阅读说明：本技术 一种可常温保存的灭活型病毒保存液及其制备方法 (Inactivated virus preservation solution capable of being preserved at normal temperature and preparation method thereof ) 是由 李杨霞 王稀莹 贾俊玲 曾丽 于 2021-08-18 设计创作，主要内容包括：本发明涉及病毒保存液领域,公开了一种可常温保存的灭活型病毒保存液及其制备方法,保存液的原料包括：异硫氰酸胍190～210g/L；柠檬酸钠2～3g/L；十二烷基肌氨酸钠1.8～2.0g/L；HAc-NaAc缓冲液55～65mL/L；二茂铁-谷胱甘肽10～30mg/L；酸碱指示剂30～50 mg/L。本发明在保存液中加入异硫氰酸胍、二茂铁-谷胱甘肽及十二烷基肌氨酸钠,通过各组分的复配可以实现对病毒迅速灭活,并可以维持样本核酸的稳定性,可以在常温下长时间保存不降解,提高了核酸检测的准确性。(The invention relates to the field of virus preservation solution, and discloses inactivated virus preservation solution capable of being preserved at normal temperature and a preparation method thereof, wherein the preservation solution comprises the following raw materials: 190-210 g/L of guanidinium isothiocyanate; 2-3 g/L of sodium citrate; 1.8-2.0 g/L of sodium dodecyl sarcosinate; 55-65 mL/L HAc-NaAc buffer solution; 10-30 mg/L of ferrocene-glutathione; and 30-50 mg/L of an acid-base indicator. According to the invention, guanidine isocyanate, ferrocene-glutathione and sodium dodecyl sarcosinate are added into the preservation solution, and through the compounding of the components, the virus can be quickly inactivated, the stability of sample nucleic acid can be maintained, the preservation solution can be preserved for a long time at normal temperature without degradation, and the accuracy of nucleic acid detection is improved.)

1. An inactivated virus preservation solution capable of being preserved at normal temperature is characterized by comprising the following raw materials:

190-210 g/L of guanidinium isothiocyanate;

2-3 g/L of sodium citrate;

1.8-2.0 g/L of sodium dodecyl sarcosinate;

55-65 mL/L HAc-NaAc buffer solution;

10-30 mg/L of ferrocene-glutathione;

and 30-50 mg/L of an acid-base indicator.

2. The preservative solution for inactivated viruses according to claim 1, wherein the HAc-NaAc buffer solution has a pH of 4.0 to 4.5.

3. The preservative solution for inactivated viruses that can be preserved at room temperature according to claim 1, wherein the acid-base indicator is methyl red.

4. A method for producing the inactivated virus preservation solution according to any one of claims 1 to 3, comprising the steps of:

(1) preparing a sodium citrate solution;

(2) preparing a sodium dodecyl sarcosinate solution;

(3) preparing HAc-NaAc buffer solution;

(4) preparing a mixed solution of guanidine isothiocyanate and ferrocene-glutathione;

(5) adding a sodium citrate solution into a mixed solution of guanidine isothiocyanate and ferrocene-glutathione according to the proportion of the raw materials, adding a sodium dodecyl sarcosinate solution, uniformly mixing, adding an HAc-NaAc buffer solution, adding an acid-base indicator, and fixing the volume by using water; filtering and sterilizing to obtain the inactivated virus preservation solution.

5. The method for preparing an inactivated virus preservation solution according to claim 4, wherein the concentration of the sodium citrate solution in the step (1) is 0.65 to 0.75 mol/L.

6. The method for preparing an inactivated virus preservation solution according to claim 4, wherein the mass fraction of the sarcosyl solution in the step (2) is 8 to 10%.

7. The method for preparing an inactivated virus preservation solution according to claim 4, wherein the concentration of guanidinium isothiocyanate in the mixed solution in the step (4) is 190 to 210g/800 mL.

8. The method for preparing an inactivated virus preservation solution according to claim 4, wherein the sterilization temperature in the step (5) is 120 to 125 ℃ and the sterilization time is 25 to 35 min.

9. The method for preparing an inactivated virus preservation solution according to claim 4, wherein the water used in the steps (1) to (5) is sterile water without an enzyme.

Technical Field

The invention relates to the field of virus preservation solution, in particular to inactivated virus preservation solution capable of being preserved at normal temperature and a preparation method thereof.

Background

Respiratory viral infection is one of the most common diseases in clinic, the etiology is complex, most respiratory viral infections show similar symptoms, a clinician has difficulty in making an accurate diagnosis according to the clinical manifestations of patients, and accurate etiology analysis is not only a basis for accurate diagnosis, but also a basis for reasonably selecting a treatment scheme. The gold standard for viral infection detection is real-time fluorescent quantitative PCR, and nucleic acids (DNA, RNA) in a suspected sample need to be extracted before detection. Due to the time or distance limitation, the sample to be tested generally needs to be stored for a certain period of time or sent to a specific place for detection. The virus preservation solution is used for collecting, preserving and transporting a virus sample, is a liquid which is used for immersing a sampling swab virus sample in a sampling tube and protecting a detected substance of the virus, can be used for collecting a throat swab, a nose swab or a tissue sample of a specific part, and can be used for subsequent clinical experiments such as nucleic acid extraction or purification and the like. The collection and storage of the sample are one of the key steps of pathogen detection, and are one of the important factors influencing the accuracy of the pathogen detection result.

The virus preservation solution mainly comprises a non-inactivated type virus preservation solution and an inactivated type virus preservation solution, wherein the non-inactivated type virus preservation solution does not contain a splitting salt, and simultaneously reserves a protein shell of the virus and virus nucleic acid DNA or RNA, so that the virus has integrity of a protein epitope and nucleic acid in vitro, the detection rate is higher, but certain infectious risk exists during operation, and the safety is low. The inactivated virus preservation solution is added with high-concentration splitting salt, can rapidly and efficiently split and inactivate virus proteins of a sample to be detected, can effectively prevent secondary infection of an operator, contains an RNase inhibitor, can protect virus nucleic acid from being degraded, and is suitable for collecting most virus samples. For example, the patent literature of China discloses "a formula of an inactivated virus preservation solution and a preparation method thereof", and the publication No. CN113186251A includes the following components and the weight of each component is: protein denaturant: 5-20 g of high-concentration guanidine salt, one or more of guanidine hydrochloride and guanidine isothiocyanate; rnase inhibitors: 0.5-2 g; buffering stabilizer: tris 2-6 g; surfactant (b): 1-4 g of sodium dodecyl sarcosinate; acid-base indicator: 0.001-0.01 g; an antifreezing agent: 0.2 to 1 g. The preservation solution is safe and nontoxic, can keep a liquid state at low temperature, is convenient to sample, and can inactivate viruses and protect RNA enzyme from degrading viruses.

However, after the sampling of the existing inactivated preservation solution, the sample is usually required to be preserved at low temperature, so that the sample cannot be preserved for a long time at normal temperature, and the sample is easily degraded at normal temperature, thereby causing false negative and being not beneficial to the preservation of the virus nucleic acid detection sample.

Disclosure of Invention

The invention aims to solve the problems that samples are usually required to be stored at low temperature after the samples of the inactivated type preservation solution in the prior art are sampled and the samples cannot be stored for a long time at normal temperature, and provides the inactivated type virus preservation solution capable of being stored at normal temperature and the preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

an inactivated virus preservation solution capable of being preserved at normal temperature comprises the following raw materials:

the guanidine isothiocyanate is added into the preservation solution, and can rapidly break and inactivate viruses, so that nucleoprotein and nucleic acid are rapidly separated, secondary infection risk is effectively prevented, released RNA enzyme activity is inhibited, integrity of a primary structure of the nucleic acid is ensured, and nucleic acid extraction efficiency is improved. Meanwhile, ferrocene-glutathione is added into the preservation solution, and the sulfhydryl in the glutathione can destroy the disulfide bond in the RNA enzyme protein, so that the RNA enzyme is denatured, and the RNA enzyme and guanidine isothiocyanate jointly act to effectively protect virus nucleic acid from being degraded, improve the detection rate of the virus and ensure the accuracy of a detection result; in addition, glutathione is modified on ferrocene, so that the antibacterial activity of the glutathione can be improved, and the preservation time of the sample at normal temperature after sampling is prolonged. According to the invention, sodium dodecyl sarcosinate is also added as a surfactant, so that the compatibility of each component in the preservation solution and the solubility of nucleic acid in the preservation solution can be improved, and meanwhile, the preservative also has an antibacterial effect; and the buffer solution and the acid-base indicator are added, so that the pH value of the preservation solution can be kept within a certain range, and the stability of the sample is ensured. According to the invention, through the matching of the components and the regulation and control of the dosage of the components, the sample can be stably stored for 30 days at normal temperature, and the accuracy of subsequent nucleic acid detection is ensured.

Preferably, the preparation method of the ferrocene-glutathione comprises the following steps:

A) dissolving ferrocenecarboxylic acid in a mixed solvent of water and acetone, cooling to 0-4 ℃, then sequentially dropwise adding an acetone solution of triethylamine and an acetone solution of ethyl chloroformate, stirring for 25-35 min, then dropwise adding an aqueous solution of sodium azide, stirring for reaction for 60-90 min, extracting an organic phase, and drying to obtain azidocarbonyl ferrocene; wherein the addition amount ratio of the ferrocenecarboxylic acid, the triethylamine, the ethyl chloroformate and the sodium azide is 1g, 0.6-0.8 mL, 0.3-0.5 mL and 0.4-0.5 g;

B) adding azidocarbonyl ferrocene into tert-butyl alcohol, wherein the mass-volume ratio of the azidocarbonyl ferrocene to the tert-butyl alcohol is 19-20 mg:1 mL; carrying out reflux reaction at 75-85 ℃ for 1-2 h, and evaporating the reaction solution to dryness to obtain tert-butyloxy-amino ferrocene;

C) dissolving tert-butyloxy-amino ferrocene in ethyl acetate, introducing dry HCl gas at 0-4 ℃ for 60-90 min, then reacting at room temperature for 30-60 min, and evaporating the reaction solution to dryness to obtain amino modified ferrocene;

D) mixing a 1, 4-dioxane solution of di-tert-butyl dicarbonate with a saturated sodium bicarbonate solution, then adding reduced glutathione, wherein the mass ratio of the di-tert-butyl dicarbonate to the sodium bicarbonate to the reduced glutathione is 1.2-1.3: 0.7-0.8: 1, stirring for reaction for 8-12 h, separating an oil phase, and drying to obtain amino-protected reduced glutathione;

E) adding amino-protected reduced glutathione into dichloromethane for dissolving, cooling to 0-4 ℃, adding triethylamine, adding HBTU, reacting for 60-90 min under heat preservation, and adding amino-modified ferrocene, wherein the addition amount ratio of the amino-protected reduced glutathione to the triethylamine to the HBTU to the amino-modified ferrocene is 310-330 mg:1mL: 400-420 mg: 300-310 mg; continuing to perform heat preservation reaction for 2-3 h, then heating to 25-30 ℃ for reaction for 8-12 h, washing with saturated sodium bicarbonate solution, hydrochloric acid and water in sequence after the reaction is finished, separating an oil phase and drying to obtain tert-butyloxy-ferrocene-glutathione;

F) dissolving tert-butyl oxy-ferrocene-glutathione in ethyl acetate, introducing dry HCl gas at 0-4 ℃ for 60-90 min, then reacting at room temperature for 30-60 min, and evaporating the reaction solution to dryness to obtain the ferrocene-glutathione.

Modifying amino on ferrocene through steps A) -C), then protecting the amino in reduced glutathione through steps D) and E), modifying the reduced glutathione on ferrocene by utilizing the reaction of hydroxyl in the glutathione and the amino on the ferrocene, and finally removing the protection of the amino through step F) to obtain the ferrocene-glutathione.

The invention modifies glutathione on ferrocene, can improve the inhibition effect of glutathione on RNase while maintaining the inhibition effect of glutathione on RNase, and also improves the solubility of ferrocene after the modification of glutathione, so that the obtained ferrocene-glutathione can be added into a preservation solution to be matched with guanidine isothiocyanate and sodium dodecyl sarcosinate, and a sample can be preserved for a long time at normal temperature.

Preferably, the HAc-NaAc buffer solution has a pH of 4.0 to 4.5.

Preferably, the acid-base indicator is methyl red.

The invention also discloses a preparation method of the inactivated virus preservation solution, which is characterized by comprising the following steps:

(1) preparing a sodium citrate solution;

(2) preparing a sodium dodecyl sarcosinate solution;

(3) preparing HAc-NaAc buffer solution;

(4) preparing a mixed solution of guanidine isothiocyanate and ferrocene-glutathione;

(5) adding a sodium citrate solution into a mixed solution of guanidine isothiocyanate and ferrocene-glutathione according to the proportion of the raw materials, adding a sodium dodecyl sarcosinate solution, uniformly mixing, adding an HAc-NaAc buffer solution, adding an acid-base indicator, and fixing the volume by using water; filtering and sterilizing to obtain the inactivated virus preservation solution.

Preferably, the concentration of the sodium citrate solution in the step (1) is 0.65-0.75 mol/L.

Preferably, the mass fraction of the sodium dodecyl sarcosinate solution in the step (2) is 8-10%.

Preferably, the concentration of the guanidinium isothiocyanate in the mixed solution in the step (4) is 190-210 g/800 mL.

Preferably, the sterilization temperature in the step (5) is 120-125 ℃, and the sterilization time is 25-35 min.

Preferably, the water used in steps (1) to (5) is enzyme-free sterile water.

Therefore, the invention has the following beneficial effects:

(1) safety: the pathogen is quickly inactivated, and the biological safety is guaranteed;

(2) maintaining stability of sample nucleic acids: the pathogen nucleic acid can be stored for 30 days at normal temperature without degradation, and the accuracy of subsequent nucleic acid detection is guaranteed;

(3) the application range is wide: is suitable for most extraction reagents in the market;

(4) the storage period is long: the effective period can reach 2 years (most of similar products sold in the market are 1 to 1.5 years).

Drawings

FIG. 1 is a graph showing the test results of the effect of the preservation solution on the detection rate of the lowest concentration sample in example 1.

Fig. 2 is a graph showing the results of testing the effect of the preservation solution of comparative example 1 on the detection rate of the lowest concentration sample.

Detailed Description

The invention is further described with reference to the following detailed description and accompanying drawings.

In the present invention, all the raw materials are commercially available or commonly used in the industry, and the methods in the following examples are conventional in the art unless otherwise specified.

Example 1:

an inactivated virus preservation solution capable of being preserved at normal temperature comprises the following raw materials:

the preparation method of the ferrocene-glutathione comprises the following steps:

A) dissolving ferrocenecarboxylic acid in a mixed solvent of water and acetone in a volume ratio of 3:10, cooling to 0 ℃, then sequentially dropwise adding an acetone solution of triethylamine and an acetone solution of ethyl chloroformate, stirring for 30min, then dropwise adding a sodium azide aqueous solution, stirring for reacting for 80min, then extracting an organic phase, and drying to obtain azidocarbonyl ferrocene; wherein the addition amount ratio of ferrocenecarboxylic acid, triethylamine, ethyl chloroformate and sodium azide is 1g:0.7mL:0.4mL:0.45 g;

B) adding azidocarbonyl ferrocene into tert-butyl alcohol, wherein the mass-volume ratio of the azidocarbonyl ferrocene to the tert-butyl alcohol is 19.5mg:1 mL; carrying out reflux reaction at 80 ℃ for 1.5h, and evaporating the reaction solution to dryness to obtain tert-butyloxy-amino ferrocene;

C) dissolving tert-butyloxy-amino ferrocene in ethyl acetate, introducing dried HCl gas at 0 ℃ for 80min, then reacting at room temperature for 40min, and evaporating the reaction solution to dryness to obtain amino modified ferrocene;

D) mixing a 1, 4-dioxane solution of di-tert-butyl dicarbonate with a saturated sodium bicarbonate solution, then adding reduced glutathione, wherein the mass ratio of the di-tert-butyl dicarbonate to the sodium bicarbonate to the reduced glutathione is 1.25:0.75:1, stirring for reaction for 10 hours, separating an oil phase, and drying to obtain amino-protected reduced glutathione;

E) adding amino-protected reduced glutathione into dichloromethane for dissolving, cooling to 0 ℃, adding triethylamine, adding HBTU, reacting for 70min under heat preservation, and adding amino-modified ferrocene, wherein the addition amount ratio of the amino-protected reduced glutathione to the triethylamine to the HBTU to the amino-modified ferrocene is 320mg:1mL:410mg:305 mg; continuing to perform heat preservation reaction for 2.5h, heating to 26 ℃ again to perform reaction for 10h, washing with saturated sodium bicarbonate solution, hydrochloric acid and water in sequence after the reaction is finished, separating oil phase and drying to obtain tert-butyloxy-ferrocene-glutathione;

F) dissolving tert-butyloxy-ferrocene-glutathione in ethyl acetate, introducing dry HCl gas at 0 ℃ for 70min, then reacting at room temperature for 40min, and evaporating the reaction solution to dryness to obtain the ferrocene-glutathione.

The preparation method of the fire extinguishing type virus preservation solution comprises the following steps:

(1) preparing 0.65mol/L sodium citrate solution: adding 191.16g of trisodium citrate dihydrate into 200mL of enzyme-free sterile water for dissolving, adjusting the pH value of the solution to be more than or equal to 7, adding the enzyme-free sterile water for fixing the volume to 1L to obtain 0.65mol/L sodium citrate solution, placing the sodium citrate solution on a label, and storing at the temperature of 2-8 ℃;

(2) preparing 8 wt% of sodium dodecyl sarcosinate solution: adding 80g of sodium dodecyl sarcosinate into enzyme-free sterile water for dissolving, and fixing the volume to 1L to obtain 8 wt% of sodium dodecyl sarcosinate solution, putting the solution on a label and storing the solution at 2-8 ℃;

(3) preparing a HAc-NaAc buffer solution with a pH of 4.0:

I) preparing a 2.0mol/L sodium acetate solution: 299.37g of sodium acetate trihydrate is added into enzyme-free sterile water to be dissolved, and the volume is determined to be 1L, so that 2.0mol/L sodium acetate solution is obtained;

II) preparing 2.0mol/L acetic acid solution: adding 118mL of glacial acetic acid into enzyme-free sterile water for dissolving, and fixing the volume to 1L to obtain 2.0mol/L acetic acid solution;

III) mixing 9mL of 2.0mol/L sodium acetate solution with 41mL of 2.0mol/L acetic acid solution, and uniformly stirring to obtain a HAc-NaAc buffer solution with the pH value of 4.0;

(4) preparing a mixed solution of guanidine isothiocyanate and ferrocene-glutathione: adding 200g of guanidinium isothiocyanate and 20mg of ferrocene-glutathione into 800mL of enzyme-free sterile water for dissolving to obtain a mixed solution of the guanidinium isothiocyanate and the ferrocene-glutathione;

(5) adding 13.6mL of 0.65mol/L sodium citrate solution into the mixed solution of the guanidinium isothiocyanate and the ferrocene-glutathione, uniformly mixing, adding 24.4mL of 8 wt% sodium dodecyl sarcosinate solution, and uniformly mixing; adding 60mL of HAc-NaAc buffer solution with the pH value of 4.0, uniformly mixing, adding methyl red, and fixing the volume to 1L by using sterile water without enzyme; filtering, and sterilizing at 121 deg.C for 30min to obtain inactivated virus preservation solution.

Example 2:

an inactivated virus preservation solution capable of being preserved at normal temperature comprises the following raw materials:

the preparation method of the ferrocene-glutathione comprises the following steps:

A) dissolving ferrocenecarboxylic acid in a mixed solvent of water and acetone in a volume ratio of 3:10, cooling to 4 ℃, then sequentially dropwise adding an acetone solution of triethylamine and an acetone solution of ethyl chloroformate, stirring for 25min, then dropwise adding a sodium azide aqueous solution, stirring for reacting for 90min, then extracting an organic phase, and drying to obtain azidocarbonyl ferrocene; wherein the addition amount ratio of ferrocenecarboxylic acid, triethylamine, ethyl chloroformate and sodium azide is 1g:0.6mL:0.3mL:0.4 g;

B) adding azidocarbonyl ferrocene into tert-butyl alcohol, wherein the mass-volume ratio of the azidocarbonyl ferrocene to the tert-butyl alcohol is 19mg:1 mL; carrying out reflux reaction for 2h at 75 ℃, and evaporating the reaction solution to dryness to obtain tert-butyloxy-amino ferrocene;

C) dissolving tert-butyloxy-amino ferrocene in ethyl acetate, introducing dried HCl gas at 4 ℃ for 90min, then reacting at room temperature for 60min, and evaporating the reaction solution to dryness to obtain amino modified ferrocene;

D) mixing a 1, 4-dioxane solution of di-tert-butyl dicarbonate with a saturated sodium bicarbonate solution, then adding reduced glutathione, wherein the mass ratio of the di-tert-butyl dicarbonate to the sodium bicarbonate to the reduced glutathione is 1.2:0.7:1, stirring for reacting for 8 hours, separating an oil phase, and drying to obtain amino-protected reduced glutathione;

E) adding amino-protected reduced glutathione into dichloromethane for dissolving, cooling to 4 ℃, adding triethylamine, adding HBTU, reacting for 90min under heat preservation, and adding amino-modified ferrocene, wherein the addition amount ratio of the amino-protected reduced glutathione to the triethylamine to the HBTU to the amino-modified ferrocene is 310mg:1mL:400mg:300 mg; continuing to perform heat preservation reaction for 3 hours, heating to 25 ℃ for reaction for 12 hours, washing with saturated sodium bicarbonate solution, hydrochloric acid and water in sequence after the reaction is finished, separating oil phase and drying to obtain tert-butyloxy-ferrocene-glutathione;

F) dissolving tert-butyloxy-ferrocene-glutathione in ethyl acetate, introducing dried HCl gas at 4 ℃ for 90min, then reacting at room temperature for 60min, and evaporating the reaction solution to dryness to obtain the ferrocene-glutathione.

The preparation method of the fire extinguishing type virus preservation solution comprises the following steps:

(1) preparing 0.7mol/L sodium citrate solution: adding 205.87g of trisodium citrate dihydrate into 200mL of enzyme-free sterile water for dissolving, adjusting the pH value of the solution to be more than or equal to 7, adding the enzyme-free sterile water for fixing the volume to 1L to obtain 0.7mol/L sodium citrate solution, placing the sodium citrate solution on a label, and storing at the temperature of 2-8 ℃;

(2) preparing 9 wt% of sodium dodecyl sarcosinate solution: adding 90g of sodium dodecyl sarcosinate into enzyme-free sterile water for dissolving, and fixing the volume to 1L to obtain 9 wt% of sodium dodecyl sarcosinate solution, putting the solution on a label and storing the solution at 2-8 ℃;

(3) preparing a HAc-NaAc buffer solution with a pH of 4.0:

I) preparing a 2.0mol/L sodium acetate solution: 299.37g of sodium acetate trihydrate is added into enzyme-free sterile water to be dissolved, and the volume is determined to be 1L, so that 2.0mol/L sodium acetate solution is obtained;

II) preparing 2.0mol/L acetic acid solution: adding 118mL of glacial acetic acid into enzyme-free sterile water for dissolving, and fixing the volume to 1L to obtain 2.0mol/L acetic acid solution;

III) mixing 9mL of 2.0mol/L sodium acetate solution with 41mL of 2.0mol/L acetic acid solution, and uniformly stirring to obtain a HAc-NaAc buffer solution with the pH value of 4.0;

(4) preparing a mixed solution of guanidine isothiocyanate and ferrocene-glutathione: adding 190g of guanidinium isothiocyanate and 30mg of ferrocene-glutathione into 800mL of enzyme-free sterile water for dissolving to obtain a mixed solution of the guanidinium isothiocyanate and the ferrocene-glutathione;

(5) adding 11mL of 0.7mol/L sodium citrate solution into the mixed solution of the guanidinium isothiocyanate and the ferrocene-glutathione, uniformly mixing, adding 20mL of 9 wt% sodium dodecyl sarcosinate solution, and uniformly mixing; adding 55mL of HAc-NaAc buffer solution with the pH value of 4.0, uniformly mixing, adding methyl red, and fixing the volume to 1L by using sterile water without enzyme; filtering, and sterilizing at 120 deg.C for 35min to obtain inactivated virus preservation solution.

Example 3:

an inactivated virus preservation solution capable of being preserved at normal temperature comprises the following raw materials:

the preparation method of the ferrocene-glutathione comprises the following steps:

A) dissolving ferrocenecarboxylic acid in a mixed solvent of water and acetone in a volume ratio of 3:10, cooling to 0 ℃, then sequentially dropwise adding an acetone solution of triethylamine and an acetone solution of ethyl chloroformate, stirring for 35min, then dropwise adding a sodium azide aqueous solution, stirring for reacting for 60min, then extracting an organic phase, and drying to obtain azidocarbonyl ferrocene; wherein the addition amount ratio of ferrocenecarboxylic acid, triethylamine, ethyl chloroformate and sodium azide is 1g:0.8mL:0.5mL:0.5 g;

B) adding azidocarbonyl ferrocene into tert-butyl alcohol, wherein the mass-volume ratio of the azidocarbonyl ferrocene to the tert-butyl alcohol is 20mg:1 mL; reflux reaction is carried out for 1h at the temperature of 85 ℃, and the reaction solution is evaporated to dryness to obtain tert-butyloxy-amino ferrocene;

C) dissolving tert-butyloxy-amino ferrocene in ethyl acetate, introducing dried HCl gas at 0 ℃ for 60min, then reacting at room temperature for 30min, and evaporating the reaction solution to dryness to obtain amino modified ferrocene;

D) mixing a 1, 4-dioxane solution of di-tert-butyl dicarbonate with a saturated sodium bicarbonate solution, then adding reduced glutathione, wherein the mass ratio of the di-tert-butyl dicarbonate to the sodium bicarbonate to the reduced glutathione is 1.3:0.8:1, stirring for reaction for 12 hours, separating an oil phase, and drying to obtain amino-protected reduced glutathione;

E) adding amino-protected reduced glutathione into dichloromethane for dissolving, cooling to 0 ℃, adding triethylamine, adding HBTU, reacting for 60min under heat preservation, and adding amino-modified ferrocene, wherein the addition amount ratio of the amino-protected reduced glutathione to the triethylamine to the HBTU to the amino-modified ferrocene is 330mg:1mL:420mg:310 mg; continuing to perform heat preservation reaction for 2 hours, heating to 30 ℃ again to perform reaction for 8 hours, washing with saturated sodium bicarbonate solution, hydrochloric acid and water in sequence after the reaction is finished, separating an oil phase, and drying to obtain tert-butyloxy-ferrocene-glutathione;

F) dissolving tert-butyloxy-ferrocene-glutathione in ethyl acetate, introducing dry HCl gas at 0 ℃ for 60min, then reacting at room temperature for 30min, and evaporating the reaction solution to dryness to obtain the ferrocene-glutathione.

The preparation method of the fire extinguishing type virus preservation solution comprises the following steps:

(1) preparing 0.75mol/L sodium citrate solution: adding 220.57g of trisodium citrate dihydrate into 200mL of enzyme-free sterile water for dissolving, adjusting the pH value of the solution to be more than or equal to 7, adding the enzyme-free sterile water for fixing the volume to 1L to obtain 0.75mol/L sodium citrate solution, placing the sodium citrate solution on a label, and storing at the temperature of 2-8 ℃;

(2) preparing a 10 wt% sodium dodecyl sarcosinate solution: adding 100g of sodium dodecyl sarcosinate into enzyme-free sterile water for dissolving, and fixing the volume to 1L to obtain 10 wt% of sodium dodecyl sarcosinate solution, putting the solution on a label, and storing at 2-8 ℃;

(3) preparing HAc-NaAc buffer solution with pH 4.5:

I) preparing a 2.0mol/L sodium acetate solution: 299.37g of sodium acetate trihydrate is added into enzyme-free sterile water to be dissolved, and the volume is determined to be 1L, so that 2.0mol/L sodium acetate solution is obtained;

II) preparing 2.0mol/L acetic acid solution: adding 118mL of glacial acetic acid into enzyme-free sterile water for dissolving, and fixing the volume to 1L to obtain 2.0mol/L acetic acid solution;

III) mixing 17.7mL of 2.0mol/L sodium acetate solution with 32.3mL of 2.0mol/L acetic acid solution, and uniformly stirring to obtain a HAc-NaAc buffer solution with the pH value of 4.5;

(4) preparing a mixed solution of guanidine isothiocyanate and ferrocene-glutathione: adding 210g of guanidinium isothiocyanate and 10mg of ferrocene-glutathione into 800mL of enzyme-free sterile water for dissolving to obtain a mixed solution of the guanidinium isothiocyanate and the ferrocene-glutathione;

(5) adding 15.5mL of 0.75mol/L sodium citrate solution into the mixed solution of the guanidinium isothiocyanate and the ferrocene-glutathione, uniformly mixing, adding 20mL of 10 wt% sodium dodecyl sarcosinate solution, and uniformly mixing; adding 65mL of HAc-NaAc buffer solution with the pH value of 4.5, uniformly mixing, adding methyl red, and fixing the volume to 1L by using sterile water without enzyme; filtering, and sterilizing at 125 deg.C for 25min to obtain inactivated virus preservation solution.

Comparative example 1 (without ferrocene-glutathione addition):

an inactivated virus preservation solution, which comprises the following raw materials:

the fire-fighting virus preservation solution was prepared in the same manner as in example 1.

Comparative example 2 (ferrocene-glutathione addition too much):

an inactivated virus preservation solution, which comprises the following raw materials:

the fire-fighting virus preservation solution was prepared in the same manner as in example 1.

Comparative example 3 (no ferrocene modification on glutathione):

an inactivated virus preservation solution, which comprises the following raw materials:

the fire-fighting virus preservation solution was prepared in the same manner as in example 1.

Comparative example 4 (ferrocene mixed directly with glutathione):

an inactivated virus preservation solution, which comprises the following raw materials:

when the fire extinguishing type virus preservation solution is prepared, 200g of guanidine isothiocyanate, 12.5mg of glutathione and 7.5mg of ferrocene are added into 800mL of enzyme-free sterile water in the step (4) to be dissolved, so as to obtain a mixed solution of guanidine isothiocyanate, ferrocene and glutathione; the rest is the same as in example 1.

Comparative example 5 (too little guanidinium isothiocyanate added):

an inactivated virus preservation solution, which comprises the following raw materials:

the fire-fighting virus preservation solution was prepared in the same manner as in example 1.

Comparative example 6 (guanidinium isothiocyanate added too much):

an inactivated virus preservation solution, which comprises the following raw materials:

the fire-fighting virus preservation solution was prepared in the same manner as in example 1.

Comparative example 7 (too little sarcosyl added):

an inactivated virus preservation solution, which comprises the following raw materials:

comparative example 8 (extra sarcosyl addition):

an inactivated virus preservation solution, which comprises the following raw materials:

the performance of the inactivated virus preservation solutions in the above examples and comparative examples was tested:

1. influence of different preserving fluids on detection effect

4 positive pharyngeal swab samples and 2 negative pharyngeal swab samples (numbers 2M-1 to 2M-6) were selected, diluted 100-fold with the preservation solutions in the examples and comparative examples, respectively, and the sample nucleic acids were extracted using a nucleic acid extraction kit and detected using a PCR kit. Differences in Ct values of diluted samples of different preservation solutions were analyzed, and the effect of each preservation solution on the detection effect was compared, with the results shown in table 1.

Table 1: sample Ct value test results (instrument model ABI 7500).

As can be seen from table 1, the Ct values of the samples of the preservation solutions prepared by using the raw materials and the mixture ratios in the invention in examples 1 to 3 are small, while the Ct values are increased by changing the types or the mixture ratios of the raw materials of the preservation solutions in the comparative examples, which affects the detection effect.

2. Effects of different preserving solutions on the detection efficiency of the minimum detection limit concentration samples, 2 positive pharyngeal swab samples (numbers 2M-7 and 2M-8) were diluted 100 times by using the preserving solutions in example 1 and comparative example 1, so that the diluted samples were at the minimum detection limit concentration, the detection was repeated 20 times respectively, the minimum detection limit was verified, the detection instrument was an ABI7500 real-time fluorescence quantitative PCR instrument, and the effects of the preserving solutions on the detection rate of the minimum detection limit concentration samples were detected, and the results are shown in Table 2, Table 3, and FIGS. 1 and 2.

Table 2: the effect of the preservative solution in example 1 on the detection effect of the sample at the lowest detection limit concentration was tested.

Table 3: the effect of the preservation solution in comparative example 1 on the detection effect of the sample with the lowest detection limit concentration was tested.

As can be seen from the results in table 2 and table 3 and fig. 1 and fig. 2, the samples with the lowest detection limit concentration diluted by the sample preservation solutions in example 1 and comparative example 1 were all detected at 100%, but the Ct of the preservation solution in example 1 was about 1 time earlier than that in comparative example 1.

3. Effect period research for maintaining nucleic acid stability by different preservation solutions

3 samples (numbers 2M-9, 2M-10 and 2M-11) of viruses with different concentration levels were selected, each sample was divided into 30 portions, each portion was divided into 20. mu.L, 180. mu.L of the sample preservative solutions of examples and comparative examples were added to each portion, and the diluted samples were placed in a 37 ℃ incubator, taken out on days 7, 14, 21 and 30, respectively, and compared with the detection results of the samples stored at-20 ℃. The results are shown in Table 4.

Table 4: the preservation solution maintains the test result (Ct value) of nucleic acid stability expiration date research.

As can be seen from table 4, the preserved sample of the preservation solution prepared by using the raw materials and the mixture ratio in the invention in example 1 can be stably stored for 30 days at 37 ℃, while the ferrocene-glutathione is not added to the preservation solution in comparative example 1, the Ct value is significantly increased compared with that in example 1, and the preserved sample can be stored for only 7 days at 37 ℃; in the comparative example 2, the addition amount of the ferrocene-glutathione is too much, and is beyond the range of the invention, the Ct value is increased, and the storage time of the sample at normal temperature is shortened to 14 days; the glutathione which is not modified by ferrocene is added into the preservation solution of the comparative example 3, so that the detection effect is reduced compared with that in the example 1, and the storage time of the sample at normal temperature can not reach 30 days; in the comparative example 4, the glutathione and the ferrocene are directly mixed and added into the preservation solution, the glutathione and the ferrocene are not bonded, the detection effect of the sample is greatly influenced due to the small solubility of the ferrocene, and the Ct value is obviously increased; the storage stability of the sample is also significantly reduced; the guanidine isothiocyanate in the preservation solution of the comparative example 5 is added too little, the Ct value is large, the detection effect of the sample is poor, and the inhibition effect of the guanidine isothiocyanate on the RNase is weakened, so that the preservation time of the sample is shortened; in comparative example 6, too much guanidinium isothiocyanate is added, which also affects the preservation time of the sample at normal temperature; in comparative example 7, the addition amount of the surfactant, namely sodium lauryl sarcosine, is too small, the solubilizing ability of nucleic acid is reduced, and the bacteriostatic performance of the preservation solution is reduced, so that the detection effect and the preservation time of the sample are reduced; in comparative example 8, the addition amount of sarcosyl was too large, which was not favorable for stable storage of the sample. In conclusion, the types and the proportions of the components in the preservation solution have great influence on the detection effect and the preservation time of the sample, and the detection effect of the sample can be effectively improved and the preservation time of the sample at normal temperature can be prolonged under the combined action of the components, so that the accuracy of nucleic acid detection is improved.