CN114958969A - Respiratory tract pathogen nucleic acid preservation solution and preparation method thereof - Google Patents
Respiratory tract pathogen nucleic acid preservation solution and preparation method thereof Download PDFInfo
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- CN114958969A CN114958969A CN202210736530.7A CN202210736530A CN114958969A CN 114958969 A CN114958969 A CN 114958969A CN 202210736530 A CN202210736530 A CN 202210736530A CN 114958969 A CN114958969 A CN 114958969A
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- 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
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses a respiratory tract pathogen nucleic acid preservative fluid and a preparation method thereof, belonging to the technical field of biological products; the invention provides a respiratory tract pathogen nucleic acid preservative fluid, which is used for treating nasal, pharyngeal swab, sputum and other specimens, can effectively keep DNA nucleic acid stable, does not influence subsequent sample DNA/RNA extraction and detection experiments, and has good preservation effect.
Description
Technical Field
The invention belongs to the technical field of biological products, and particularly relates to a nucleic acid preservation solution, and a preparation method and application thereof.
Background
Nucleic acid is a biological macromolecular compound synthesized by many nucleotides, widely exists in all animal and plant cells and microorganisms, and nucleic acid in organisms can be divided into ribonucleic acid (DNA) and deoxyribonucleic acid (RNA) according to different chemical compositions.
Accurate detection of nucleic acids is an important aspect of analysis of molecular biological samples, and the high cost and high specificity of nucleic acid detection usually separate the acquisition and detection of samples, and most of the samples to be detected need to be transported over short distance or long distance before entering the detection process. However, as carriers of biogenetic information, nucleic acids are unstable in performance and are subject to external physical factors such as temperature, humidity, ultraviolet rays, etc.; chemical factors such as PH, hydrolysis, oxidation, etc.; biological factors such as enzymolysis and microbial infection are easy to cause degradation and denaturation of nucleic acid, so that strict limitation needs to be made on the transportation time of a sample, and the difficulty in storing the sample in the transportation process is greatly increased.
Therefore, there is a need to develop a preservation solution that can provide a stable environment for the sample under transportation conditions, prevent cell disruption and nucleic acid degradation, and eliminate adverse effects on subsequent testing.
Therefore, it is an urgent need to solve the problem of providing a preservation solution for respiratory pathogen nucleic acid and a preparation method thereof.
Disclosure of Invention
In order to solve the problems, the invention discloses a preservation solution for nucleic acid of a respiratory tract pathogen and a preparation method thereof.
In order to achieve the purpose, the technical scheme of the invention is as follows:
the invention provides a nucleic acid preservation solution which comprises the following components in percentage by weight: 10-40% of citric acid, 201-5% of tween, 10-40% of EDTA disodium, 35-70% of sodium sulfate, 1-2.50% of isopropanol and the balance of DEPC water.
Further, the paint comprises the following components in percentage by weight: 20% of citric acid, 203% of tween, 35% of EDTA disodium, 40% of sodium sulfate, 2% of isopropanol and the balance of DEPC water.
Further, the pH of the nucleic acid preservation solution is 4.5 to 6.5.
The invention also provides a preparation method of the nucleic acid preservation solution, which comprises the following steps:
(1) weighing citric acid, tween 20, EDTA disodium, sodium sulfate and isopropanol according to the weight percentage, and dissolving in DEPC water;
(2) adjusting the pH of the dissolved solution to 4.5-6.5; if the reagent is slightly acidic, adjusting the pH value by using NaOH; if the pH value is alkaline, adjusting the pH value by using concentrated HCl;
(3) the pH-adjusted solution was sterilized with a 0.22. mu.M aqueous membrane to obtain a nucleic acid-preserving solution. The obtained nucleic acid preservation solution is subpackaged to a sampling tube aseptically without enzyme, and stored at the temperature of 2-8 ℃ for 72 hours and at the temperature of-80 ℃ for 1 year.
The nucleic acid preservation solution is applied to preservation and transportation of biological samples, and the preservation and transportation temperature is 2-8 ℃.
Further, the biological sample is a respiratory pathogen nucleic acid.
Further, the biological sample is human sputum, saliva and nasopharyngeal swab collection fluid.
The using method comprises the following steps: taking a fresh sample, immediately putting the fresh sample into 3ml of the nucleic acid preservation solution, and preserving the sample at the temperature of 2-8 ℃ for 72 hours.
The invention has the beneficial effects that:
according to the technical scheme, compared with the prior art, the invention discloses the respiratory tract pathogen nucleic acid preservation solution and the preparation method thereof, the nucleic acid preservation solution has the advantages of low cost of used raw materials, less component quantity, simple and quick preparation method, and reduction of production cost while more accurate control of the additive dosage; the preservation solution provides a stable environment for the sample, prevents cell rupture and nucleic acid degradation, does not influence subsequent sample DNA/RNA extraction and detection experiments, and has a good preservation effect.
Drawings
FIG. 1 is a QPCR amplification plot of the extracted DNA of examples 1-4 of the present invention;
FIG. 2 is a QPCR amplification plot of RNA after extraction in examples 1-4 of the present invention;
FIG. 3 is a QPCR amplification plot of the extracted DNA of examples 5-8 of the present invention;
FIG. 4 is the QPCR amplification plot of RNA after extraction in examples 5-8 of the present invention;
FIG. 5 is a QPCR amplification plot of extracted DNA/RNA of examples 9-11 of the present invention;
FIG. 6 is a QPCR amplification graph of example 3 of the present invention and comparative examples 1 to 3.
Detailed Description
The present invention will be further illustrated with reference to the accompanying drawings and specific embodiments, which are to be understood as merely illustrative of the invention and not as limiting the scope of the invention.
In the following examples, the DNA sample was extracted from parainfluenza virus type III, and the RNA sample was extracted from Streptococcus pneumoniae.
Example 1
The parainfluenza virus culture and the Streptococcus pneumoniae culture were extracted without being placed in a storage solution by adding physiological saline, and QPCR was performed using the extracted DNA/RNA as a template.
Example 2
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, immediately extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preserving fluid comprises the following components in percentage by mass: pH6.0, 10% of citric acid, 203% of tween, 35% of EDTA disodium, 40% of sodium sulfate, 2% of isopropanol and the balance of DEPC water.
Example 3
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, immediately extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preserving fluid comprises the following components in percentage by mass: pH6.0, 20% of citric acid, 203% of tween, 35% of EDTA disodium, 40% of sodium sulfate, 2% of isopropanol and the balance of DEPC water.
Example 4
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, immediately extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preserving fluid comprises the following components in percentage by mass: pH6.0, 30% of citric acid, 203% of tween, 35% of EDTA disodium, 40% of sodium sulfate, 2% of isopropanol and the balance of DEPC water.
Example 5
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, immediately extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preserving fluid comprises the following components in percentage by mass: pH6.0, 40% of citric acid, 203% of tween, 35% of EDTA disodium, 40% of sodium sulfate, 2% of isopropanol and the balance of DEPC water.
Example 6
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, immediately extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preserving fluid comprises the following components in percentage by mass: pH4.5, citric acid 20%, Tween 203%, EDTA disodium 35%, sodium sulfate 40%, isopropanol 2%, and the balance DEPC water.
Example 7
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, immediately extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preserving fluid comprises the following components in percentage by mass: pH5.0, 20% of citric acid, 203% of tween, 35% of EDTA disodium, 40% of sodium sulfate, 2% of isopropanol and the balance of DEPC water.
Example 8
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, immediately extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preserving fluid comprises the following components in percentage by mass: pH5.5, 20% of citric acid, 203% of tween, 35% of EDTA disodium, 40% of sodium sulfate, 2% of isopropanol and the balance of DEPC water.
The above examples were performed in 3 replicates per sample, and the Ct values were averaged over 3 runs and are shown in Table 1.
TABLE 1
| Ct value of DNA | Ct value of RNA | ||
| Example 1 | 23.44 | Example 1 | 22.73 |
| Example 2 | 23.13 | Example 2 | 22.55 |
| Example 3 | 22.04 | Example 3 | 21.57 |
| Example 4 | 22.09 | Example 4 | 21.35 |
| Example 5 | 22.21 | Example 5 | 21.51 |
| Example 6 | 22.28 | Example 6 | 21.39 |
| Example 7 | 22.93 | Example 7 | 21.50 |
| Example 8 | 23.66 | Example 8 | 20.86 |
From the results of fig. 1 to fig. 4 and table 1, it can be seen that the effects of examples 3 to 7 are not very different, the effects of examples 3 to 7 are significantly better than those of examples 1 and 2, and the performance of example 3 is slightly better than those of other examples, so that the formula of the preservative fluid of example 3 is selected as the optimal scheme.
Example 9
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, standing for 12h, extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preservative solution composition was the same as in example 3.
Example 10
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, standing for 24h, extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preservative solution composition was the same as in example 3.
Example 11
And (3) uniformly mixing the parainfluenza virus culture and the streptococcus pneumoniae culture in a preservation solution, standing for 36h, extracting, and performing QPCR by using the extracted DNA/RNA as a template. The preservative solution composition was the same as in example 3.
The post-experiment Ct values are shown in Table 2, and the results are graphically shown in FIG. 5.
TABLE 2
| Ct value of DNA | Ct value of RNA | ||
| Example 9 | 22.60 | Example 9 | 22.51 |
| Example 10 | 22.43 | Example 10 | 22.57 |
| Example 11 | 22.55 | Example 11 | 22.45 |
Comparative example 1
The parainfluenza virus culture and the streptococcus pneumoniae culture are put into a preservation solution and are uniformly mixed, then extraction is carried out immediately, and QPCR is carried out by taking the extracted DNA as a template. The preserving fluid comprises the following components in percentage by mass: pH6.0, citric acid 20%, EDTA disodium 35%, sodium sulfate 40%, and the balance DEPC water. Comparative example 2
This comparative example differs from comparative example 1 in that 203% tween was added, and the remainder was the same as in comparative example 1.
Comparative example 3
This comparative example differs from comparative example 1 in that 2% isopropanol was added, and the remainder was the same as comparative example 1.
The results are shown in FIG. 6, and the Ct values are shown in Table 3.
TABLE 3
| Ct value of DNA | |
| Example 3 | 23.13 |
| Comparative example 1 | 36.16 |
| Comparative example 2 | 30.97 |
| Comparative example 3 | 32.07 |
It should be noted that the above-mentioned contents only illustrate the technical idea of the present invention, and the protection scope of the present invention is not limited thereby, and it is obvious to those skilled in the art that several modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations fall within the protection scope of the claims of the present invention.
Claims (8)
1. The nucleic acid preservation solution is characterized by comprising the following components in percentage by mass:
10-40% of citric acid, 201-5% of tween, 10-40% of EDTA disodium, 35-70% of sodium sulfate, 1-2.50% of isopropanol and the balance of DEPC water.
2. The nucleic acid preservation solution according to claim 1, which is composed of the following components in percentage by mass: 20% of citric acid, 203% of tween, 35% of EDTA disodium, 40% of sodium sulfate, 2% of isopropanol and the balance of DEPC water.
3. The nucleic acid preservation solution according to claim 1 or 2, wherein the pH of the nucleic acid preservation solution is 4.5 to 6.5.
4. A method for producing a nucleic acid preservation solution according to any one of claims 1 to 3, comprising the steps of:
(1) weighing citric acid, tween 20, EDTA disodium, sodium sulfate and isopropanol according to the weight percentage, and dissolving in DEPC water;
(2) adjusting the pH of the dissolved solution to 4.5-6.5;
(3) the pH-adjusted solution was sterilized with a 0.22. mu.M aqueous membrane to obtain a nucleic acid-preserving solution.
5. The method for preparing a nucleic acid preservation solution according to claim 4, wherein the step (3) further comprises aseptically and enzymatically dispensing the obtained nucleic acid preservation solution into a sampling tube, and storing at 2-8 ℃ for 72 hours and at-80 ℃ for 1 year.
6. Use of a nucleic acid preservation solution according to any one of claims 1 to 3 for preserving and transporting biological samples at a temperature of 2 to 8 ℃.
7. The use of the nucleic acid preservation solution according to claim 6 for preserving and transporting biological samples, wherein the biological samples are respiratory pathogen nucleic acids.
8. The use of the nucleic acid preservation solution according to claim 6 for preserving and transporting biological samples, wherein the biological samples include human sputum, saliva and nasopharyngeal swab collection.
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