CN111718927A - Preservation solution for improving stability of nucleic acid and application thereof - Google Patents
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Abstract
The invention discloses a preservation solution for improving the stability of nucleic acid and application thereof, and the preservation solution comprises one or more of 10 mM-500 mM Tris (hydroxymethyl) aminomethane (Tris), 1-20 mg/ml nonionic surfactant, 1 mM-0.5 MEDTA, 10 mM-500 mM inorganic salt and 0.1-20 mg/ml sorbitol. The preservation solution can be directly used for the subsequent extraction step without centrifugal removal, does not affect the adsorption of silica gel plasma membranes and magnetic beads on nucleic acids, can promote the adsorption of the nucleic acids on centrifugal columns and magnetic beads during the subsequent nucleic acid extraction, improves the extraction yield and saves the time.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preservation solution for improving the stability of nucleic acid and application thereof.
Background
Microorganisms (microorganisms) are a group of minute and simple microorganisms existing in nature. The microorganisms are invisible to the naked eye and must be magnified with the aid of an optical microscope or an electron microscope for observation. Microorganisms are widely present in nature, and some of them invade the human body to cause infection and cause infectious diseases, and such microorganisms are called pathogenic microorganisms. The pathogenic microorganisms comprise prions, parasites (protozoa, worms, medical insects), fungi, bacteria, spirochetes, mycoplasma, rickettsia, chlamydia, viruses, wherein the harmfulness of bacteria and viruses is greatest.
After the pathogenic microorganisms invade the human body, the human body is the place where the pathogenic microorganisms live, and is medically called as a host of the pathogenic microorganisms. Pathogenic microorganisms grow and reproduce in hosts, release toxic substances and the like to cause pathological changes of organisms to different degrees, and the process is called infection. After the pathogenic microorganism infects the host, the virulence or pathogenicity of the pathogenic microorganism is enhanced through continuous reproduction, variation and evolution, so that the immunological rejection reaction of the organism is initiated, and when the normal physiological bearing range of the organism is exceeded, the symptom is shown, and the disease or even serious infection and infectious diseases are initiated. Infectious diseases and infectious diseases caused by pathogenic microorganisms are one of the most common diseases in clinical face, and account for about 50% of the common diseases. Pathogenic microorganisms are highly variable due to rapid propagation and unstable nucleic acid, so that the pathogenic microorganisms are diversified; in addition, with the influence of factors such as the change of the crowd structure, environmental pollution, drug abuse and the like, diseases caused by pathogenic microorganisms have a complicated trend, and the human health is seriously threatened.
At present, the commonly used detection methods for pathogenic microorganisms include smear microscopy, biochemical reaction, immunodetection, culture methods, conventional PCR methods, qPCR, gene chip technology, high throughput sequencing (NGS), mass spectrometry and other technologies; the nucleic acid-based detection is a diagnostic gold standard due to its high sensitivity, high accuracy, and high speed, and includes the methods introduced above, such as conventional PCR, qPCR, gene chip technology, and high throughput sequencing (NGS).
Nucleic acids include both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), which are known to be genetic material of all organisms, and are found primarily in the nucleus of cells and in the virus coating. The nucleic acid is easily decomposed by nuclease existing in the environment, so the time for completely storing the nucleic acid under the general environment is short; particularly, the storage difficulty of RNA is higher, on one hand, the RNA is single-stranded and extremely unstable, and on the other hand, RNase (RNase) widely exists in the conventional experimental environment and the RNase is very stable and is difficult to remove. Therefore, how to obtain high quality and complete nucleic acid from a sample is a problem to be solved in the field of biology.
In the context of nucleic acid preservation, Ambion, Inc., USA (now purchased from Thermo Fisher Inc.) developed a solution RNAlater for preserving intact cells, which consists of sodium citrate, ethylenediaminetetraacetic acid (EDTA) and ammonium sulfate. The RNAlater-preserved nucleic acid sample can be stably preserved for one day at 37 ℃ without obvious degradation. In addition, guanidine salts (e.g., guanidine isothiocyanate or guanidine hydrochloride) are commonly used in cell lysis in nucleic acid extraction as a typical protein denaturant, and thus many nucleic acid preservation solutions developed based on guanidine salts are commercially available.
In the aspect of nucleic acid extraction, the traditional methods are phenol/chloroform organic solvent extraction, a silica gel membrane adsorption centrifugal column method and a magnetic bead method which is compatible with an automatic platform and widely applied. The principle of phenol/chloroform extraction is mainly that phenol denatures proteins and simultaneously inhibits degradation of nuclease; chloroform can produce a phase separation, with protein molecules dissolved in the phenol phase and nucleic acids in the aqueous phase. In addition, isoamyl alcohol is often added in the extraction process to promote phase separation, and air bubbles generated in the protein denaturation operation process are reduced by reducing the surface tension, so that the phase separation is maintained to be stable. The silica gel membrane adopted by the centrifugal column method is modified, and the surface of the silica gel membrane is provided with a large number of anion groups, so that nucleic acid can be efficiently adsorbed at high salt and low pH. The magnetic bead method for extracting nucleic acid adopts superparamagnetic silicon oxide nanometer magnetic beads (magnetic beads for short) as carriers, and the magnetic beads adsorb nucleic acid in high-salt solution without adsorbing protein and other impurities; the nucleic acid is desorbed from the surface of the magnetic beads in a low-salt solution, thereby achieving the purpose of quickly separating and purifying the nucleic acid. At present, the centrifugal column purification method and the magnetic bead purification method are widely applied because the extraction purity is high, the yield is high, the influence of human operation factors is small, and the method can be compatible with automation (magnetic bead method).
The basis of the detection of pathogenic microorganisms based on nucleic acid is the safe storage and effective extraction of nucleic acid, and the final nucleic acid yield becomes a restriction factor for success or failure of downstream nucleic acid research.
In terms of nucleic acid preservation, RNAlater-like preservation solutions developed by Ambion, USA, in use, must be centrifuged to remove the supernatant before nucleic acid extraction can be performed for subsequent experiments. The storage solution based on guanidine salt on the market also has a plurality of limitations because the instability of the guanidine salt component changes the structure after oxidation, so that the storage effect is extremely unstable, the yield is low and false negative is easy to detect. In the aspect of nucleic acid extraction, although the centrifugal column method and the magnetic bead method are widely applied, the method is only limited to a large number of samples and samples with high integrity, and when a trace amount of samples of pathogenic microorganisms are extracted, the method is often limited by the quality of the preserved samples, and has the defects of low yield and poor repeatability.
Disclosure of Invention
The invention aims to provide a preservation solution which can improve the stability of nucleic acid, has good compatibility with subsequent nucleic acid extraction and improves the yield of nucleic acid and application thereof. The preservation solution can effectively preserve nucleic acid including but not limited to pathogenic microorganism samples without degradation, and can be perfectly compatible with subsequent nucleic acid extraction without removal during use.
The purpose of the invention is realized by the following technical scheme:
the invention provides a preservation solution for improving the stability of nucleic acid, which comprises one or more of 10 mM-500 mM of Tris (hydroxymethyl) aminomethane (Tris), 1-20 mg/ml of nonionic surfactant, 1 mM-0.5M of EDTA, 10 mM-500 mM of inorganic salt and 0.1-20 mg/ml of sorbitol.
In some embodiments, the preservation solution of the present invention further comprises 0.1-10 mg/ml polydimethylsiloxane.
In some embodiments, the remaining ingredient of the preservation solution is water.
In some embodiments, the non-ionic surfactant in the preservation solution of the present invention is selected from one or more of Triton X-100, Tween-20, ethylphenylpolyethylene glycol (NP-40), digitonin, and IGEPALCA-630.
In some embodiments, the inorganic salt in the preservation solution according to the invention is selected from sodium chloride (NaCl), potassium chloride (KCl), ammonium chloride (NH)4Cl), sodium sulfate (Na)2SO4) Potassium sulfate (K)2SO4) Ammonium sulfate [ (NH)4)2SO4]One or more of (a).
In some embodiments, the preservation solution of the present invention comprises one or more of trihydroxymethyl aminomethane (Tris) 10-200 mM, a nonionic surfactant 1-20 mg/ml, EDTA 0.1-0.5M, an inorganic salt 10 mM-200 mM, sorbitol 0.1-20 mg/ml, and polydimethylsiloxane 0.1-10 mg/ml.
In some embodiments, the preservation solution of the present invention comprises one or more of trihydroxymethyl aminomethane (Tris) 10-100 mM, a nonionic surfactant 5-20 mg/ml, EDTA 0.1-0.5M, an inorganic salt 10 mM-100 mM, sorbitol 0.5-5 mg/ml, and polydimethylsiloxane 0.5-5 mg/ml.
In some embodiments, the preservation solution of the present invention comprises one or more of Tris (hydroxymethyl) aminomethane (Tris) 20-80 mM, a nonionic surfactant 5-15 mg/ml, EDTA 0.1-0.4M, an inorganic salt 20 mM-50 mM, sorbitol 0.5-2 mg/ml, and polydimethylsiloxane 0.5-2 mg/ml.
In some embodiments, the preservation solution of the present invention comprises one or more of Tris (hydroxymethyl) aminomethane (Tris) 40-60 mM, a nonionic surfactant 8-12 mg/ml, EDTA 0.2-0.3M, an inorganic salt 40 mM-50 mM, sorbitol 0.5-1.5 mg/ml, and polydimethylsiloxane 0.5-1.5 mg/ml.
In some embodiments, the inorganic salt is preferably potassium chloride (KCl) and ammonium sulfate [ (NH)4)2SO4]Combining; in some more specific embodiments, potassium chloride (KCl) and ammonium sulfate [ (NH)4)2SO4]The molar ratio of (1-5): 1.
the invention also provides a kit which comprises the preservation solution, wherein the kit comprises or does not comprise a swab or/and a nucleic acid extraction reagent.
The preservation solution of the invention has good compatibility with subsequent extraction steps, so if the kit of the invention contains a nucleic acid extraction reagent, the kit can be a nucleic acid extraction reagent commonly used in the prior art, including but not limited to an extraction reagent with higher requirements on samples such as a centrifugal column method or a magnetic bead method.
The invention also provides application of the preservation solution in preservation of a sample containing nucleic acid.
The sample containing nucleic acid of the present invention may be a tissue sample containing nucleic acid, including human and various animals and plants, or a sample containing microorganisms such as cultured cells, fungi, bacteria, viruses, or the like; human or animal samples include, but are not limited to, saliva, nasopharyngeal mucosa, lower respiratory secretions, liver, brain, lung, spleen, kidney, heart, muscle, blood, stool, and the like; plants include, but are not limited to, roots, stems, leaves, and the like.
In some embodiments, the virus or virus sample is porcine epidemic diarrhea virus.
In some embodiments, the virus or virus sample is COVID-19.
In some embodiments, the invention further provides a specific application of the preservation solution in preservation of a sample containing nucleic acid, wherein the sample containing nucleic acid is added into the preservation solution and preserved at 1-60 ℃; preferably 4-56 ℃; more preferably 4 to 37 ℃.
In some embodiments, the volume ratio of the nucleic acid-containing sample to the preservation solution is 1: (3-20); or 1-100 ten thousand cells/ml; in some specific embodiments, the volume ratio of the nucleic acid-containing sample to the preservation solution is 1: (3-10); or 30 to 70 ten thousand cells/ml.
The invention has the beneficial effects that:
(1) the preserving fluid has good compatibility with subsequent nucleic acid extraction, and can be suitable for various nucleic acid extraction methods, including extraction methods which have certain requirements on sample scale and integrity, such as a centrifugal column method or a magnetic bead method.
(2) The preservation solution can be directly used for the subsequent extraction step without centrifugal removal, does not affect the adsorption of silica gel plasma membranes and magnetic beads on nucleic acids, can promote the adsorption of the nucleic acids on centrifugal columns and magnetic beads during the subsequent nucleic acid extraction, improves the extraction yield and saves the time.
(3) The preservation solution can rapidly crack cells, promote the penetration of the cells and pathogenic microorganisms, rapidly penetrate the components of the preservation solution into the cells or the pathogenic microorganisms, and inhibit the degradation of nucleic acid to keep stable; the addition of sorbitol further improves the nucleic acid stability; the polydimethylsiloxane can effectively prevent the pollution among the bubble rupture primer samples and improve the operation experience.
(4) The preservation solution disclosed by the invention is wider in applicable temperature range, can be suitable for preservation at 1-60 ℃, can be suitable for preservation of samples in different temperature environments, and has more important practical value.
Unless otherwise specified, the terms used in the present invention have the following meanings:
pathogenic microorganisms: pathogenic microorganisms refer to microorganisms, or pathogens, that can invade the body and cause infections and even infectious diseases.
Deoxyribonucleic acid (DNA): DeoxyriboNucleic Acid (abbreviated as DNA) is one of the four kinds of nucleic acids contained in biological cells. DNA carries the genetic information necessary for the synthesis of RNA and proteins, and is a biological macromolecule essential for the development and proper functioning of an organism. DNA is a macromolecular polymer composed of deoxynucleotides. Deoxynucleotides are composed of bases, deoxyribose, and phosphate. Wherein, the basic groups are 4 types: adenine (a), guanine (G), thymine (T) and cytosine (C).
Ribonucleic acid (RNA): ribonucleic Acid (abbreviated as RNA, or ribonuclear Acid), a genetic information carrier present in biological cells and in parts of viruses, viroids. RNA is a long chain molecule formed by the condensation of ribonucleotides via phosphodiester bonds. One ribonucleotide molecule consists of a phosphate, a ribose and a base. RNA has 4 kinds of bases, namely A adenine, G guanine, C cytosine, U uracil, wherein U (uracil) replaces T in DNA.
Drawings
FIG. 1 shows the detection of the integrity of nucleic acids according to example 1;
FIG. 2 is the PEDV amplification curve (4 ℃ VS 56 ℃ for 60min and 120min) for each treatment group of the preservation solution corresponding to example 2;
FIG. 3 is a PEDV amplification curve (4 ℃ VS 37 ℃ C. for 3d 5d 7d) for each treatment group of the preservation solution corresponding to example 2;
FIG. 4 is a PEDV amplification curve (4 ℃ VS 56 ℃ for 60min and 120min) for each treatment group of Hank's solution corresponding to example 2;
FIG. 5 is a PEDV amplification curve (4 ℃ VS 37 ℃ C. for 3d 5d 7d) for each treatment group of Hank's solution corresponding to example 2.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
The chemicals described in the examples below were from the national pharmaceutical group, Inc. (national drug group) or Sigma Aldrich.
Unless otherwise specified, the sample control preservative solution used in the following examples is Hank's solution: 8g/L NaCl, 0.4g/L KCl, 0.14g/L CaCl2，0.2g/L MgSO4·7H2O，0.048g/L Na2HPO4，0.06g/L KH2PO4,0.35g/L NaHCO30.01g/L phenol red.
Example 1:
this example uses normally cultured HEK293 cells as a model to study the effect of preservation solutions on nucleic acid preservation and extraction promotion. The nucleic acid extraction reagent used in this example was a magnetic bead method nucleic acid extraction reagent of Nanjing Novozam Biotechnology Ltd, cat # RM 101.
The ratio of the preservation solution in this embodiment is: 50mM Tris, 10mg/ml Tween-20, 0.2M EDTA, 30mM KCl, 15mM (NH)4)2SO41mg/ml sorbitol and 1mg/ml silicone oil, the balance being water. The specific experimental procedures are as follows:
1. sample collection and preservation:
collecting freshly cultured HEK293 cells; cell activity detection is carried out, and the cell activity is more than 95 percent; counting the collected cells, adding 1ml of the preservation solution of the embodiment or the conventional sample control preservation solution (Hank's solution) into each of the 50 ten thousand cell volumes, screwing a tube cover, and slightly swirling the discrete cells;
2. sample processing
For the preservation solution and the Hank's solution of the present example, 4 ℃ preservation (as a control), oven heating at 56 ℃ for 30min and 120min, and oven heating at 37 ℃ for 3 days, 5 days, and 7 days were set for different treatment groups, respectively.
3. Nucleic acid extraction (using Vazyme # RM101)
3.1 Add the above different processed samples to 1.5ml Nuclear-free Low sorption EP tube (self-contained), then add 20. mu.l proteinase K, mix by gentle vortex or up-down reversal, add 20. mu.l magnetic beads and 600. mu.l lysis solution, vortex and mix for 15sec, lyse for 5min at room temperature, mix twice by up-down reversal.
3.2 instantaneous centrifugation, place the EP tube on a magnetic rack, stand for 1min, remove the supernatant with a pipettor.
3.3 washing: taking the sample off the magnetic frame, adding 700 μ l of washing solution, vortexing for 15sec, mixing, centrifuging instantaneously, placing the EP tube on the magnetic frame, standing for 1min, and removing the supernatant.
3.4 rinsing: taking the sample off the magnetic frame, adding 700 μ l of rinsing liquid, vortexing for 15sec, mixing, centrifuging instantaneously, placing the EP tube on the magnetic frame, standing for 1min, and removing the supernatant.
3.5 after the instantaneous centrifugation, the magnetic frame is put on again, and the residual supernatant is removed. And opening the cover and airing at room temperature for 3-5 min until the surface of the magnetic beads does not reflect light.
Note: in order to ensure the purity of nucleic acid, the rinsing liquid is removed completely; at the same time, excessive drying (cracking) of the beads can affect the final yield.
3.6 adding 50 μ l of eluent, mixing gently for 15sec, standing at room temperature for 3min, shaking and mixing for 2 times. And (3) instantly centrifuging to the bottom of the EP tube, putting the sample on a magnetic frame again, standing for 1min, and sucking the supernatant into a new nucleic-free centrifuge tube (self-contained) for subsequent detection.
4. Nucleic acid extraction yield integrity detection
4.1 cellular nucleic acid concentration and purity OneDrop detection
The detection probe was washed with the eluent and calibrated, and 1. mu.L of each sample was tested for RNA concentration, DNA concentration and purity according to the instructions of the OneDrop apparatus, with the results shown in Table 1.
4.2 cellular genomic DNA and RNA integrity assays
4.2.1 preparation of 1.2% agarose gel using 1 XTAE;
4.2.2 mu.L of each of the extracts was taken, and 1. mu.L of 10 × DNA Loading buffer (Vazyme # P022) and 4. mu.L of RNase-free H were added2O, vortex and mix evenly, put in PCR instrument 72 degrees centigrade, 2min denaturalization;
4.2.3 spotting together 5. mu.L of DL15000 Marker (Vazyme # MD 103);
4.2.4 agarose gel electrophoresis in an electrophoresis apparatus, 220V, 10-12 min;
4.2.5 gel imager pictures are shown in FIG. 1.
TABLE 1 cellular nucleic acid concentration and purity Onedrop assay
As can be seen from table 1, in terms of the yield of nucleic acid extraction, the samples stored in the storage solution configured in this example were treated differently, and the yields were significantly higher than those of the Hank's solution storage group, regardless of DNA or RNA; the yield of the nucleic acid preserved by the preservation solution in the 4-degree extraction control group is higher than that in the Hank's solution preservation group, which shows that the preservation solution has the function of improving the efficiency of nucleic acid extraction.
According to the analysis of the integrity of the nucleic acid shown in fig. 1, in terms of the integrity of RNA, the sample stored in the preservation solution prepared in the present case always keeps the integrity of the nucleic acid after different treatments, and the electrophoresis band is not dispersed; whereas the samples preserved with the Hank's solution showed significant degradation upon oven heating at 56 ℃ for 60min and 37 ℃ for 1 day, and showed worse subsequent longer pressing times.
The results collectively show that the nucleic acid preservation solution disclosed in the embodiment can effectively protect cellular nucleic acid from degradation and can promote the yield of nucleic acid extraction by a magnetic bead method.
Example 2:
this example is used to preserve pathogenic microorganisms in saliva samples. The Porcine Epidemic Diarrhea Virus (PEDV) live vaccine adopted in this example (purchased from warrior, formerly veterinary drug, japanese alphabet 170041133) belongs to the genus α coronavirus of the family coronaviridae, is a single-stranded positive-strand RNA virus, can cause Porcine Epidemic Diarrhea (PED), and can effectively simulate pathogenic microorganisms of clinical RNA virus types. The nucleic acid extraction reagent used in this example was a Nanjing NuoZan Biotechnology Inc. spin column nucleic acid extraction reagent, cat # RC 311-C1.
The ratio of the preservation solution in this embodiment is: 50mM Tris, 10mg/ml NP-40, 0.3M EDTA, 30mM KCl, 15mM (NH)4)2SO41mg/ml sorbitol and 1mg/ml silicone oil, the balance being water. The specific experimental procedures are as follows:
1. sample collection and model construction:
1.1 preparation of Porcine Epidemic Diarrhea (PEDV) Virus model
Purchasing a porcine transmissible gastroenteritis and porcine epidemic diarrhea bigeminal live vaccine (WH-1R strain + AJ1102-R strain) from Wuhan Ke's Probiotics GmbH (Wuhan Ke's Probiotics Co., Ltd) (Wuhan Ke's Probiotics, veterinary medicine word 170041133), 10 heads/bottle, and virus content more than or equal to 105TCID50Each bottle is fully dissolved by 10ml of normal saline (0.9 percent NaCl);
1.2 preparation of profound expectoration samples
According to the standard operation, after deep inhalation, the sputum in the deep part of the respiratory tract is expectorated with force, and the sample volume is about 3 ml; after vortex mixing, dividing into two parts, sucking 1ml of sputum from each part, adding 5ml of the preservation solution prepared in the embodiment into one part, and adding 5ml of Hank's solution into the other part; 6ul of PEDV vaccine diluted in the previous step was added separately.
2. Sample processing
The samples are subpackaged, and the preservation solution and the Hank's solution are respectively preserved at 4 ℃ (used as a control), heated in a 56 ℃ oven for 60min and 120min, and heated in a 37 ℃ oven for 3 days, 5 days and 7 days to form different treatment groups.
3. Nucleic acid extraction (using Vazyme # RC311-C1)
3.1 Add 500. mu.l lysis buffer into 1.5ml RNase-free centrifuge tube (self-contained);
3.2 Add 200. mu.l of the above differently treated sample and vortex to mix.
3.3 Place the adsorption column in a 2ml collection tube, transfer the mixture to the adsorption column, and centrifuge at 12,000 Xg for 1 min.
3.4 discard the filtrate, put the adsorption column back into the 2ml collection tube, add 600. mu.l of the rinse solution 12,000 Xg, centrifuge for 30sec, and discard the filtrate.
3.5 repeat step 3.4 once.
3.6 the adsorption column was returned to the collection tube and centrifuged through a 12,000 Xg empty column for 2 min.
3.7 transfer the adsorption column to a new 1.5ml collection tube (provided in the kit), add 50. mu.l eluent to the center of the adsorption column membrane, and let stand at room temperature for 1 min. Centrifuge at 12,000 Xg for 1 min.
3.8 discard the adsorption column, the resulting DNA/RNA can be used directly for subsequent detection.
4. Extracting micro sample RNA for detection (adopting Vazyme # Q222-CN)
4.1 mix solutions (as ABI StepOnePlus) as given in Table 2 below were prepared in RNase-free centrifuge tubesTMFor tester type)
[ TABLE 2 ]
4.2 one-step qRT-PCR reactions performed under the conditions of Table 3 below
[ TABLE 3 ]
The sequences of the primers and probes used in the experiment are shown in Table 4, and the results of the experiment are shown in Table 5 and FIGS. 2-5.
TABLE 4 primers and probes specific for PEDV design
According to the results in table 5 and fig. 2-5, it can be seen that, by designing specific primers and probes for PEDV and then detecting with nucleic acids processed in different groups, it can be found that the Δ CT difference of the storage solution sample group of the present example is within 0.5 compared with that of the storage solution group at 4 ℃ after being processed at various temperatures; and the difference between the delta CT values of the Hank's solution group after being heated in a 37 ℃ oven and the corresponding storage at 4 ℃ is obviously increased along with the time, and the difference is more than 0.5. The above results collectively demonstrate that the nucleic acid preservation solution of the present example can effectively inhibit the degradation of viral nucleic acid.
Table 5 difference in Δ CT for each treatment group versus the corresponding control group in example 2
Claims (10)
1. A preservation solution for improving the stability of nucleic acid is characterized by comprising one or more of 10 mM-500 mM of Tris (hydroxymethyl) aminomethane (Tris), 1-20 mg/ml of a nonionic surfactant, 1 mM-0.5M of EDTA, 10 mM-500 mM of an inorganic salt and 0.1-20 mg/ml of sorbitol.
2. The preservation solution according to claim 1, characterized in that the preservation solution further comprises 0.1-10 mg/ml polydimethylsiloxane.
3. The preservation solution according to claim 1, wherein the preservation solution residual component is water.
4. The preservation solution according to claim 1, wherein the non-ionic surfactant in the preservation solution is selected from one or more of Triton X-100, Tween-20, ethylphenylpolyethylene glycol (NP-40), digitonin, IGEPALCA-630; the inorganic salt is selected from sodium chloride (NaCl), potassium chloride (KCl), and ammonium chloride (NH)4Cl), sodium sulfate (Na)2SO4) Potassium sulfate (K)2SO4) Ammonium sulfate [ (NH)4)2SO4]One or more of; preferably, the inorganic salt is a combination of potassium chloride and ammonium sulfate; more preferably, the molar ratio of the potassium chloride to the ammonium sulfate is (1-5): 1.
5. the preservation solution according to claim 1, comprising one or more of 10mM to 200mM Tris, 1 to 20mg/ml EDTA, 10mM to 200mM inorganic salts, 0.1 to 20mg/ml sorbitol, and 0.1 to 10mg/ml dimethicone; preferably, the cleaning agent comprises one or more of 10 mM-100 mM Tris (hydroxymethyl) aminomethane (Tris), 5-20 mg/ml nonionic surfactant, 0.1M-0.5M EDTA, 10 mM-100 mM inorganic salt, 0.5-5 mg/ml sorbitol and 0.5-5 mg/ml polydimethylsiloxane; further preferably, the detergent composition comprises one or more of 20 mM-80 mM Tris (hydroxymethyl) aminomethane (Tris), 5-15 mg/ml nonionic surfactant, 0.1M-0.4M EDTA, 20 mM-50 mM inorganic salt, 0.5-2 mg/ml sorbitol and 0.5-2 mg/ml polydimethylsiloxane; most preferably, the surfactant comprises one or more of trihydroxymethyl aminomethane (Tris) of 40 mM-60 mM, nonionic surfactant of 8-12 mg/ml, EDTA of 0.2M-0.3M, inorganic salt of 40 mM-50 mM, sorbitol of 0.5-1.5 mg/ml and polydimethylsiloxane of 0.5-1.5 mg/ml.
6. A kit comprising the preservation solution according to any one of claims 1 to 5; preferably, the kit includes or does not include a swab or/and nucleic acid extraction reagents.
7. Use of the preservation solution according to any one of claims 1 to 5 for preserving a sample containing nucleic acids.
8. The use according to claim 7, wherein the sample containing nucleic acid is a tissue sample containing nucleic acid, including human and various animals and plants, or a cultured cell, fungus, bacterium, virus sample; preferably, the virus sample is porcine epidemic diarrhea virus or COVID-19.
9. The use according to claim 7, wherein the sample containing nucleic acid is added to a preservation solution and preserved at 1-60 ℃; preferably 4-56 ℃; more preferably 4 to 37 ℃.
10. The use according to claim 7, wherein the volume ratio of the sample containing nucleic acids to the preservation solution is 1: (3-20); or 1-100 ten thousand cells/ml; preferably, the volume ratio of the sample containing nucleic acid to the preservation solution is 1: (3-10); or 30 to 70 ten thousand cells/ml.
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