阅读说明：本技术 一种含有绿色染料的灭活型病毒核酸保存液 (Inactivated virus nucleic acid preservation solution containing green dye ) 是由 张冬平 张珉艳 黄艳 于 2021-07-26 设计创作，主要内容包括：一种含有绿色染料的灭活型病毒核酸保存液,主要包含以下主要成分：缓冲稳定剂10-1000mM/L,蛋白变性剂100-8000mM/L,核酸酶抑制剂1-100mM/L,表面活性剂1-100mM/L,绿色染料0.05-1mM/L。本保存液在pH6-8范围内呈现绿色,可以清晰地指示液面位置,并且可以与现有的非灭活型病毒核酸保存液区分开来。本保存液可直接灭活病毒,保护医务人员,减少感染风险,能够抑制核酸酶活性,保存病毒核酸片段。可采集咽拭子,鼻拭子或特定部位组织样本,可储存和运输样本用于后续的核酸提取。(An inactivated virus nucleic acid preservation solution containing a green dye mainly comprises the following main components: 10-1000mM/L of buffer stabilizer, 100mM/L of protein denaturant, 8000mM/L of nuclease inhibitor, 1-100mM/L of surfactant and 0.05-1mM/L of green dye. The preservation solution is green in pH6-8, can clearly indicate the liquid surface position, and can be distinguished from the existing non-inactivated virus nucleic acid preservation solution. The preservative fluid can directly inactivate viruses, protect medical staff, reduce infection risks, inhibit nuclease activity and preserve virus nucleic acid fragments. Pharyngeal swabs, nasal swabs or site-specific tissue samples can be collected, and samples can be stored and transported for subsequent nucleic acid extraction.)

1. An inactivated virus nucleic acid preservation solution containing a green dye, which is characterized in that: mainly comprises 10-1000mM/L of buffer stabilizer, 100-8000mM/L of protein denaturant, 1-100mM/L of nuclease inhibitor, 1-100mM/L of surfactant and 0.05-1mM/L of green dye.

2. The preservation solution for inactivated viral nucleic acid according to claim 1, wherein the preservation solution comprises a green dye, and the green dye is selected from the group consisting of: the buffer stabilizer is one or more of sodium acetate, trihydroxymethyl aminomethane, sodium citrate, glycine and mannose.

3. The preservation solution for inactivated viral nucleic acid according to claim 1, wherein the preservation solution comprises a green dye, and the green dye is selected from the group consisting of: the protein denaturant is one or more of urea, guanidinium isothiocyanate, guanidinium hydrochloride, and ammonium sulfate.

4. The preservation solution for inactivated viral nucleic acid according to claim 1, wherein the preservation solution comprises a green dye, and the green dye is selected from the group consisting of: the surfactant is one or more of sodium dodecyl sulfate, Triton X-100, and Tween 20.

5. The preservation solution for inactivated viral nucleic acid according to claim 1, wherein the preservation solution comprises a green dye, and the green dye is selected from the group consisting of: the nuclease inhibitor is one or more of Ethylene Diamine Tetraacetic Acid (EDTA), ethylene glycol-bis- (2-aminoethylether) tetraacetic acid (EGTA) and Cyclohexane Diamine Tetraacetic Acid (CDTA).

6. The preservation solution for inactivated viral nucleic acid according to claim 1, wherein the preservation solution comprises a green dye, and the green dye is selected from the group consisting of: the green dye is fast green (FCF).

Technical Field

The invention relates to the field of biomedical technology, in particular to an inactivated virus nucleic acid preservation solution containing a green dye.

Background

At present, the most important detection method for infection of new coronavirus and the like is nucleic acid detection. The first step of nucleic acid detection is sample collection, which is to put nasopharyngeal swab, sputum, alveolar lavage fluid and feces of a subject into a virus preservation solution for transportation or preservation. At present, virus preservation liquid mainly comprises two types: the non-inactivated virus preservation solution mainly ensures the integrity of the virus; the inactivated virus preservation solution mainly inactivates viruses but ensures the integrity of nucleic acid thereof. In the actual operation process, the inactivated virus preservation solution is popular with first-line medical care personnel because of high safety.

Originally, inactivated virus preservation solutions were generally colorless liquids, and it was not easy to observe the liquid surface position, and even leakage sometimes occurred without detection. Therefore, Wei-Jian-Wei-Jian-Wei has a requirement for the new crown nucleic acid storage solution to have a color that is easy to observe and identify. Based on the requirement, manufacturers add phenol red reagent into the inactivated virus preservation solution to change the color of the preservation solution into red. However, the addition of phenol red to an inactivated virus preservation solution has the following disadvantages: first, the color of the non-inactivated virus preservation solution is the same, and the solution is easy to be confused. The main component of the prior non-inactivated virus preservation solution is Hanks solution, which contains phenol red as an instant pH indicator. Many manufacturers do not explicitly indicate on the collection tube whether the preservation fluid is inactivated or not, and confusion is likely to occur if both are red in color. The two preservation solutions have some differences in subsequent nucleic acid extraction and PCR reaction operations, for example, the sample preserved in the inactivated virus preservation solution must be subjected to nucleic acid extraction, and a non-extraction means such as a nucleic acid releasing agent cannot be used. Secondly, the color of the inactivated virus preservation solution is unstable when phenol red is added. Phenol red acts as a ready-to-use pH indicator, and in non-inactivated virus preservation solutions: if the storage tube is used or contaminated, the pH of the solution in the tube drops, causing the phenol red color to change from red to yellow. The inactivated virus preservation solution can work in a wide pH range. For example, the inactivated virus preservation solution with guanidine isocyanate as the main component can well inactivate viruses and preserve nucleic acid within the pH range of 5-8. In this pH range, phenol red will appear yellow, orange, pink and red. Although such a color difference does not affect the use, it may cause troubles to first-line medical staff and subjects.

Therefore, there is an urgent need to develop a novel inactivated virus preservation solution having the following characteristics: (1) effectively inactivating viruses and preserving viral nucleic acids; (2) the dye is added to meet the requirement of Weijian of 'color easy to observe and identify', the color of the dye is different from the red color of phenol red, and the color of the dye is not changed by the change of pH; (3) the nucleic acid extraction and the fluorescent quantitative PCR reaction are not influenced after the dye is added.

Disclosure of Invention

Therefore, the inactivated virus nucleic acid preservation solution containing the green dye can be transported at normal temperature without low temperature, the sample is immediately inactivated after entering the preservation solution after the sample is collected, and RNA cannot be degraded within several days at normal temperature. And further, the technical improvement is made, the preservation solution is green, the liquid level position and the liquid leakage can be conveniently observed, and the preservation solution effect is not influenced.

The preserving fluid of the invention comprises: 10-1000mM/L of buffer stabilizer, 100mM/L of protein denaturant, 8000mM/L of nuclease inhibitor, 1-100mM/L of surfactant and 0.05-1mM/L of green dye.

In the above technical solution, further elaboration is as follows: the buffer stabilizer is one or more of sodium acetate, trihydroxymethyl aminomethane, sodium citrate, glycine and mannitol.

In the above technical solution, further elaboration is as follows: the protein denaturant is one or more of guanidine hydrochloride, guanidine isothiocyanate and urea.

In the above technical solution, further elaboration is as follows: the nuclease inhibitor is one or more of Ethylene Diamine Tetraacetic Acid (EDTA), ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA), diethyl pyrophosphate, vanadyl ribonucleic acid complex, DTT and diatomite.

In the above technical solution, further elaboration is as follows: the surfactant is one or more of Sodium Dodecyl Sulfate (SDS), ethyl phenyl polyethylene glycol (NP-40), Cetyl Trimethyl Ammonium Bromide (CTAB) and triton X-100.

In the above technical solution, further elaboration is as follows: the green dye is fast green (FCF).

The preservation solution can effectively inactivate viruses, protect the safety of medical personnel, prevent nucleic acid degradation in a normal-temperature environment, and is suitable for normal-temperature transportation after sampling. This liquid that preserves contains green dyestuff and admittedly green, can help medical personnel to discern the liquid level position clearly, the follow-up experiment operation of being convenient for.

Drawings

FIG. 1 shows the virus-inactivating effect of the inactivated virus nucleic acid preservation solution containing a green dye according to the present invention. From left to right: the normal saline and the IBV are inoculated to the chicken embryo, the preservation solution and the IBV are inoculated to the chicken embryo in the embodiment, and the preservation solution and the IBV are inoculated to the chicken embryo in the embodiment.

FIG. 2 shows the effect of the preservation solution for inactivated viral nucleic acid containing a green dye according to the present invention on preservation of viral nucleic acid.

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.

The first embodiment is as follows:

preparation of a preservation solution: 472.64g of guanidinium isothiocyanate, 7.5g of disodium citrate, 0.75g of disodium ethylene diamine tetraacetate, Triton X-1002 mL and 0.079g of fast green are weighed, double distilled water is added to the mixture to be constant volume of 1L, so that the final concentration of the guanidinium isothiocyanate is 4M/L, the final concentration of the disodium citrate is 25mM/L, the final concentration of the disodium ethylene diamine tetraacetate is 2mM/L, the final concentration of the Triton X-100 is 8mM/L and the final concentration of the fast green is 0.1 mM/L. After preparation, the solution was sterilized through a 0.25 μm filter.

Example two:

preparation of a preservation solution: 24g of urea, 32.8g of anhydrous sodium acetate, 3.7g of ethylene diamine tetraacetic acid, 2.88g of lauryl sodium sulfate, 2.42g of tris (hydroxymethyl) aminomethane and 0.079g of fast green are weighed, and double distilled water is added to the mixture to a constant volume of 1L, so that the final concentration of urea is 400mM/L, the final concentration of anhydrous sodium acetate is 400mM/L, the final concentration of disodium ethylene diamine tetraacetic acid is 10mM/L, the final concentration of lauryl sodium sulfate is 10.4mM/L, the final concentration of tris (hydroxymethyl) aminomethane is 20mM/L and the final concentration of fast green is 0.1 mM/L. After preparation, the solution was sterilized through a 0.25 μm filter.

Example three:

evaluation of the effect of the preservative fluid of the present invention in inactivating viruses: adding the prepared avian Infectious Bronchitis Virus (IBV) QXL87 strain seed virus into a preservation solution, and mixing the solution according to the ratio of 1 (virus stock solution): 10 (preservation solution), and inactivating at room temperature of 18-26 deg.C for 30 min; and inoculating the inactivated virus solution to SPF chick embryos for inactivation test. As shown in fig. 1, the inactivated virus nucleic acid preservation solutions containing green dyes in the first and second embodiments of the present invention can well inactivate IBV viruses, so that chick embryos can normally develop, while normal saline in a control group cannot inactivate IBV viruses, so that chick embryos die after inoculation, and the development of the chick embryos stops.

Example four:

evaluation of the effect of the preservative solution of the present invention in preserving viral nucleic acid: the IBV virus culture stock solution was diluted with 3 different kinds of preservation solutions (colorless guanidine salt nucleic acid preservation solution, preservation solution of the inventive example II) by a factor of 10 for 3 gradients. And respectively storing the diluted virus samples at room temperature for 7 days, then sampling, directly extracting RNA, and detecting by using an IBV virus nucleic acid detection kit after extraction. As shown in fig. 2, the colorless guanidine salt nucleic acid preservative solution and the preservative solution according to the embodiment of the present invention can effectively inactivate viruses in both the preservative solution according to the embodiment of the present invention and the green dye has no influence on the virus inactivation effect of the inactivated preservative solution.