises virus culture detection, an immune gold-labeled method, antibody detection, nucleic acid detection and the like, and has overlong virus culture detection period and low sensitivity; the immune gold labeling method has low sensitivity; the antibody detection is also carried out after a certain scale of antibody formation in a host reaches the detection limit, the window period is not short, about 3-5 weeks, and the time is very long for a patient who needs to know the disease result urgently; with the development of biological detection technology, HIV nucleic acid detection has been widely popularized and applied in China due to its advantages of relatively short window period (about 1 week), rapid detection efficiency (about 2 hours), higher sensitivity, etc.
In nucleic acid detection, when the detection object is a mixed nucleic acid, a method of nucleic acid mixed detection is used, for example, blood screening requires simultaneous detection of a plurality of viruses, and compatibility is required; when the detection target is a single nucleic acid, the detection is performed using a single nucleic acid, and therefore, the detection is targeted. At present, the HIV-1 nucleic acid extraction method on the market has a large optimization space, such as redundant steps, redundant reaction time, a formula and proportion with higher biological risk, rough reaction details, higher detection limit and the like.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a reagent combination product for extracting human immunodeficiency virus HIV-1 nucleic acid, all components are cooperated, the extraction sensitivity, the nucleic acid quality and the extraction efficiency are improved, and the extraction stability is good.
The second purpose of the invention is to provide a method for extracting human immunodeficiency virus HIV-1 nucleic acid, which integrates cracking and combination into one step, optimizes washing eluent, strictly controls the operation time of each step, and can obtain target nucleic acid by only 3 steps, thereby greatly improving the extraction efficiency of nucleic acid.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a reagent combination for extracting human immunodeficiency virus HIV-1 nucleic acid comprising:
lysis solution: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 2-6mol/L of guanidinium isothiocyanate, 3-20mmol/L of chelating agent, 1-10% of mass concentration of anionic surfactant, 300mmol/L of nonionic surfactant, 1-4mol/L of isopropanol and 0.1-5% of mass concentration of CTAB;
washing liquid: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 15-45mmol/L of anhydrous citric acid, 10-40mmol/L of sodium citrate dihydrate, 5-3.0ml/L of Proclin 9500.5, and the pH value is 4.7-5.3;
eluent: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 10-40mmol/L of trihydroxymethyl aminomethane, 1-15mmol/L of trihydroxymethyl aminomethane hydrochloride, 1.5-3.0 ml/L of Proclin 9500, and 0.3-2.0 μ g/μ L of Carrier RNA.
The reagent combination product for extracting the HIV-1 nucleic acid provided by the invention has the advantages that the lysis solution contains the anionic surfactant and CTAB, impurities generated in cell lysis are easier to digest due to the two components, the nucleic acid quality and the extraction efficiency are improved, and the lysis and the combination are integrated into one step; the reagents are cooperated with each other, the quality of the extracted nucleic acid is better, clinical verification proves that the detection limit of the extracted nucleic acid is not less than 30IU/ml, and compared with the extracted product on the existing market, the detection limit is 60IU/ml, and the sensitivity is higher. In addition, the stability of reagent extraction is good.
Further, the chelating agent is sodium citrate dihydrate.
Further, the anionic surfactant is dodecylaminopropanesulfonic acid.
Further, the nonionic surfactant is triton X-100.
Further, the reagent also comprises proteinase K, magnetic beads and mineral oil.
Further, the concentration of proteinase K is 20 + -5 mg/ml.
Further, the magnetic beads are silicon-based magnetic beads.
Further, the particle size of the magnetic beads is 1.2 +/-0.2 μm.
Further, the mineral oil is paraffin oil.
Preferably, the lysate: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 4.2 +/-0.2 mol/L of guanidinium isothiocyanate, 10 +/-1 mmol/L of chelating agent, 5% +/-0.5% of anionic surfactant mass concentration, 167.6 +/-5 mmol/L of nonionic surfactant, 2.6 +/-0.2 mol/L of isopropanol and 0.5% +/-0.05% of CTAB mass concentration.
Preferably, the washing solution: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 30.7 plus or minus 2mmol/L of anhydrous citric acid, 19.2 plus or minus 2mmol/L of sodium citrate dihydrate, 9501 plus or minus 0.2ml/L of Proclin and pH of 5 plus or minus 0.2.
Preferably, the eluent: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 22.5 plus or minus 0.5mmol/L of trihydroxymethyl aminomethane, 7.4 plus or minus 0.2mmol/L of trihydroxymethyl aminomethane hydrochloride, 9501 plus or minus 0.2ml/L of Proclin and 1 plus or minus 0.2 mu g/mu L of Carrier RNA.
The invention also provides an extraction method of the human immunodeficiency virus HIV-1 nucleic acid, which adopts the reagent combination product for extraction and comprises the following steps:
sequentially adding protease K, an internal standard, a sample to be detected, a lysis solution, mineral oil and magnetic beads, mixing and lysing, and performing magnetic adsorption;
adding the washing solution for rinsing, and removing liquid after magnetic adsorption;
then adding the eluent to elute the nucleic acid adsorbed on the magnetic beads to obtain a liquid, namely the extracting solution containing the target nucleic acid.
The operation steps of the extraction reagent of the invention integrate cracking and combination into one step, and optimize the washing eluent, and the operation time of each step is strictly controlled, and the target nucleic acid can be obtained only by 3 steps, thereby greatly improving the extraction efficiency of the nucleic acid.
Preferably, the volume ratio of the proteinase K, the internal standard (the concentration range is 500-10000IU/ml, preferably 8000IU/ml), the sample to be detected, the lysate, the mineral oil and the magnetic beads (the concentration is 50 +/-10 mg/ml) is 8 +/-0.5: 1:100 +/-1: 96 +/-1: 8 +/-0.5: 2 +/-0.2.
In the present invention, the sample to be tested is generally a blood sample.
The lysis solution prepared aiming at HIV nucleic acid has high lysis force, and the lysis solution can be fully lysed at a ratio lower than that of a sample.
Further, the mixing is uniformly mixed by blowing, and the blowing time is 120 +/-10 s.
Further, magnetic adsorption is carried out after the blowing, beating and uniformly mixing are finished, and the magnetic adsorption time is 360 +/-30 s.
Further, the blowing volume is 80% of the total volume of the solution, and the blowing mode is blowing along with the liquid level.
The special blow-beating range, the blow-beating method and the mineral oil sealing cover for the reaction liquid can greatly reduce the generation of fine and small bubbles and improve the efficiency and the stability.
The volume ratio of the washing liquid to the sample to be detected is 80-150: 1, the blow-beating time is 60 +/-10 s, and the magnetic adsorption time is 60 +/-10 s.
Further, the washing solution is washed only once.
Further, the volume ratio of the eluent to the sample to be detected is 10-15: 1, elution time 60. + -.10 s.
Compared with the prior art, the invention has the beneficial effects that:
(1) clinical detection shows that the reagent combination product for extracting the human immunodeficiency virus HIV-1 nucleic acid has a real and reliable detection limit of not less than 30IU/ml, and has a detection limit of 60IU/ml and higher sensitivity compared with the extraction products on the existing market.
(2) The reagent combination product for extracting the human immunodeficiency virus HIV-1 nucleic acid provided by the invention has high nucleic acid quality and extraction efficiency and good extraction stability.
(3) The operation steps of the extraction reagent of the invention integrate cracking and combination into one step, and optimize the washing eluent, and the operation time of each step is strictly controlled, and the target nucleic acid can be obtained only by 3 steps, thereby greatly improving the extraction efficiency of the nucleic acid.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the three extractant formulations of example 2 for 5X 104A curve diagram of real-time fluorescent quantitative PCR is carried out after HIV samples with IU/ml concentration are extracted;
FIG. 2 is a graph showing the PCR of the reagent of the present invention in example 3 after 20 replicates of 100IU/ml HIV specimen;
FIG. 3 is a graph showing the PCR of the reagent of the present invention in example 3 after 20 replicates of a 50IU/ml HIV specimen;
FIG. 4 is a graph showing the PCR of the reagent of the present invention in example 3 after 20 replicates of a 30IU/ml HIV specimen;
FIG. 5 is a graph showing real-time fluorescent quantitative PCR of nucleic acid extracted after adding 160. mu.l of lysis buffer in example 4;
FIG. 6 is a graph showing real-time fluorescent quantitative PCR of nucleic acid extracted after adding 200. mu.l of lysis buffer in example 4;
FIG. 7 is a graph showing real-time fluorescent quantitative PCR of nucleic acids extracted after adding 300. mu.l of lysate in example 4.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The invention provides an optimized extraction reagent capable of quickly and stably extracting HIV-1 nucleic acid from blood. The following detailed description is provided to illustrate the present technology, and it should be particularly pointed out that the embodiments are illustrative of the present disclosure and are not to be construed as limiting the invention, and that the skilled artisan, in light of the present disclosure, may make insubstantial modifications and improvements within the scope of the invention. The steps which are not described in detail in the following embodiments belong to the general technology of the industry.
Example 1
Configuration of nucleic acid extraction reagent
The reagent formula comprises lysis solution A, washing solution B, eluent C, proteinase K, magnetic beads and mineral oil A:
lysate a components were as follows: 4.2mol/L guanidinium isothiocyanate, 10mmol/L sodium citrate dihydrate, 5% dodecylaminopropanesulfonic acid, 167.6mmol/L triton X-100, 2.6mol/L isopropanol and 0.5% CTAB.
The washing solution B comprises the following components: 30.7mmol/L anhydrous citric acid, 19.2mmol/L sodium citrate dihydrate, 1ml Proclin950, pH 5.
Eluent C comprises the following components: 22.5mmol/L Tris, 7.4mmol/L Tris-HCl, 1ml Proclin950, 1. mu.g/. mu.l Carrier RNA.
And (3) protease K: 20 mg/ml.
Magnetic beads: silicon-based magnetic beads with the particle size of 1.2 mu m and the concentration of 50 mg/ml.
Mineral oil A: a paraffinic oil.
Internal standard: armor RNA.
Example 2
Three formulations of nucleic acid extraction kit X, Y and Z were prepared.
The kit X, Y and Z are both composed of lysis solution A, washing solution B, eluent C, proteinase K, magnetic beads, mineral oil A and internal standard.
The lysate A, the washing solution B, the eluent C, the protease K, the magnetic beads and the mineral oil A in the X kit are shown in example 1.
Y kit:
lysate a: 3.5mol/L guanidine isothiocyanate, 2.5mmol/L sodium citrate dihydrate, 166.1mmol/L triton X-100 and 3.3mol/L isopropanol.
Washing solution B: 31.1mmol/L anhydrous citric acid, 19.0mmol/L sodium citrate dihydrate and 1ml Proclin950, and the pH value is 4.0.
Washing solution C: 5.7mmol/L anhydrous citric acid, 3.7mmol/L sodium citrate dihydrate and 1ml Proclin950, and the pH value is 4.0.
Eluent C: 14.8mmol/L Tris-hydroxymethyl, 5.0mmol/L Tris-hydroxymethyl hydrochloride, 1. mu.g/. mu.l Carrier RNA.
And (3) protease K: 20 mg/ml.
Magnetic beads: silicon-based magnetic beads with the particle size of 1.2 mu m and the concentration of 50 mg/ml.
Mineral oil A: a paraffinic oil.
Internal standard: armor RNA.
Z kit:
lysate a: 2.0mol/L guanidine isothiocyanate, 5mmol/L sodium citrate dihydrate, 150.0mmol/L triton X-100 and 3.0mol/L isopropanol.
Washing solution B: 32.1mmol/L anhydrous citric acid, 20.0mmol/L sodium citrate dihydrate and 1ml Proclin950, and the pH value is 4.5.
Washing solution C: 5.7mmol/L anhydrous citric acid, 3.7mmol/L sodium citrate dihydrate and 1ml Proclin950, and the pH value is 4.5.
Eluent C: 14.8mmol/L Tris-hydroxymethyl, 5.0mmol/L Tris-hydroxymethyl hydrochloride, 1. mu.g/. mu.l Carrier RNA.
And (3) protease K: 20 mg/ml.
Magnetic beads: silicon-based magnetic beads with the particle size of 1.2 mu m and the concentration of 50 mg/ml.
Mineral oil A: a paraffinic oil.
Internal standard: armor RNA.
Use ofThe prepared reagent kit X, Y and Z are respectively corresponding to 5 multiplied by 104The HIV-1 sample at IU/ml concentration was subjected to nucleic acid extraction as follows (note that the X kit, i.e., the kit washing step of the present invention, was only once):
(1) sequentially adding protease K, an internal standard, a sample to be detected, lysate A, mineral oil A and magnetic beads into a 2ml round-bottom centrifugal tube according to a fixed sequence, wherein the adding sequence cannot be changed, then blowing and uniformly mixing, and then performing magnetic adsorption in equipment with magnetic adsorption;
(2) adding a washing solution B into a 2ml round-bottom centrifuge tube for rinsing after the solution in the tube is fully cracked, placing the tube on a device with magnetic adsorption for adsorption after the tube is fully blown, and then discarding the waste liquid to retain magnetic beads;
(3) (the step is additionally needed by the Y and Z kits), adding washing liquid C diluted by 10 times into a 2ml round-bottom centrifuge tube for secondary rinsing, fully blowing and beating, performing magnetic adsorption, and keeping magnetic beads in waste liquid;
(4) and adding the eluent C into a 2ml round-bottom centrifuge tube to elute the nucleic acid adsorbed on the magnetic beads, fully blowing, uniformly mixing and storing the solution to obtain the solution, namely the nucleic acid corresponding to the three samples A, B and C.
The results of real-time fluorescent quantitative PCR reaction after mixing the nucleic acids of samples with different concentrations, respectively extracted from X, Y and Z kits, with the pre-prepared reaction solution are shown in FIG. 1. In FIG. 1, the CT value is shown in the front of the sample extracted with the reagent of example 1 of the present invention.
From the PCR graph of FIG. 1, it can be seen that the CT value of the PCR curve of the kit X, which is identified by the present invention, is the first, indicating that the quality of the extracted nucleic acid is better.
Example 3
Comparing sensitivity and stability with those of main stream products on the market
HIV nucleic acid samples were selected and diluted to two concentrations, 100IU/ml and 50IU/ml, respectively. The samples with the two concentrations were extracted and detected by using a quantitative detection kit for human immunodeficiency virus (hiv-1) nucleic acid from a mainstream company (Shenzhen Qianjie) in the actual clinical market, and the concentration and CT value shown in the following table 1 were obtained after 20 times of repetition.
TABLE 1 results of the extraction of the existing reagents
The specification of the clinical kit indicates that the detection sensitivity of the reagent is 100IU/ml, and particularly indicates that the hypersensitivity detection with 50IU/ml sensitivity can be carried out under the limit condition, as can be seen from the table 1, in the actual detection, a sample with the concentration of 100IU/ml can be stably detected by the kit, only one sample is missed, the detection rate reaches 95%, the hypersensitivity detection rate is only 70%, and 6 samples are missed, so that the true and reliable detection limit of the clinical reagent is more than 50 IU/ml.
Meanwhile, in order to verify the sensitivity and stability of the reagent formula developed by the invention, the same HIV sample stock solution is diluted to 3 concentrations: 100IU/ml, 50IU/ml and 30IU/ml, and the detection results are shown in Table 2 and FIGS. 2-4.
TABLE 2 results of the detection of three concentrations of HIV samples
As can be seen from Table 2 and FIGS. 2-4, the reagent of the present invention has a detection rate of 100%, and the stability and reproducibility of the curve are good in 20 repetitions of concentrations of 100IU/ML and 50IU/ML, while at a concentration of 30IU/ML, one sample is not detected, the detection rate is 95%, and the coincidence of the curve is loose, so that the detection limit of the HIV extraction reagent formula of the present invention is greater than 30 IU/ML. The data can be very intuitively seen, and the reagent provided by the invention is superior to a clinical mainstream HIV extraction kit used as a comparison in sensitivity and stability.
Example 4
Lysate efficiency detection
By using the reagent formula provided by the invention, the volume of the added lysate is taken as a variable, other factors are unchanged, and the reagent formula can be used for three concentrations of 1 multiplied by 104，1×103，1×102The samples of (2) were subjected to an extraction experiment using 200. mu.l of the HIV-1 sample as a template, and the volumes of the added lysates were 160. mu.l (8:10), 200. mu.l (1:1), and 300. mu.l (3:2), respectively.
The remaining specific procedures were as in example 2, and three replicates were performed for each concentration.
The nucleic acid obtained after extraction was mixed with the reaction solution prepared in advance, and real-time fluorescent quantitative PCR detection was carried out, and the obtained results are shown in FIGS. 5 to 7 in sequence. In FIGS. 5-7, the curves other than the internal standard are graphs of the results of different lysate addition volumes.
As can be seen from the figure, the fluorescence curves are not significantly different, indicating that the nucleic acid quality is consistent, indicating that the lysis efficiency is sufficient even if the amount of the lysate is less than the sample amount.
In addition, the proportion of the lysis solution A, the washing solution B and the eluent C in the kit X is changed, and if the following reagents are selected:
extraction reagent combination 1:
lysis solution: water is used as a solvent, and the components of the water-soluble organic solvent are as follows: 2mol/L of guanidinium isothiocyanate, 20mmol/L of chelating agent, 10% of anionic surfactant mass concentration, 100mmol/L of nonionic surfactant, 1mol/L of isopropanol and 0.1% of CTAB mass concentration.
Washing liquid: water is used as a solvent, and the components of the water-soluble organic solvent are as follows: 15mmol/L of anhydrous citric acid, 40mmol/L of sodium citrate dihydrate, and Proclin9500.5ml/L of pH 4.7.
Eluent: water is used as a solvent, and the components of the water-soluble organic solvent are as follows: 10mmol/L of trihydroxymethyl aminomethane, 15mmol/L of trihydroxymethyl aminomethane hydrochloride, Proclin 9500.5 ml/L and 0.3 mu g/mu L of Carrier RNA.
Extraction reagent combination 2:
lysis solution: water is used as a solvent, and the components of the water-soluble organic solvent are as follows: 6mol/L of guanidinium isothiocyanate, 3mmol/L of chelating agent, 1% of anionic surfactant by mass, 300mmol/L of nonionic surfactant, 4mol/L of isopropanol and 5% of CTAB by mass.
Washing liquid: water is used as a solvent, and the components of the water-soluble organic solvent are as follows: 45mmol/L of anhydrous citric acid, 10mmol/L of sodium citrate dihydrate, and 10 ml/L of Proclin9503.0 ml/L, wherein the pH value is 5.3.
Eluent: water is used as a solvent, and the components of the water-soluble organic solvent are as follows: tris-hydroxymethyl aminomethane 40mmol/, Tris-hydroxymethyl aminomethane hydrochloride 1mmol/L, Proclin 9503.0 ml/L, Carrier RNA 2.0. mu.g/. mu.l.
The combination of these reagents also achieves substantially the same extraction results as the previous examples.
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (11)
1. A reagent combination for the extraction of human immunodeficiency virus HIV-1 nucleic acid comprising:
lysis solution: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 2-6mol/L of guanidinium isothiocyanate, 3-20mmol/L of chelating agent, 1-10% of mass concentration of anionic surfactant, 300mmol/L of nonionic surfactant, 1-4mol/L of isopropanol and 0.1-5% of mass concentration of CTAB;
washing liquid: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 15-45mmol/L of anhydrous citric acid, 10-40mmol/L of sodium citrate dihydrate, 5-3.0ml/L of Proclin 9500.5, and the pH value is 4.7-5.3;
eluent: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 10-40mmol/L of trihydroxymethyl aminomethane, 1-15mmol/L of trihydroxymethyl aminomethane hydrochloride, 1.5-3.0 ml/L of Proclin9500, and 0.3-2.0 mug/mul of Carrier RNA;
the anionic surfactant is dodecylaminopropanesulfonic acid;
the nonionic surfactant is triton X-100;
the reagent combination product also comprises proteinase K, magnetic beads and mineral oil; the concentration of the proteinase K is 20 +/-5 mg/ml; the magnetic beads are silicon-based magnetic beads; the particle size of the magnetic beads is 1.2 +/-0.2 mu m;
the mineral oil is paraffin oil.
2. The reagent combination according to claim 1, wherein the lysis solution: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 4.2 +/-0.2 mol/L of guanidinium isothiocyanate, 10 +/-1 mmol/L of chelating agent, 5% +/-0.5% of anionic surfactant mass concentration, 167.6 +/-5 mmol/L of nonionic surfactant, 2.6 +/-0.2 mol/L of isopropanol and 0.5% +/-0.05% of CTAB mass concentration.
3. The reagent combination of claim 1, wherein the wash solution: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 30.7 plus or minus 2mmol/L of anhydrous citric acid, 19.2 plus or minus 2mmol/L of sodium citrate dihydrate, 9501 plus or minus 0.2ml/L of Proclin and pH of 5 plus or minus 0.2.
4. The reagent combination according to any one of claims 1 to 3, wherein the eluent: water is used as a solvent, and the components of the water-soluble organic solvent comprise: 22.5 +/-0.5 mmol/L of trihydroxymethyl aminomethane, 7.4 +/-0.2 mmol/L of trihydroxymethyl aminomethane hydrochloride, 9501 +/-0.2 ml/L of Proclin, and 1 +/-0.2 mug/microliter of Carrier RNA.
5. A method for extracting nucleic acid of human immunodeficiency virus HIV-1, comprising the steps of using the reagent combination of claim 1 to extract:
sequentially adding protease K, an internal standard, a sample to be detected, a lysis solution, mineral oil and magnetic beads, mixing and lysing, and performing magnetic adsorption;
adding the washing solution for rinsing, and removing liquid after magnetic adsorption;
then adding the eluent to elute the nucleic acid adsorbed on the magnetic beads to obtain an extracting solution containing the target nucleic acid;
the volume ratio of the protease K, the internal standard, the sample to be detected, the lysate, the mineral oil and the magnetic beads is 8 +/-0.5: 1:100 +/-1: 96 +/-1: 8 +/-0.5: 2 +/-0.2;
the mixing is carried out by adopting blow beating for mixing uniformly, and the blow beating time is 120 +/-10 s; carrying out magnetic adsorption after blowing, beating and uniformly mixing, wherein the magnetic adsorption time is 360 +/-30 s; the elution time was 60. + -.10 s.
6. The extraction method according to claim 5, wherein the concentration range of the internal standard is 500-10000 IU/ml; the concentration of the magnetic beads is 50 +/-10 mg/ml.
7. The extraction method according to claim 5, wherein the concentration of the internal standard is within the range of 7000-8000 IU/ml.
8. The extraction method according to claim 5, wherein the blowing volume is 80% of the total volume of the solution, and the blowing is performed by following the liquid level.
9. The extraction method according to any one of claims 5 to 8, wherein the volume ratio of the washing solution to the sample to be tested is 80 to 150: 1, the blow-beating time is 60 +/-10 s, and the magnetic adsorption time is 60 +/-10 s.
10. The extraction process according to claim 9, wherein the washing liquid is washed only once.
11. The extraction method according to claim 5, wherein the volume ratio of the eluent to the sample to be tested is 10-15: 1.
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