CN112592961A - Nucleic acid sample preservation solution and preparation method and application thereof - Google Patents
Nucleic acid sample preservation solution and preparation method and application thereof Download PDFInfo
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
Abstract
The invention provides a nucleic acid sample preservation solution and a preparation method and application thereof, and belongs to the technical field of biomedicine. The nucleic acid sample preservation solution consists of sodium acetate, EDTA sodium salt, Tris, protease K, SDS, Triton X-100, SLS and DEPC double distilled water. The invention fully mixes the collected sample with the virus lysate and the virus nucleic acid preservation solution to inactivate the virus in the sample, effectively ensures the integrity of the virus nucleic acid in the sample, and can transport and preserve the collected sample at normal temperature for a long time. It can instantly crack pathogen to release nucleic acid, and the protective agent can prevent nucleic acid from being degraded, so it has good practical production application value.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a nucleic acid sample preservation solution as well as a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
The virus is a microorganism with a simple structure, the method for completing the self-replication process needs to help host cells and utilize the nutrition of the host to complete the virus replication. The more common viruses causing human infection are very many and can cause respiratory tract diseases, such as common cold viruses, influenza viruses, new coronavirus, SARS and other coronavirus. In the clinical detection process, short-term storage and transportation are often required, and a sample protective liquid medium after sampling is required, which is generally called virus storage solution (VTM solution or UTM solution) in China. In general, in nucleic acid detection, nucleic acid PCR cannot be directly performed at a sample collection site, and a VTM needs to be added for transportation and detection of a sample collected by a swab. The sample preserving fluid can be used for collecting, preserving and transporting nasopharyngeal pathogen specimens of new coronavirus, influenza, avian influenza, hand-foot-and-mouth disease, measles and the like. The preserved virus RNA sample can be widely applied to gene detection, enzyme-linked immunosorbent assay (ELISA), PCR detection and the like.
At present, the sample preservation solution has two types of inactivation type and non-inactivation type, and the different specific effects of the sample preservation solution types are different. For viruses with stronger infectivity, the characteristic that nucleic acid is easy to degrade is realized, so that the inventor finds that the conventional sampling and preserving solution cannot ensure that the nucleic acid in a sample is not degraded and can not inactivate the viruses with strong infectivity possibly existing in the sample in time, so that the sample causes secondary propagation during preservation, transportation and uncovering detection, and extremely high infection risk is caused to medical staff.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a nucleic acid sample preservation solution and a preparation method and application thereof. The invention fully mixes the collected sample with the virus lysate and the virus nucleic acid preservation solution to inactivate the virus in the sample, effectively ensures the integrity of the virus nucleic acid in the sample, and can transport and preserve the collected sample at normal temperature for a long time. It can instantly cleave pathogens to release nucleic acids, and the protective agent can prevent the nucleic acids from being degraded.
Specifically, the invention relates to the following technical scheme:
in a first aspect of the present invention, there is provided a nucleic acid sample preservation solution, which comprises sodium salt, proteolytic enzyme, surfactant, biological buffer and water.
In a second aspect of the present invention, there is provided a method for preparing the nucleic acid sample preservation solution, the method comprising: mixing the above materials.
In a third aspect of the present invention, there is provided use of the above-mentioned nucleic acid sample preservation solution for in vitro preservation of a nucleic acid sample. The prepared nucleic acid sample preservation solution is suitable for preserving pharynx swab, nose swab or specific part tissue samples after sampling, and the stored samples can be used for subsequent clinical experiments such as nucleic acid extraction or purification. The nucleic acids include ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Is particularly suitable for the preservation of nucleic acid samples of viruses (such as RNA viruses).
The beneficial technical effects of one or more technical schemes are as follows:
the nucleic acid sample preservation solution prepared by the technical scheme has double functions of virus splitting and nucleic acid preservation, can inactivate viruses in the sample, effectively ensures the integrity of the virus nucleic acid in the sample, and can be transported and preserved for a long time under the normal temperature condition. The RNA sample can be subjected to instantaneous lysis to release nucleic acid, the protective agent component can prevent the nucleic acid from being degraded, and the purity of the extracted RNA sample is still high after the RNA sample is stored for 3 months at normal temperature, so that the RNA sample meets the requirements of LAMP and PCR methods, and the RNA quality requirement after cell storage is met, and therefore the RNA sample has good practical application value.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph showing a comparison of the concentrations of RNAs extracted before and after storage in an example of the present invention.
FIG. 2 is a diagram showing the expression change of STC-1 gene detected by the PCR method according to the embodiment of the present invention.
FIG. 3 shows the sensitivity of the PCR method to detect STC-1 gene after three months of storage according to the embodiment of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 application belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
The present invention is further illustrated by reference to specific examples, which are intended to be illustrative only and not limiting. If the experimental conditions not specified in the examples are specified, they are generally according to the conventional conditions, or according to the conditions recommended by the sales companies; materials, reagents and the like used in examples were commercially available unless otherwise specified.
As mentioned above, the conventional sampling and preserving fluid cannot ensure that nucleic acid in the sample is not degraded, and simultaneously cannot inactivate viruses with strong infectivity possibly existing in the sample in time, so that the sample causes secondary propagation during preservation, transportation and uncapping detection, thereby causing extremely high infection risk to medical staff.
In view of the above, in one embodiment of the present invention, a nucleic acid sample preservation solution is provided, which comprises sodium salt, proteolytic enzyme, surfactant, biological buffer and water.
In another embodiment of the present invention, the sodium salt is any one or more of sodium acetate and sodium EDTA.
In yet another embodiment of the invention, the proteolytic enzyme comprises proteinase K.
In yet another embodiment of the present invention, the surfactant is any one or more of SDS (sodium dodecyl sulfate), Triton X-100 (polyethylene glycol octyl phenyl ether), and SLS (sodium lauryl sulfate).
In yet another embodiment of the present invention, to avoid the introduction of impurities, the water is preferably DEPC triple distilled water.
In still another embodiment of the present invention, the nucleic acid sample preservation solution is composed of the following components: sodium acetate, EDTA sodium salt, Tris, protease K, SDS, Triton X-100, SLS and DEPC triple distilled water.
In still another embodiment of the present invention, the nucleic acid sample preservation solution is composed of the following components in concentration:
0.1-0.5M sodium acetate, 2-6mM EDTA sodium salt, 2-6mM Tris, 0.5-2 μ g/ml proteinase K, 0.1-0.5% SDS, 0.1-0.5% Triton X-100, 0.3-0.6% SLS, and DEPC triple distilled water as solvent. According to the invention, through optimizing the components, the nucleic acid sample preservation solution can inactivate viruses in the sample, meanwhile, the integrity of the virus nucleic acid in the sample is effectively ensured, and the collected sample can be transported and preserved for a long time under the normal temperature condition.
In still another embodiment of the present invention, the nucleic acid sample preservation solution is composed of the following components in concentration:
0.3M sodium acetate, 5mM EDTA sodium salt, 5mM Tris, 1. mu.g/ml proteinase K, 0.2% SDS, 0.2% Triton X-100, 0.5% SLS, and DEPC triple distilled water as solvent.
In still another embodiment of the present invention, there is provided a method for preparing the nucleic acid sample preservation solution, the method comprising: mixing the above materials.
In still another embodiment of the present invention, there is provided a use of the above-mentioned nucleic acid sample preservation solution for in vitro preservation of a nucleic acid sample. The prepared nucleic acid sample preservation solution is suitable for preserving pharynx swab, nose swab or specific part tissue samples after sampling, and the stored samples can be used for subsequent clinical experiments such as nucleic acid extraction or purification. The nucleic acids include ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Is particularly suitable for the preservation of nucleic acid samples of viruses (such as RNA viruses).
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
A nucleic acid sample preservation solution comprises the following components:
0.3M sodium acetate, 5mM EDTA sodium salt, 5mM Tris, 1. mu.g/ml proteinase K, 0.2% SDS, 0.2% Triton X-100, 0.5% SLS, and DEPC triple distilled water as solvent.
The composition is used by mixing the above components.
Effect verification
Cell culture and passage
Taking HL-60 cells out of the liquid nitrogen bottle, and putting the cells into a constant-temperature water bath kettle at 37 ℃ for rapid melting. Adding the thawed cells into three centrifugal tubes (4-5 ml of cell culture solution is added into the three centrifugal tubes in advance), and centrifuging at the speed of 800 rpm for 3 minutes. Discarding the supernatant, adding 4-5 ml of cell culture solution into each centrifuge tube again, uniformly blowing and beating cell precipitates by using a suction tube, respectively transferring the three cells into a cell culture bottle or a cell culture dish, and culturing in a 5% CO2 incubator at 37 ℃.
Cell liquid change and passage
The ultraviolet lamp is turned on to irradiate the clean bench forty minutes in advance. Before entering a cell room, a mask cap is worn, part of a glass window of the superclean workbench, a fan and a lighting lamp are turned on, and self-inspection is carried out for three minutes. The new benzalkonium bromide wipes the workbench, and articles entering and exiting the superclean workbench, including both hands, need to be disinfected every time. The normal operation is performed at the vent. When the suspension cells are changed, the old culture solution in the culture bottle is transferred to a pipetteCentrifuge in the heart tube at 800 rpm for 3 minutes. Discard the supernatant, add new culture solution with pipette, blow gently to make the cell distribute evenly. Transferring the culture solution with cells in the centrifuge tube to a culture flask by pipette, and placing at 37 deg.C and 5% CO2Culturing in an incubator. If the cells are enough, the cells can be subcultured, the original culture solution containing the cells is evenly distributed into two or three culture bottles, and a proper amount of culture solution is supplemented until the logarithmic phase is ready for use.
Cell RNA extraction and identification
Quality evaluation of nucleic acid extraction before and after 3 months of normal temperature storage using the nucleic acid sample storage solution prepared in the above example using HL60 cells as storage cells. Dissolving RNA in 20 mu L of 1% DEPC water, blowing, mixing uniformly, centrifuging, measuring the concentration, and evaluating and identifying the RNA: purity (OD260/OD 280). Taking the RNA sample, taking DEPC water as a blank tube for zero adjustment, and using an ultraviolet spectrophotometer (ND1000) to determine the concentration (ng/mu L) of the RNA sample, wherein the absorbance of OD260 nm and OD280 nm is 1.8-2.0, the ratio indicates that the RNA purity is higher, the protein pollution is indicated when the ratio is less than 1.8, and the partial degradation is indicated when the ratio is more than 2.0. The concentration of the extracted RNA is between 500 and 2000 ng/mu L through the detection of a micro-spectrophotometer, and the requirements of LAMP and PCR methods are met. The results are shown in FIG. 1.
Change in Gene expression before and after preservation (RT-PCR)
(1) RNA extraction was performed according to the experimental methods and procedures described above. Mainly 10ml of lymphocyte separation solution is added into a test tube, then 2ml of bone marrow cells are slowly added into the test tube, and the mixture is centrifuged for 20min at 4000 r/min. The mononuclear cell is taken and transferred into another test tube, and then the RNA is extracted by the one-step method of guanidine hydrogen isothiolate-phenol-chloroform. During extraction, attention is paid to avoid cross contamination among samples.
(2) RT-PCR experiments: detecting AML patients STC-1 by nested RT-PCR: the primer is STC-1PCR (NM003155),
outer forward primer (201-:
5′-CTTCACTCAAGCCAGGAGAGGGAAA-3′(SEQ ID NO.1)；
outer reverse primer (1063-1090):
5′-TGGTGTGTCAACACCCCTAAAATGATA-3′(SEQ ID NO.2)；
inner forward primer (374) -407):
5′-GTGGCGGCCCAAAACTCAGCTGAA-3′(SEQ ID NO.3)；
inner reverse primer (1003-:
5'-TTATGCACTCTCATGGGATGTGCGTT-3' (SEQ ID NO.4) (used in the preliminary experiments).
(3) Analysis of PCR products: mu.l of the 2 nd round PCR product was electrophoresed on a 1.5% agarose gel, stained with 1. mu.g/ml ethidium bromide, and photographed. The final volume of the reaction mixture for the first PCR was 50. mu.l, and included 2. mu.l of cDNA, 2.5U of Taq DNA polymerase (Toyobo, Tokyo), 1 XPCR buffer (10mM Tris-HCl (pH8.3), 50mM KCl), 1.5mM MgCl20.2mM dNTP and 0.5mM of each primer. The mixture was subjected to 1 amplification for 25 minutes at 94 ℃, 2 minutes at 65 ℃ and 3 minutes at 72 ℃ by using a DNA thermal cycler (Astec PC-700, Fukuoka). A second PCR was performed using 2. mu.l of the first round product as template, in the same procedure as the first PCR. The PCR products of the second amplification were run on a 1.5% agarose gel and 645bp was confirmed as a single band with UV light and stained with ethidium bromide.
PCR detection results
FIG. 2 shows that the target gene expression of the cells is still positive within 3 months of normal temperature storage, and the expression level has no obvious change. FIG. 3 shows that the lowest detection sensitivity of the target gene still reached 0.5 ng/. mu.l within 3 months of storage at room temperature. The result shows that the preservation solution has higher cell cryopreservation effect and meets the RNA quality requirement after cell preservation.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Shandong Keshuo Biotechnology Co., Ltd
<120> nucleic acid sample preservation solution and preparation method and application thereof
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<170> PatentIn version 3.3
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Claims (10)
1. The preservation solution for the nucleic acid sample is characterized by comprising sodium salt, proteolytic enzyme, a surfactant, a biological buffer and water.
2. The nucleic acid sample preservation solution according to claim 1, wherein the sodium salt is one or more of sodium acetate and sodium EDTA.
3. The nucleic acid sample storage solution according to claim 1, wherein the proteolytic enzyme comprises proteinase K.
4. The nucleic acid sample preservation solution according to claim 1, wherein the surfactant is any one or more of SDS, Triton X-100, and SLS.
5. The nucleic acid specimen preservation solution according to claim 1, wherein the water is DEPC triple-distilled water.
6. The nucleic acid sample preservation solution according to any one of claims 1 to 5, which is composed of the following components: sodium acetate, EDTA sodium salt, Tris, protease K, SDS, Triton X-100, SLS and DEPC triple distilled water.
7. The nucleic acid sample preservation solution according to claim 6, which is composed of:
0.1-0.5M sodium acetate, 2-6mM EDTA sodium salt, 2-6mM Tris, 0.5-2 μ g/ml proteinase K, 0.1-0.5% SDS, 0.1-0.5% Triton X-100, 0.3-0.6% SLS, and DEPC triple distilled water as solvent;
preferably, the nucleic acid sample preservation solution consists of the following components:
0.3M sodium acetate, 5mM EDTA sodium salt, 5mM Tris, 1. mu.g/ml proteinase K, 0.2% SDS, 0.2% Triton X-100, 0.5% SLS, and DEPC triple distilled water as solvent.
8. The method for preparing a nucleic acid sample preservation solution according to any one of claims 1 to 7, wherein the method comprises: mixing the above materials.
9. Use of the nucleic acid sample preservation solution according to claim 8 for in vitro preservation of a nucleic acid sample.
10. The use of claim 9, wherein the nucleic acid sample preservation solution is suitable for the preservation of a pharyngeal swab, a nasal swab or a tissue sample of a specific site after sampling, and the stored sample is used for subsequent nucleic acid extraction or purification;
the nucleic acid comprises ribonucleic acid and deoxyribonucleic acid;
preferably, the nucleic acid sample is a viral nucleic acid sample.
Priority Applications (1)
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| CN114958969A (en) * | 2022-06-27 | 2022-08-30 | 爱科睿特生物医疗科技(南京)有限公司 | Respiratory tract pathogen nucleic acid preservation solution and preparation method thereof |
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