CN112704062A

CN112704062A - Cell preservation solution and using method thereof

Info

Publication number: CN112704062A
Application number: CN202011621777.1A
Authority: CN
Inventors: 赵国栋; 熊尚岷
Original assignee: Suzhou Weishan Biotechnology Co ltd
Current assignee: Suzhou Weishan Biotechnology Co ltd
Priority date: 2020-12-30
Filing date: 2020-12-30
Publication date: 2021-04-27

Abstract

The invention belongs to the field of biomedicine, and particularly relates to a cell preservation solution and a using method thereof, wherein the preservation solution contains 0.1-10% of a nucleic acid stabilizer, 0.05-1% of an osmotic pressure stabilizer, 0.01-0.1% of a pH stabilizer, 0.05-1% of a freeze-drying protective agent and the balance of water. The cell preservation solution has better time stability and temperature stability, and can save sample collection cost and accelerate nucleic acid detection efficiency for areas which cannot be delivered to a detection site on the same day and lack effective transportation equipment.

Description

Cell preservation solution and using method thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a cell preservation solution and a using method thereof.

Background

Both the confirmed gold standard and the standard of the. And the specimen related to virus or nucleic acid detection should be stored in a refrigerator at 4 ℃ for no more than 24 h. Therefore, the sensitivity of the sample collection and preservation solution to the subsequent detection result is greatly influenced. The sensitivity of the existing nucleic acid detection is about 30-50%, and in order to avoid false negative, at least more than 2 times of detection are needed for accurate diagnosis; therefore, the nucleic acid detection ability is limited to some extent, and the consumption of manpower and material resources is extremely high. In the case of an area where the daily delivery to the test site is impossible and there is a lack of effective transportation equipment, a preservation solution excellent in both time stability and temperature stability is urgently required. And the preservation solution capable of ensuring better room temperature stability has important significance for saving cost of sample collection and accelerating nucleic acid detection efficiency.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a cell preservation solution and a using method thereof.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

a cell preservation solution comprises the following components in percentage by mass: 0.1-10% of nucleic acid stabilizer, 0.05-1% of osmotic pressure stabilizer, 0.01-0.1% of pH stabilizer, 0.05-1% of freeze-drying protective agent and the balance of water.

Further, the nucleic acid stabilizer is one or more of sodium dodecyl sulfate, guanidine isothiocyanate and ethylene diamine tetraacetic acid.

Further, the osmotic pressure stabilizer is one or more of sodium chloride, potassium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate.

Further, the pH stabilizer is Tris-HCl.

Further, the freeze-drying protective agent is one or more of sucrose, glucose and trehalose.

Further, the cell preservation solution also contains antibiotics, and the concentration of the antibiotics is 20-30 ug/mL.

Further, the antibiotic is benzyl penicillin.

Further, the concentration of the antibiotic is 20-30 ug/mL.

The invention also provides application of the cell preservation solution in nucleic acid sampling, wherein the nucleic acid is deoxyribonucleic acid and ribonucleic acid.

The invention also provides a using method of the cell preservation solution, the sampling swab is used for sampling and then is immersed into the cell preservation solution, the transportation time is not more than 7 days at room temperature, the storage time at 4 ℃ is not more than 28 days, and the cell preservation solution is stored at-80 ℃ after being stored for more than 28 days.

Compared with the prior art, the application has the following beneficial effects:

the cell preservation solution has better time stability and temperature stability, can save sample collection cost and accelerate nucleic acid detection efficiency for areas which cannot be delivered to a detection site on the same day and lack effective transportation equipment, can be simultaneously used for DNA and RNA preservation solutions, does not need a cracking step in subsequent detection, can be directly used for nucleic acid purification, and accelerates the detection process.

Drawings

FIG. 1 shows RNA stability at 37 ℃ for 7 days;

FIG. 2 shows the stability of DNA stored at 4 ℃ and room temperature for 28 days.

FIG. 3 is a comparison of the preservation effect of the preservation solution of the present invention on the preservation of RNA by using the Cytoprep preservation solution and the Pierce preservation solution;

FIG. 4 is a comparison of preservation effects of different component preservation solutions.

Detailed Description

Example 1

A cell preservation solution was prepared using 1% sodium lauryl sulfate, 0.1% guanidine isothiocyanate, 0.1% ethylenediaminetetraacetic acid, 0.05% sodium chloride, 0.05% potassium chloride, 0.05% disodium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.05% Tris-HCl buffer (pH 8.0), 0.1% sucrose, and 25ug/ml benzylpenicillin. Fresh HeLa cell lines are placed in cell preservation solution and stored in samples of 0 day, 1 day, 3 days and 7 days at 37 ℃, RNA is extracted by adopting RNeasy Mini kit of Qiagen company, four genes such as ACTB, GADPH, IL1B and TLR4 are detected by fluorescence quantitative PCR, and each group of samples is repeated for 3 times. As shown in FIG. 1, the Ct values of RNA detection of 4 genes were not significantly changed when fresh RNA and RNA treated with cell preservation solution were stored at 37 ℃ for 0-7 days.

Example 2

A cell preservation solution was prepared using 1% guanidinium isothiocyanate, 0.1% ethylenediaminetetraacetic acid, 0.05% sodium chloride, 0.05% potassium chloride, 0.05% disodium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.01% Tris-HCl buffer (pH 8.0), 0.1% glucose, and 25ug/ml benzylpenicillin. A high-risk HPV in-vitro molecular diagnostic kit of Suzhou national science and technology Limited is adopted, samples of a fresh HeLa cell line and samples preserved by cell preservation solution at 4 ℃ and Room Temperature (RT) for 0 day, 7 days, 14 days, 21 days and 28 days are extracted by a genome extraction kit of Suzhou national science and technology Limited, and then fluorescence quantitative PCR detection is carried out, and each group of samples are repeated for 3 times.

As shown in FIG. 2, the Ct values of fresh DNA and DNA treated by the cell preservation solution have no obvious difference after being preserved for 28 days at 4 ℃ and room temperature, and reach the standard of protecting DNA stability.

Example 3

A cell preservation solution was prepared using 1% sodium lauryl sulfate, 0.1% guanidine isothiocyanate, 0.1% ethylenediaminetetraacetic acid, 0.05% sodium chloride, 0.05% potassium chloride, 0.05% disodium hydrogen phosphate, 0.05% potassium dihydrogen phosphate, 0.01% Tris-HCl buffer (pH 8.0), 0.1% sucrose, and 25ug/ml benzylpenicillin. And (3) placing a fresh HeLa cell line in a cell preservation solution to preserve samples at 37 ℃ for 0 day, 1 day, 3 days and 7 days, and simultaneously preserving the fresh HeLa cell line in the same way by adopting a Cytoprep preservation solution based on alcohol groups of Versabio company and a Pierce preservation solution of Sammarzel company to preserve samples at 37 ℃ for 0 day, 1 day, 3 days and 7 days. RNA is extracted by RNeasy Mini kit of Qiagen company, and then fluorescent quantitative PCR is carried out to detect four genes such as ACTB, GADPH, IL1B, TLR4 and the like, and each group of samples is repeated for 3 times. As shown in FIG. 3, the Ct values of RNA detection of 4 genes have no obvious change when fresh RNA and RNA treated by the cell preservation solution of the invention are preserved at 37 ℃ for 0-7 days, but the RNA treated by the Cytoprep preservation solution and the Pierce preservation solution is obviously degraded in the first day, the Ct values are delayed by nearly 10, namely the degraded RNA only remains 1/1000.

Example 4

Component 1 is prepared by using 0.1% guanidine isothiocyanate, 0.1% ethylene diamine tetraacetic acid, 0.05% disodium hydrogen phosphate, 0.05% monopotassium phosphate, 0.1% sucrose and 25ug/ml benzyl penicillin, and component 2 is prepared by using 1% sodium dodecyl sulfate, 0.05% sodium chloride, 0.05% potassium chloride and 0.01% Tris-HCl buffer (pH 8.0), and then the component 1 and the component 2 are mixed to prepare the cell preservation solution (combined components). The fresh HeLa cell line was placed in fraction 1, fraction 2, cell preservation solution (pooled fractions) and stored at room temperature for 7 days. After RNA was extracted using RNeasy Mini kit from Qiagen, ACTB gene was detected by fluorescent quantitative PCR, and each group was repeated 3 times. As shown in FIG. 4, although fraction 1 and fraction 2 can protect RNA to some extent, there is some degradation and the remaining DNA is 1/10 as it is, but there is only a slight delay in Ct after mixing fraction 1 and fraction 2.

Claims

1. The cell preservation solution is characterized by comprising the following components in percentage by mass: 0.1-10% of nucleic acid stabilizer, 0.05-1% of osmotic pressure stabilizer, 0.01-0.1% of pH stabilizer, 0.05-1% of freeze-drying protective agent and the balance of water.

2. The cell preservation solution according to claim 1, wherein the nucleic acid stabilizer is one or more of sodium dodecyl sulfate, guanidine isothiocyanate, and ethylenediaminetetraacetic acid.

3. The cell preservation solution according to claim 1, wherein the osmotic pressure stabilizer is one or more of sodium chloride, potassium chloride, disodium hydrogen phosphate, and potassium dihydrogen phosphate.

4. The cell preservation solution according to claim 1, wherein the pH stabilizer is Tris-HCl.

5. The cell preservation solution according to claim 1, wherein the lyoprotectant is one or more of sucrose, glucose, and trehalose.

6. The cell preservation solution according to claim 1, further comprising an antibiotic.

7. The cell preservation solution according to claim 6, wherein the antibiotic is benzylpenicillin.

8. The cell preservation solution according to claim 6, wherein the concentration of the antibiotic is 20-30 ug/mL.

9. Use of a cell preservation solution according to any one of claims 1 to 8 in nucleic acid sampling, wherein the nucleic acid is deoxyribonucleic acid or ribonucleic acid.

10. The method for using a cell preservation solution according to any one of claims 1 to 8, wherein the cell preservation solution is immersed in the cell preservation solution after sampling with a sampling swab, and the cell preservation solution is stored and transported at room temperature for not more than 7 days, stored at 4 ℃ for not more than 28 days, and stored at-80 ℃ for more than 28 days.