CN112280823A - Novel coronavirus RNA sample preservation solution, preparation method and application thereof - Google Patents
Novel coronavirus RNA sample preservation solution, preparation method and application thereof Download PDFInfo
- Publication number
- CN112280823A CN112280823A CN202011217058.3A CN202011217058A CN112280823A CN 112280823 A CN112280823 A CN 112280823A CN 202011217058 A CN202011217058 A CN 202011217058A CN 112280823 A CN112280823 A CN 112280823A
- Authority
- CN
- China
- Prior art keywords
- novel coronavirus
- rna sample
- coronavirus rna
- rna
- sample
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- 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
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a novel coronavirus RNA sample preservation solution, a preparation method and application thereof. The novel coronavirus RNA sample preservation solution comprises the following components: 0.1-2 v/v% of surfactant, 1-100 mmol/L of citric acid-sodium citrate buffer solution, 0.1-5 w/v% of trehalose, 0.2-2 mol/L of betaine and 0.5-5 w/v% of sorbitol, and the balance of solvent. The formula combination of the novel coronavirus RNA sample preservative fluid provided by the invention can rapidly crack viruses to release RNA, stabilize an RNA structure, protect the RNA structure, and still keep the integrity of the RNA after being placed at high temperature for 10 days. The formula combination of the invention can be directly used for RT-PCR amplification, each component can not inhibit the amplification process, RNA extraction is avoided, and the virus detection steps are simpler and shorter.
Description
Technical Field
The invention relates to a novel coronavirus RNA sample preservation solution, in particular to a preservation solution for sample RNA of a novel coronavirus oral swab, a nasopharyngeal swab and the like, a preparation method and application thereof, and belongs to the technical field of biological products.
Background
The novel coronavirus (2019-nCoV) brings great threat to the health of human beings, and the early diagnosis and early treatment of patients have great significance to the prevention and control of epidemic situations. The novel coronavirus is an RNA virus, the current early diagnosis method is nucleic acid detection, the method mainly comprises two detection modes of real-time fluorescence quantitative RT-PCR and gene sequencing, and the integrity of the RNA structure plays an important role in the efficient and accurate diagnosis process. Due to the degradation effect of cell endogenous RNA lytic enzymes and exogenous RNA lytic enzymes in the environment on RNA and the sensitivity of RNA to temperature and humidity in the environment, the existing preservation solution for RNA samples needs to be preserved at low temperature (-20 ℃ to-80 ℃) or room temperature (20 ℃ to-25 ℃) to ensure the integrity of RNA structures, and is used for next experimental study. The existing sample preservation solution has the following defects: 1. the sample can be stored at room temperature for a short time, and the sample is rapidly stored at low temperature after sampling; 2. the preservation solution has poor effect, so that RNA is easy to degrade, and certain difficulty is brought to subsequent experiments; 3. after sampling, the sample cannot be cracked and quickly inactivated, so that the risks of transworkers and experimenters after sampling are increased; 4. cannot be directly used for amplification detection, and needs RNA extraction.
Disclosure of Invention
The invention aims to provide a novel coronavirus RNA sample preservation solution and a preparation method thereof, thereby overcoming the defects of the prior art.
The invention also aims to provide application of the novel coronavirus RNA sample preservative fluid.
In order to achieve the purpose, the technical scheme adopted by the invention comprises the following steps:
the embodiment of the invention provides a novel coronavirus RNA sample preservation solution, which comprises the following components in part by weight: the components are as follows: 0.1-2 v/v% of surfactant, 1-100 mmol/L of citric acid-sodium citrate buffer solution, 0.1-5 w/v% of trehalose, 0.2-2 mol/L of betaine and 0.5-5 w/v% of sorbitol, and the balance of solvent.
In some preferred embodiments, the surfactant includes NP-40, but is not limited thereto.
Further, the novel coronavirus RNA sample preservative solution can keep the RNA structural integrity for more than 10 days at 40 ℃.
The embodiment of the invention also provides a preparation method of the novel coronavirus RNA sample preservation solution, which comprises the following steps: and (2) uniformly mixing a surfactant, a citric acid-sodium citrate buffer solution, trehalose, betaine, sorbitol and a solvent, adjusting the pH value to 5.8-7.0, and filtering to obtain the novel coronavirus RNA sample preservation solution.
Correspondingly, the embodiment of the invention also provides a method for preserving the novel coronavirus RNA sample, which comprises the following steps:
and (3) placing the novel coronavirus RNA sample to be preserved in the novel coronavirus RNA sample preservation solution, uniformly mixing and preserving.
The embodiment of the invention also provides a novel device for preserving the coronavirus RNA sample, which comprises a device body and the novel coronavirus RNA sample preserving fluid arranged in the device body.
In conclusion, the formula combination of the novel coronavirus RNA sample preservative fluid provided by the invention can rapidly crack viruses to release RNA, stabilize an RNA structure, protect the RNA structure, and still keep the integrity of the RNA after being placed at high temperature for 10 days. The formula combination can be directly used for RT-PCR amplification, each component can not inhibit the amplification process, RNA extraction is omitted, and the virus detection steps are simpler and shorter.
Compared with the prior art, the invention has the beneficial effects that:
1) the novel coronavirus RNA sample preservation solution can directly crack a sample, quickly inactivate viruses, reduce the infection risk of transportation personnel and experimental personnel, and has the characteristics of safety, simplicity, stable performance and the like;
2) the formula components in the novel coronavirus RNA sample preservation solution can not inhibit the RT-PCR detection process, and the obtained sample can be directly used for RT-PCR nucleic acid detection without purifying sample RNA, so that the detection steps are simpler and more convenient, and the detection time is saved;
3) the novel coronavirus RNA sample preservation solution disclosed by the invention can well keep the structural integrity of RNA for 10 days in a high-temperature (40 ℃) environment, and reduces the harsh requirements on the storage and transportation conditions of the sample.
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, it is obvious that the drawings in the following description are only some embodiments described in the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graphical representation of the results of different formulation specimen preservative fluid tests prepared in example 1 of the present invention;
FIG. 2 is a graph showing the results of 1 day storage of the novel coronavirus RNA specimen preservation solution prepared in example 2 of the present invention;
FIG. 3 is a graph showing the results of 3 days of storage of the novel coronavirus RNA sample storage solution prepared in example 2 of the present invention;
FIG. 4 is a graph showing the results of 5 days of storage of the novel coronavirus RNA specimen preservation solution prepared in example 2 of the present invention;
FIG. 5 is a graph showing the results of 7 days of storage of the novel coronavirus RNA sample storage solution prepared in example 2 of the present invention;
FIG. 6 is a graph showing the results of 10 days of storage of the novel coronavirus RNA specimen preservation solution prepared in example 2 of the present invention;
FIG. 7 is a schematic diagram showing the detection result of the 2019-nCoV pseudovirus sample by double RT-PCR in example 3 of the present invention;
FIG. 8 is a schematic diagram showing the comparative results of the amplified GAPDH gene of B lymphoma cells in the same type of specimen preservation solution in the market in example 4 of the present invention;
FIG. 9 is a schematic diagram showing the comparative results of the double RT-PCR of the GAPDH gene of a certain kind of the commercial products of example 5 of the present invention and the ORF1ab gene of the novel coronavirus.
Detailed Description
The inventor of the present invention has made a long-term study and a great deal of practice to provide a technical scheme of the present invention, which mainly provides a novel coronavirus sample preservation solution capable of lysing a sample, rapidly inactivating viruses, directly performing RT-PCR detection, and preserving RNA at a high temperature (40 ℃) for several days. The technical solution, the implementation process and the principle thereof will be further explained with reference to the attached drawings, but it should not be understood as the limitation of the scope of the present invention, and the insubstantial modifications and adjustments made by those skilled in the art according to the above disclosure still belong to the scope of the present invention.
In one aspect of the technical scheme of the invention, the novel coronavirus RNA sample preservation solution comprises the following components: 0.1-2 v/v% of surfactant, 1-100 mmol/L of citric acid-sodium citrate buffer solution, 0.1-5 w/v% of trehalose, 0.2-2 mol/L of betaine and 0.5-5 w/v% of sorbitol, and the balance of solvent.
In some preferred embodiments, the surfactant includes NP-40, but is not limited thereto.
Further, the pH value of the citric acid-sodium citrate buffer solution is 5.6-6.6.
Further, the solvent includes sterilized water.
Further, the pH value of the novel coronavirus RNA sample preservation solution is 5.8-7.0.
Further, the novel coronavirus RNA sample preservative solution can keep the RNA structural integrity for more than 10 days at 40 ℃.
In some more preferred embodiments, the formula of the novel coronavirus RNA sample preservation solution provided by the invention is shown in the following Table 1:
TABLE 1 sample preservative fluid formulation
| Composition (I) | Final concentration |
| NP-40 | 0.1％～2％(v/v) |
| Citric acid-sodium citrate buffer | 1mmol/L～100mmol/L |
| Trehalose | 0.1％～5％(w/v) |
| Betaine | 0.2mol/L～2mol/L |
| Sorbitol | 0.5％～5％(w/v) |
In the formula of the novel coronavirus RNA sample preservation solution, NP-40 is a surfactant, so that the virus can be rapidly split, and RNA contained in the virus can be released; the citric acid-sodium citrate buffer solution provides a stable buffer environment for the preservation of RNA in the sample preservation solution, and can also chelate divalent metal ions and inhibit the activity of RNase; trehalose, betaine and sorbitol can stabilize the RNA structure and reduce the degradation of RNA, and the trehalose and betaine can also improve the RT-PCR reaction efficiency.
In conclusion, the formula combination of the novel coronavirus RNA sample preservative fluid provided by the invention can rapidly crack viruses to release RNA, stabilize an RNA structure, protect the RNA structure, and still keep the integrity of the RNA after being placed at high temperature for 10 days. The formula combination of the invention can be directly used for RT-PCR amplification, each component can not inhibit the amplification process, RNA extraction is avoided, and the virus detection steps are simpler and shorter.
In one aspect of the present invention, there is provided a method for preparing a novel coronavirus RNA sample storage solution, comprising: and (2) uniformly mixing a surfactant, a citric acid-sodium citrate buffer solution, trehalose, betaine, sorbitol and a solvent, adjusting the pH value to 5.8-7.0, and filtering to obtain the novel coronavirus RNA sample preservation solution.
In some preferred embodiments, the preparation method comprises: and carrying out suction filtration treatment on the liquid phase obtained by mixing by adopting a filter membrane.
Further, the pore size of the filter membrane is 0.22 μm.
In some preferred embodiments, the preparation method specifically comprises: mixing the components in the table 1, adding a plurality of milliliters of RNase-free water, stirring and uniformly mixing, adjusting the pH value to 5.8-7.0, performing suction filtration on a 0.22-micron filter membrane, and standing at room temperature for later use.
Correspondingly, the other aspect of the embodiment of the invention also provides the application of the novel coronavirus RNA sample preservation solution.
For example, another aspect of the embodiments of the present invention also provides a method for preserving a novel coronavirus RNA sample, which comprises: and (3) placing the novel coronavirus RNA sample to be preserved in the novel coronavirus RNA sample preservation solution, uniformly mixing and preserving.
Further, the preservation temperature is-20 ℃ to 40 ℃.
Further, the novel coronavirus RNA sample includes, but is not limited to, a buccal swab, a pharyngeal swab, a cultured cell, and the like.
For example, another aspect of the embodiments of the present invention further provides a device for preserving a novel coronavirus RNA sample, which includes a device body, and the novel coronavirus RNA sample preservation solution provided in the device body.
By the preparation process, compared with the prior products of the same type, the novel coronavirus RNA sample preservative fluid has the following advantages: the novel coronavirus RNA sample preservation solution can directly crack a sample, quickly inactivate viruses, reduce the infection risk of transportation personnel and experimental personnel, and has the characteristics of safety, simplicity, stable performance and the like; the formula components in the novel coronavirus RNA sample preservation solution can not inhibit the RT-PCR detection process, and the obtained sample can be directly used for RT-PCR nucleic acid detection without purifying sample RNA, so that the detection steps are simpler and more convenient, and the detection time is saved; the novel coronavirus RNA sample preservation solution disclosed by the invention can well keep the structural integrity of RNA for 10 days in a high-temperature (40 ℃) environment, and reduces the harsh requirements on the storage and transportation conditions of the sample.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are described in further detail below with reference to the accompanying drawings and several preferred embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. The test methods in the following examples are carried out under conventional conditions without specifying the specific conditions. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
Example 1 different formulations sample preservative fluid testing
1. Instruments, reagents, samples
1) The main apparatus is as follows: BSC-1300-IIA2 biological safety cabinet; an OSE-MCBmini centrifuge; 3K15 refrigerated centrifuge; c1000Touch rapid automatic programming PCR instrument; SW-CJ-1FD single-person super clean bench; electrophoresis apparatus, GenoSen gel imaging system.
2) The main reagents are as follows: a GAPDH primer pair; one-step RT-PCR reaction detection kit.
3) Sample preparation: oral swab
2. The experimental scheme is as follows:
1) according to the following formula, sample preservation solutions with different formulas are prepared:
2) and (3) preserving the oral swab samples by using the prepared sample preserving fluid, respectively treating 2 samples by using each sample preserving fluid, and detecting after placing for 20min at room temperature.
3) Performing RT-PCR amplification reaction on the sample
The reaction system is as follows:
| reaction reagent | Volume (μ L) |
| 2 x one-step reaction mixture | 10 |
| |
2 |
| Enzyme mixture | 0.4 |
| Sample(s) | 7.6 |
| Total volume | 20 |
The reaction procedure was as follows:
4) performing agarose gel electrophoresis on the amplification product:
putting the prepared 2% agarose gel into an electrophoresis apparatus, adding 2 muL of 6 Xloading buffer solution into 10 muL of samples, uniformly mixing, adding all the mixed solution into a loading hole, carrying out electrophoresis at 140V for 30min, and observing the electrophoresis result under a gel imaging apparatus.
3. The experimental results are as follows:
FIG. 1 is a graph showing the test results of different formulations of specimen preservation solutions prepared in this example.
4. And (4) experimental conclusion:
clear sight zone can be seen in the ultraviolet detection of a gel imager, which shows that the sample preservation solution can realize the lysis of oral cells under different final concentration formulas and has no inhibition to one-step RT-PCR reaction. .
Example 2 buccal swab sample preservation test
1. Instruments, reagents, samples
1) The main apparatus is as follows: BSC-1300-IIA2 biological safety cabinet; an OSE-MCBmini centrifuge; 3K15 refrigerated centrifuge; c1000Touch rapid automatic programming PCR instrument; SW-CJ-1FD single-person super clean bench; electrophoresis apparatus, GenoSen gel imaging system.
2) The main reagents are as follows: a GAPDH primer pair; one-step RT-PCR reaction detection kit.
3) Sample preparation: oral swab
2. The experimental scheme is as follows:
1) through optimization, the final concentration of NP-40, the final concentration of citric acid-sodium citrate buffer solution and trehalose in the sample preservation solution is 0.6% (V/V), the final concentration of the citric acid-sodium citrate buffer solution is 30mmol/L, the final concentration of the trehalose is 3% (W/V), the final concentration of the betaine is 0.55mol/L, the final concentration of the sorbitol is 2(W/V), and the pH value is adjusted to be 6.0.
2) The sample preservation solution is used for preserving the oral swab samples, and the oral swab samples are respectively placed in the environment (1 tube) at room temperature (20-25 ℃) and the environment (2 tubes) at 40 ℃. The design of this experiment is as follows: marking the stored samples, respectively placing the samples into specified environments, and carrying out detection according to specified periods, wherein the specific steps are as follows: firstly, placing the sample in a room temperature (20-25 ℃) environment, and detecting the sample once every 1 day, 3 days, 5 days, 7 days and 10 days for 10 days; ② the detection is carried out once respectively in the environment of 40 ℃ for 1 day, 3 days, 5 days, 7 days and 10 days, and the detection lasts 10 days.
Control group: the sample is placed in the environment of room temperature (20-25 ℃) to be preserved by the sample preservation solution.
Experimental groups: the sample preserved by the sample preservative fluid is placed in an environment with the temperature of 40 ℃.
3) And carrying out RT-PCR amplification reaction on the sample.
The reaction system is as follows:
| reaction reagent | Volume (μ L) |
| 2 x one-step reaction mixture | 10 |
| |
2 |
| Enzyme mixture | 0.4 |
| Sample(s) | 7.6 |
| Total volume | 20 |
The reaction procedure was as follows:
4) performing agarose gel electrophoresis on the amplification product:
putting the prepared 2% gel into an electrophoresis apparatus, adding 2 muL of 6 Xloading buffer solution into 10 muL of sample, mixing uniformly, adding all the mixed solution into a sample adding hole, carrying out electrophoresis at 140V for 30min, and observing the electrophoresis result under a gel imaging apparatus.
3. The experimental results are as follows:
FIG. 2 is a graph showing the results of the preservation of the novel coronavirus RNA specimen prepared in this example for 1 day, FIG. 3 is a graph showing the results of the preservation for 3 days, FIG. 4 is a graph showing the results of the preservation for 5 days, FIG. 5 is a graph showing the results of the preservation for 7 days, and FIG. 6 is a graph showing the results of the preservation for 10 days.
4. And (4) experimental conclusion:
clear ground bands can be seen in the ultraviolet detection of a gel imager, which shows that the sample preservation solution can ensure that the quality of the RNA of the oral cells can be up to 10 days at the temperature of 40 ℃.
Example 3 novel coronavirus (2019-nCoV) pseudovirus sample preservation test
1. Instruments, reagents, samples
1) The main apparatus is as follows: BSC-1300-IIA2 biological safety cabinet; an OSE-MCBmini centrifuge; 3K15 refrigerated centrifuge; c1000Touch rapid automatic programming PCR instrument; SW-CJ-1FD single-person super clean bench; GenoSen gel imaging system.
2) The main reagents are as follows: ORF1ab primer pair; e gene primer pair; one-step RT-PCR reaction detection kit.
3) Sample preparation: novel coronavirus (2019-nCoV) pseudovirus
2. The experimental scheme is as follows:
1) a novel coronavirus (2019-nCoV) pseudovirus is added into a sample preservation solution, the sample preservation solution is placed in an environment at 40 ℃ until the final concentration is 500copies/uL, 10 mu L of the sample preservation solution is taken out after the sample preservation solution is placed for 1 day, 3 days, 5 days, 7 days and 10 days respectively, the sample preservation solution is placed at 20 ℃ immediately, and then double RT-PCR detection is carried out together.
2) And carrying out double RT-PCR amplification reaction on the sample.
The reaction system is as follows:
| reaction reagent | Volume (μ L) |
| 2 x one-step reaction mixture | 10 |
| RNase-free water | 1.6 |
| ORF1ab primer pair | 1.5 |
| E gene primer pair | 1.5 |
| Enzyme mixture | 0.4 |
| Sample(s) | 5 |
| Total volume | 20 |
The reaction procedure was as follows:
3) performing agarose gel electrophoresis on the amplification product:
putting the prepared 2% gel into an electrophoresis apparatus, adding 2 muL of 6 Xloading buffer solution into 10 muL of samples respectively, mixing uniformly, adding the mixed solution into the loading holes, performing electrophoresis at 140V for 30min, and observing the electrophoresis result under a gel imager.
3. The experimental results are as follows:
FIG. 7 is a schematic diagram showing the results of the two-fold RT-PCR detection of 2019-nCoV pseudovirus samples in the present example.
4. And (4) experimental conclusion:
two clear ground bands can be seen in the ultraviolet detection of a gel imager, which shows that the sample preservation solution can stably preserve the virus RNA at the temperature of 40 ℃.
Example 4 comparison of GAPDH Gene of B-lymphocytoma cells amplified by the same product in the market
1) The main apparatus is as follows: BSC-1300-IIA2 biological safety cabinet; an OSE-MCBmini centrifuge; 3K15 refrigerated centrifuge; c1000Touch rapid automatic programming PCR instrument; SW-CJ-1FD single-person super clean bench; GenoSen gel imaging system.
2) The main reagents are as follows: GAPDH primer pair, one-step RT-PCR reaction detection kit and similar sample preservation solution in certain market
3) Sample preparation: b lymphoma cells
2. The experimental scheme is as follows:
1) b lymphoma cells are added into the sample preservation solution and the similar sample preservation solution in a certain market to enable the final concentration of the cells to be 70/uL, 2 samples are respectively treated, and the samples are placed for 20min at room temperature;
2) and carrying out one-step RT-PCR amplification reaction on the sample.
The reaction system is as follows:
| reaction reagent | Volume (μ L) |
| 2 x one-step reaction mixture | 10 |
| |
2 |
| Enzyme mixture | 0.4 |
| Sample(s) | 7.6 |
| Total volume | 20 |
The reaction procedure was as follows:
3) performing agarose gel electrophoresis on the amplification product:
putting the prepared 2% gel into an electrophoresis apparatus, adding 2 muL of 6 Xloading buffer solution into 10 muL of samples respectively, mixing uniformly, adding the mixed solution into a sample adding hole, performing electrophoresis at 140V for 30min, and observing the electrophoresis result under a gel imaging apparatus.
3. The experimental results are as follows:
FIG. 8 is a schematic diagram showing the comparative results of the amplified GAPDH gene of B lymphoma cells in the preservation solution of the same type of samples in the market.
4. And (4) experimental conclusion:
the amplification effect of the sample preservative solution is obviously superior to that of a certain similar product in the market under the condition of the same cell content.
EXAMPLE 5A commercial dual RT-PCR comparison of the GAPDH gene of a certain generic product with the ORF1ab gene of a novel coronavirus
1. Instruments, reagents, samples
1) The main apparatus is as follows: BSC-1300-IIA2 biological safety cabinet; an OSE-MCBmini centrifuge; 3K15 refrigerated centrifuge; c1000Touch rapid automatic programming PCR instrument; SW-CJ-1FD single-person super clean bench; GenoSen gel imaging system.
2) The main reagents are as follows: ORF1ab primer pair; GAPDH gene primer pair; one-step RT-PCR reaction detection kit and preservation solution for similar samples in certain markets.
3) Sample preparation: b-lymphoma cell, novel coronavirus (2019-nCoV) pseudovirus
2. The experimental scheme is as follows:
1) b lymphoma cells and novel coronavirus pseudoviruses are added into the sample preservation solution and the similar sample preservation solution in a certain market, so that the final concentration of the cells is 100/uL, the final concentration of the pseudoviruses is 500copies/uL, the two sample preservation solutions are used for respectively treating 3 samples, and the samples are placed for 20min at room temperature;
2) and carrying out double RT-PCR amplification reaction on the sample.
The reaction system is as follows:
| reaction reagent | Volume (μ L) |
| 2 x one-step reaction mixture | 10 |
| RNase-free water | 1.6 |
| ORF1ab primer pair | 1.5 |
| GAPDH gene primer pair | 1.5 |
| Enzyme mixture | 0.4 |
| Sample(s) | 5 |
| Total volume | 20 |
The reaction procedure was as follows:
3) performing agarose gel electrophoresis on the amplification product:
putting the prepared 2% gel into an electrophoresis apparatus, adding 2 muL of 6 Xloading buffer solution into 10 muL of samples respectively, mixing uniformly, adding the mixed solution into the loading holes, performing electrophoresis at 140V for 30min, and observing the electrophoresis result under a gel imager.
3. The experimental results are as follows:
FIG. 9 is a diagram showing the comparative results of the double RT-PCR of GAPDH gene and novel coronavirus ORF1ab gene of a same type of products on the market in this example.
4. And (4) experimental conclusion:
the amplification effect of the sample preservative fluid is obviously superior to that of a certain similar product in the market under the condition of the same cell content and pseudovirus content.
The aspects, embodiments, features and examples of the present invention should be considered as illustrative in all respects and not intended to be limiting of the invention, the scope of which is defined only by the claims. Other embodiments, modifications, and uses will be apparent to those skilled in the art without departing from the spirit and scope of the claimed invention.
The use of headings and chapters in this disclosure is not meant to limit the disclosure; each section may apply to any aspect, embodiment, or feature of the disclosure.
Throughout this specification, where a composition is described as having, containing, or comprising specific components or where a process is described as having, containing, or comprising specific process steps, it is contemplated that the composition of the present teachings also consist essentially of, or consist of, the recited components, and the process of the present teachings also consist essentially of, or consist of, the recited process steps.
It should be understood that the order of steps or the order in which particular actions are performed is not critical, so long as the teachings of the invention remain operable. Further, two or more steps or actions may be performed simultaneously.
In addition, the inventors of the present invention have also made experiments with other materials, process operations, and process conditions described in the present specification with reference to the above examples, and have obtained preferable results.
While the invention has been described with reference to illustrative embodiments, it will be understood by those skilled in the art that various other changes, omissions and/or additions may be made and substantial equivalents may be substituted for elements thereof without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed for carrying out this invention, but that the invention will include all embodiments falling within the scope of the appended claims. Moreover, unless specifically stated any use of the terms first, second, etc. do not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another.
Claims (10)
1. The novel coronavirus RNA sample preservation solution is characterized by comprising the following components: 0.1-2 v/v% of surfactant, 1-100 mmol/L of citric acid-sodium citrate buffer solution, 0.1-5 w/v% of trehalose, 0.2-2 mol/L of betaine and 0.5-5 w/v% of sorbitol, and the balance of solvent.
2. The novel coronavirus RNA sample storage solution according to claim 1, wherein the storage solution comprises: the surfactant includes NP-40.
3. The novel coronavirus RNA sample storage solution according to claim 1, wherein the storage solution comprises: the solvent comprises sterilized water; and/or the pH value of the citric acid-sodium citrate buffer solution is 5.6-6.6.
4. The novel coronavirus RNA sample storage solution according to claim 1, wherein the storage solution comprises: the pH value of the novel coronavirus RNA sample preservation solution is 5.8-7.0.
5. The novel coronavirus RNA sample storage solution according to claim 1, wherein the storage solution comprises: the novel coronavirus RNA sample preservation solution can keep RNA structural integrity for more than 10 days at 40 ℃.
6. The method for preparing a novel coronavirus RNA specimen preservation solution according to any one of claims 1 to 5, which comprises: and (2) uniformly mixing a surfactant, a citric acid-sodium citrate buffer solution, trehalose, betaine, sorbitol and a solvent, adjusting the pH value to 5.8-7.0, and filtering to obtain the novel coronavirus RNA sample preservation solution.
7. The production method according to claim 6, characterized by comprising: and carrying out suction filtration treatment on the liquid phase obtained by mixing by adopting a filter membrane.
8. A method for preserving a novel coronavirus RNA sample, characterized by comprising:
placing the novel coronavirus RNA sample to be preserved in the novel coronavirus RNA sample preservation solution of any one of claims 1-5, uniformly mixing and preserving.
9. The saving method according to claim 8, wherein: the preservation temperature is-20 ℃ to 40 ℃; preferably, the novel coronavirus RNA sample comprises any one or a combination of more than two of oral swab, pharyngeal swab and cultured cells.
10. A device for preserving a novel coronavirus RNA specimen, characterized by comprising a device body and the novel coronavirus RNA specimen preservative solution according to any one of claims 1 to 5 provided in the device body.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011217058.3A CN112280823A (en) | 2020-11-04 | 2020-11-04 | Novel coronavirus RNA sample preservation solution, preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011217058.3A CN112280823A (en) | 2020-11-04 | 2020-11-04 | Novel coronavirus RNA sample preservation solution, preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112280823A true CN112280823A (en) | 2021-01-29 |
Family
ID=74351688
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011217058.3A Pending CN112280823A (en) | 2020-11-04 | 2020-11-04 | Novel coronavirus RNA sample preservation solution, preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112280823A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115747303A (en) * | 2022-11-25 | 2023-03-07 | 中国人民解放军西部战区总医院 | Sample preservation solution, reagent and sample preservation method |
| WO2023098620A1 (en) * | 2021-12-02 | 2023-06-08 | 圣湘生物科技股份有限公司 | Washing-free nucleic acid extraction composition, kit, method and use |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102575286A (en) * | 2009-04-20 | 2012-07-11 | 长角牛疫苗和诊断有限责任公司 | Biological specimen collection/transport compositions and methods |
| CN111304175A (en) * | 2020-02-15 | 2020-06-19 | 南京尧顺禹生物科技有限公司 | Virus sample preservation solution for clinical detection of virus nucleic acid and use method thereof |
| CN111635960A (en) * | 2020-05-06 | 2020-09-08 | 温州医科大学附属眼视光医院 | Protective sequence, primer, probe, composition, kit, application and method for steady-state quick-acting detection of novel coronavirus |
| CN111676216A (en) * | 2020-06-18 | 2020-09-18 | 合肥铼科生物科技有限公司 | Extraction-free inactivated virus sample nucleic acid preservation solution and preparation method and application thereof |
| CN111718908A (en) * | 2020-08-11 | 2020-09-29 | 杭州博日科技有限公司 | Virus sample preserving fluid and preparation method and application thereof |
-
2020
- 2020-11-04 CN CN202011217058.3A patent/CN112280823A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102575286A (en) * | 2009-04-20 | 2012-07-11 | 长角牛疫苗和诊断有限责任公司 | Biological specimen collection/transport compositions and methods |
| CN111304175A (en) * | 2020-02-15 | 2020-06-19 | 南京尧顺禹生物科技有限公司 | Virus sample preservation solution for clinical detection of virus nucleic acid and use method thereof |
| CN111635960A (en) * | 2020-05-06 | 2020-09-08 | 温州医科大学附属眼视光医院 | Protective sequence, primer, probe, composition, kit, application and method for steady-state quick-acting detection of novel coronavirus |
| CN111676216A (en) * | 2020-06-18 | 2020-09-18 | 合肥铼科生物科技有限公司 | Extraction-free inactivated virus sample nucleic acid preservation solution and preparation method and application thereof |
| CN111718908A (en) * | 2020-08-11 | 2020-09-29 | 杭州博日科技有限公司 | Virus sample preserving fluid and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| EDWARD I PATTERSON ET AL.: "Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays", J INFECT DIS, vol. 222, no. 9, pages 192 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023098620A1 (en) * | 2021-12-02 | 2023-06-08 | 圣湘生物科技股份有限公司 | Washing-free nucleic acid extraction composition, kit, method and use |
| CN115747303A (en) * | 2022-11-25 | 2023-03-07 | 中国人民解放军西部战区总医院 | Sample preservation solution, reagent and sample preservation method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112626169B (en) | Sample preservation solution for preserving nucleic acid in biological sample and use method thereof | |
| CN111718908B (en) | Virus sample preserving fluid and preparation method and application thereof | |
| CA2428864C (en) | Method and device for collecting and stabilizing a biological sample | |
| CN112760318B (en) | Reagent composition for stabilizing nucleic acid molecules and application thereof | |
| US6617170B2 (en) | Method and device for collecting and stabilizing a biological sample | |
| CN111560417B (en) | Freeze-drying protective agent and freeze-drying method for RNA amplification reaction reagent | |
| CN111690640B (en) | Virus preservation solution of highly compatible magnetic bead method virus nucleic acid extraction kit | |
| CN105506129B (en) | A kind of RNA class Sample preservation dilution and its preparation | |
| CN112280823A (en) | Novel coronavirus RNA sample preservation solution, preparation method and application thereof | |
| EP4023767A1 (en) | Method, composition and kit for fluorescent quantitative pcr, and use thereof | |
| CN109371008B (en) | Nucleic acid extraction amplification detection kit | |
| CN111088319A (en) | Inactivated virus sample RNA preservation solution and preparation method thereof | |
| CN112725406A (en) | Nucleic acid extraction-free inactivated virus preservation solution and application thereof | |
| CN113736748A (en) | Sample preservation solution and application thereof | |
| CN112608978A (en) | Novel nucleic acid hand-free storage solution | |
| CN112704062A (en) | Cell preservation solution and using method thereof | |
| CN117546837A (en) | Blood preservation solution suitable for NK cell culture and preparation method and application thereof | |
| WO2023011162A1 (en) | Biological sample nucleic acid release preservative | |
| US20230284611A1 (en) | Aqueous solution to preserve nucleic acids | |
| CN115558704A (en) | RNA sample preservation solution and application thereof | |
| CN113999840B (en) | Nucleic acid sample preservation solution and use method and application thereof | |
| CN112342317A (en) | Nucleic acid sequence combination, kit and detection method for LAMP-CRISPR (loop-mediated isothermal amplification-CRISPR) isothermal detection of IHHNV (infectious bronchitis Virus) | |
| CN111808842A (en) | Nucleic acid protection reagent and use method thereof | |
| KR102459785B1 (en) | Composition for extracting nucleic acids and lysing cell, method for extracting nucleic acids and molecular diagnostic method using the same | |
| CN117770233A (en) | Sample retention and preservation solution for cell products |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |