CN113755556A - One-step inactivated new coronavirus preserving fluid - Google Patents

Abstract

The invention discloses one-step inactivated new coronavirus preserving fluid. The formula comprises components such as a buffering agent, a chloride salt, a calcium-magnesium ion chelating agent, proteinase K, a non-ionic surfactant, a phenol red indicator, an RNase inhibitor and the like. By utilizing the combined action of the nonionic surfactant and the proteinase K, the coat protein of the new coronavirus is cracked and RNA nucleic acid is exposed in the preservation process, and further the protein combined on the RNA is also dissociated. Therefore, the preservation solution not only has the functions of inhibiting and inactivating viruses, but also omits the steps of extracting and purifying nucleic acid, can be directly adapted to the nucleic acid detection reagent of the domestic one-step method after sampling and preservation, and directly carries out quantitative detection on nucleic acid amplification without purification after the release of the nucleic acid. Effectively shortens the detection time, prevents the problem of biological infectious hazard in the waste treatment process after detection, and reduces the epidemic prevention cost.

Description

One-step inactivated new coronavirus preserving fluid

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a one-step inactivated new coronavirus preservation solution.

Background

2019 the novel coronavirus (2019-nCoV, named after the world health organization in 2020 and 1 month; SARS-CoV-2, named after the international committee for viral classification in 2020 and 2 months and 11 days) is a new strain of coronavirus which has never been found in human body before, and has high infectivity and high concealment. After people are infected with coronavirus, the common signs of the person are respiratory symptoms, fever, cough, shortness of breath, dyspnea and the like. In more severe cases, the infection can lead to pneumonia, severe acute respiratory syndrome, renal failure, and even death. At present, fluorescent quantitative PCR method is mainly adopted in all countries in the world, and gene sequencing method is rarely adopted.

Nucleic acid detection is mainly carried out by adopting a two-step method internationally: firstly, extracting and purifying nucleic acid, and then carrying out nucleic acid amplification quantitative detection.

The early sampling, conveying and storing reagent matched with the nucleic acid detection reagent of the two-step method comprises the following steps: the disposable virus sampling tube containing the non-inactivated virus preservation solution is required to have the characteristic of inactivating viruses aiming at unprotected operators and the prevention of inadvertent infectivity in the sampling, conveying and detecting processes in the later period, and a plurality of inactivated virus preservation solutions are introduced by a plurality of manufacturers in China later, wherein the inactivated virus preservation solutions utilize guanidine salt (guanidine hydrochloride or guanidine isothiocyanate) to extract nucleic acid and inactivate viruses.

Because the two-step nucleic acid detection takes up to several hours, and a user needs to be equipped with a nucleic acid extraction and purification instrument, the new positive suspicion is that a patient is difficult to investigate in time in a place with limited medical resources, and the large-scale screening speed is also influenced under the condition of urgent situation of epidemic situation, so that the epidemic prevention effect is greatly reduced. Later, many manufacturers in China creatively develop a one-step nucleic acid detection kit which does not need a nucleic acid extraction and purification instrument to perform the extraction and purification steps, and at the moment, the guanidine salt inactivation type sample storage solution originally adapted to the two-step nucleic acid detection kit cannot be adapted to the one-step nucleic acid detection reagent. In practical application, the disposable virus sampling tube containing the guanidine salt inactivated sample preserving fluid purchased by domestic medical institutions is matched with a one-step nucleic acid detection reagent, so that false negative and serious consequences of repeated detection test failure are caused in epidemic situations.

Chinese patent CN 112280823A discloses a novel coronavirus RNA sample preservation solution, the formula comprises 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, 0.5-5 w/v% of sorbitol and the balance of solvent. The RNA structure can be stabilized and protected while the RNA is released by the quick splitting virus, and the integrity of the RNA can still be maintained after the RNA structure is placed at high temperature for 10 days. 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. However, the above patent formula only uses a surfactant to achieve the purpose of releasing the RNA nucleic acid of the virus sufficiently in theory, and the insufficient release of the RNA nucleic acid, especially the insufficient dissociation of the protein combined on the RNA, can easily cause the omission of the detection of the positive sample with low virus content, thereby causing serious epidemic prevention accidents. Further, there is no component which effectively inhibits RNA nuclease in the above formulation, and theoretically, the effect of inhibiting degradation of RNA nucleic acid is questionable. Therefore, RNases in the surrounding environment also cause RNA degradation during storage, which affects the RNA nucleic acid storage effect claimed in the patent.

Disclosure of Invention

Aiming at the defects in the prior art, the invention provides a one-step inactivated new coronavirus preserving fluid, which comprises the following components in the formula:

buffering agent

Chlorinated salt

Chelating agent for calcium and magnesium ions

Protease enzyme

Nonionic surfactant

Indicator agent

RNase inhibitors

Ultrapure water

Preferably, the formula of the one-step inactivated new coronavirus preservative solution comprises the following components:

buffer 0.16 wt% -0.8 wt%

Chloride salt 0.37 wt% -0.85 wt%

0.029-0.058 wt% of calcium-magnesium ion chelating agent

Protease 50ppm-100ppm

0.05 wt% -0.3 wt% of nonionic surfactant

Indicator 0.03 wt% -0.06 wt%

The RNase inhibitor concentration is 40-200U

The balance being ultrapure water

The buffer is tris-HCl buffer, PB buffer or citric acid buffer.

Preferably, the buffer is tris-HCl buffer.

The chloride salt is any one of sodium chloride and potassium chloride.

Preferably, the chloride salt is potassium chloride.

The calcium-magnesium ion chelating agent is any one of EDTA, EDTA disodium salt and EDTA tetrasodium salt.

Preferably, the calcium-magnesium ion chelating agent is EDTA.

The protease is any one of proteinase K, trypsin and pepsin.

Preferably, the protease is proteinase K.

The nonionic surfactant is any one of NP-40, tween-20 or TritonX-100.

Preferably, the nonionic surfactant is NP-40.

The indicator is any one of phenol red indicator, neutral red indicator and cresol red indicator.

Preferably, the indicator is a phenol red indicator.

The RNase inhibitor is RNase.

The buffer provides a stable pH environment for the whole new coronavirus nucleic acid; potassium chloride provides potassium ions and a proper osmotic pressure environment for subsequent nucleic acid amplification reaction; the proteinase K has activity in a wider pH range (4-12.5) and at a higher temperature (50-70 ℃), is suitable for separating RNA nucleic acid of the new coronavirus, and has the main function of enzymolysis of structural protein combined on the nucleic acid in RNA extraction so that the RNA is fully released and dissociated in a solution; NP-40 belongs to a mild nonionic surfactant, is mainly used for cracking capsid protein outside the new coronavirus to expose RNA nucleic acid of the new coronavirus, and can further dissociate protein components combined on the RNA by proteinase K so as to achieve the effect of fully exposing RNA nucleic acid monomers and greatly facilitate the quantitative amplification detection of subsequent nucleic acid. RNA nucleases exert RNA nucleic acid degradation destruction effects and must rely on divalent cations (Ca)²⁺With Mg²⁺) EDTA is used as a calcium-magnesium ion chelating agent, so that the activity of RNA nuclease can be blocked by EDTA, and the EDTA has the function of inhibiting RNA nuclease; the RNase is an effective RNase inhibitor, and the EDTA chelating agent and the RNase in the formula jointly act on the RNase to achieve the effect of deeply inhibiting the RNase, effectively prevent the RNA nucleic acid of the new coronavirus from being degraded by the RNase filled in the surrounding environment, greatly improve the storage life of the RNA nucleic acid and greatly prolong the storage time of a positive sample.

Phenol red has a pH indicating function, and is yellow when the pH of the preservation solution is lower than 7.0, which indicates that the performance of the preservation solution is reduced. Meanwhile, the operator can observe a plurality of sampling swabs in the storage tube conveniently (in order to reduce large-scale detection cost in the actual use process, one storage tube is needed to store a plurality of sampling swabs of the examinees).

The invention has the beneficial effects that: the invention relates to a one-step inactivated new coronavirus preservative fluid, which comprises the components of a buffering agent, chloride, a calcium-magnesium ion chelating agent, proteinase K, a non-ionic surfactant, a phenol red indicator, an RNA enzyme inhibitor and the like. Mainly utilizes the combined action of the nonionic surfactant and the proteinase K, and in the preservation process, the coat protein of the new coronavirus is firstly cracked, RNA nucleic acid is exposed, and further the protein combined on the RNA is also dissociated. Therefore, the preservation solution not only has the functions of inhibiting and inactivating viruses, but also omits the steps of extracting and purifying nucleic acid, can be directly adapted to the nucleic acid detection reagent of the domestic one-step method after sampling and preservation, and directly carries out quantitative detection on nucleic acid amplification without purification after the release of the nucleic acid. Effectively shortens the detection time, prevents the problem of biological infectious hazard in the waste treatment process after detection, and reduces the epidemic prevention cost.

Detailed Description

The raw materials used in the examples were as follows:

tris-HCl buffer with a purity of greater than or equal to 99%, Wuhan de sanden Biochemical technology Ltd.

EDTA, ethylenediaminetetraacetic acid, CAS number: 60-00-4, cargo number: s860021, Shandong Nu Ju Biotech Co., Ltd.

Proteinase K, 40U/mg, product number: p92303, shanghai ge to biochemistry technologies, ltd.

Phenol red indicator, CAS number: 143-74-8, Hubei Xin run chemical Co., Ltd.

RNase, 40U/. mu.L, Beijing Sorboard technologies, Inc.

Nonionic surfactant NP-40, Jinan Chengxuan chemical Co., Ltd.

Example 1

The formula of the one-step inactivated new coronavirus preservative fluid comprises the following components:

tris-HCl buffer 0.48 wt%

0.57 wt% of potassium chloride

EDTA 0.029wt％

Proteinase K75 ppm

Nonionic surfactant NP-400.15 wt%

Phenol Red indicator 0.04 wt%

RNase inhibitor concentration was 100U

The balance being ultrapure water

Example 2

The formula of the one-step inactivated new coronavirus preservative fluid comprises the following components:

tris-HCl buffer 0.48 wt%

0.57 wt% of potassium chloride

EDTA 0.029wt％

Nonionic surfactant NP-400.15 wt%

Phenol Red indicator 0.04 wt%

RNase inhibitor concentration was 100U

The balance being ultrapure water

Example 3

The formula of the one-step inactivated new coronavirus preservative fluid comprises the following components:

tris-HCl buffer 0.48 wt%

0.57 wt% of potassium chloride

EDTA0.029wt％

Proteinase K75 ppm

Phenol Red indicator 0.04 wt%

RNase inhibitor concentration was 100U

The balance being ultrapure water

Example 4

The formula of the one-step inactivated new coronavirus preservative fluid comprises the following components:

tris-HCl buffer 0.48 wt%

0.57 wt% of potassium chloride

EDTA 0.029wt％

Proteinase K75 ppm

Nonionic surfactant NP-400.15 wt%

Phenol Red indicator 0.04 wt%

The balance being ultrapure water

Example 5

The formula of the one-step inactivated new coronavirus preservative fluid comprises the following components:

tris-HCl buffer 0.48 wt%

0.57 wt% of potassium chloride

Proteinase K75 ppm

Nonionic surfactant NP-400.15 wt%

Phenol Red indicator 0.04 wt%

RNase inhibitor concentration was 100U

The balance being ultrapure water

Test example 1

And (3) respectively preserving the new coronavirus sample by using 3mL of the inactivated new coronavirus preserving fluid prepared in each embodiment, preserving at room temperature for 3 days and 7 days, respectively performing quantitative fluorescence PCR test detection, and judging the release effect of the inactivated new coronavirus preserving fluid of each embodiment on nucleic acid according to the CT value. Each example tested two different viral loads of a sample of new coronavirus: the viral load of sample a was 200 and the viral load of sample B was 100. Each sample was run in 3 replicates and the average was taken. The test results are shown in Table 1.

Table 1: test of release effect of inactivated new coronavirus preservative fluid on nucleic acid

The smaller the CT value in a certain range, the higher the nucleic acid content in the sample in example 1, the difference between the CT values of the sample A and the sample B is not large, the change rate of the CT value when the sample A and the sample B are stored for 3 days is also the minimum, and the comprehensive effect of the effect of releasing the nucleic acid of the formula storage solution and the stability of the stored sample nucleic acid is the optimal. Compared with example 1, the example 3 has no cleavage of the outer capsid protein of the new coronavirus by NP-40, and the CT value is higher than that of example 1; example 2 does not add proteinase K, RNA binding protein dissociation is not sufficient, so the CT value is also high, embodiment 2 and example 3 release RNA nucleic acid effect is not good. Compared with example 1, example 4 has the highest CT value without adding RNA inhibitory enzyme, and the rate of change between the CT value when stored for 7 days and the CT value when stored for 3 days is the greatest, indicating that the stability of the stored sample nucleic acid is the worst. Example 5 no EDTA was added, which also had an effect on stability.

Claims (10)

1. The one-step inactivated new coronavirus preservative fluid is characterized by comprising the following components in a formula: buffer, chloride salt, calcium-magnesium ion chelating agent, protease, nonionic surfactant, indicator, RNase inhibitor and ultrapure water.

2. The one-step inactivated new coronavirus preservative solution according to claim 1, wherein the formula comprises the following components: 0.16-0.8 wt% of buffering agent, 0.37-0.85 wt% of chloride, 0.029-0.058 wt% of calcium-magnesium ion chelating agent, 50-100 ppm of protease, 0.05-0.3 wt% of nonionic surfactant, 0.03-0.06 wt% of indicator, 40-200U of RNase inhibitor concentration and the balance of ultrapure water.

3. The one-step inactivated type preservation solution for new coronavirus according to claim 1 or 2, wherein the buffer is tris-HCl buffer, PB buffer or citric acid buffer.

4. The one-step inactivated type neocoronaviruse preservation solution according to claim 1 or 2, wherein the chloride salt is any one of sodium chloride and potassium chloride.

5. The one-step inactivated type new coronavirus preservation solution according to claim 1 or 2, wherein the calcium-magnesium ion chelating agent is any one of EDTA, disodium EDTA, and tetrasodium EDTA.

6. The one-step inactivated new coronavirus preservation solution according to claim 1 or 2, wherein the protease is any one of proteinase K, trypsin and pepsin.

7. The one-step inactivated new coronavirus preservative solution according to claim 6, wherein the protease is proteinase K.

8. The one-step inactivated coronavirus storage solution according to claim 1 or 2, wherein the nonionic surfactant is any one of NP-40, tween-20 and triton x-100.

9. The one-step inactivated new coronavirus preservation solution according to claim 1 or 2, wherein the indicator is any one of a phenol red indicator, a neutral red indicator and a cresol red indicator.

10. The one-step inactivated coronavirus storage solution according to claim 1 or 2, wherein the rnase inhibitor is RNasin.