A virus sample preservative and a method of preparing the same

Abstract

The application discloses a virus sample preservative and a preparation method thereof. The virus sample preservative comprises the following components: 50-60 mM of lithium dodecyl sulfate, 9-20 mM of anhydrous sodium dihydrogen phosphate, 9-20 mM of disodium hydrogen phosphate dodecahydrate, 0.5-1.5 mM of ethylenediaminetetraacetic acid, 0.5-3 mM of thio-geraniol, 0.005-0.03 v / v % of phenol red and water. The sample preservative can be used for collecting, transporting and storing virus samples, can effectively avoid degradation of virus nucleic acid, is beneficial to subsequent nucleic acid extraction or purification and other clinical tests, and can store the sample for more than 72 hours under the condition of 37 DEG C, can store the sample for more than 7 days under normal temperature, and can store the sample for a long time under the condition of-20 DEG C or below.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a virus sample preservative and a preparation method thereof. BACKGROUND

[0002] The virus sample preservative is divided into inactivated type and non-inactivated type. For virus samples with high infectivity and high pathogenicity such as COVID-19, the inactivated virus sample preservative should be selected to avoid the problem of personnel infection caused by virus leakage in the process of transportation and detection.

[0003] At present, the inactivated virus sample preservative has the following problems worthy of attention: 1. When the virus sample is inactivated, the components of the preservative may cause the degradation or mutation of free viral nucleic acid, affecting the accuracy of viral nucleic acid detection. At the same time, metal ions are released after the lysis of viral proteins, which affect the quality of nucleic acid. 2. The stability problem of the preservative itself. With the increase of factory time, the stability of the preservative itself decreases, and discoloration occurs, which will affect the nucleic acid detection results if it is continued to be used. SUMMARY

[0004] In view of this, the present application provides a virus sample preservative. The present application not only solves the problem of nucleic acid degradation and mutation in the preservation process which is generally concerned in the industry, but also solves the stability problem of the preservative product itself which is easily overlooked.

[0005] The present application is as follows:

[0006] A virus sample preservative, comprising the following components: lithium dodecyl sulfate 50-60 mM, sodium dihydrogen phosphate 9-20 mM, disodium hydrogen phosphate 9-20 mM, ethylenediaminetetraacetic acid 0.5-1.5 mM, thio geraniol 0.5-3 mM, phenol red 0.005 v / v% to 0.03 v / v%, and the balance of water.

[0007] Preferably, the sodium dihydrogen phosphate is anhydrous sodium dihydrogen phosphate.

[0008] Preferably, the disodium hydrogen phosphate is disodium hydrogen phosphate dodecahydrate.

[0009] In the present application, lithium dodecyl sulfate as a virus lysis solution can quickly inactivate the virus, effectively prevent secondary infection, and protect the safety of transportation and detection personnel. Anhydrous sodium dihydrogen phosphate and disodium hydrogen phosphate dodecahydrate as buffer components are used to maintain the buffer environment of nucleic acid and maintain the stability of nucleic acid. Ethylenediaminetetraacetic acid (EDTA) as a divalent metal chelating agent inhibits RNase activity, reduces cell membrane stability, lyses cells, and stabilizes RNA. Phenol red as a pH indicator can observe the performance of the preservative by its color change.

[0010] Further, the applicant found that the addition of thio geraniol in the formula can play a nucleic acid protection function, avoid nucleic acid degradation or mutation, and reduce the influence of metal ions such as Mg 2+ , Ca 2+ , etc. on the quality of nucleic acid, while improving the stability of other components in the preservation solution and avoiding discoloration during preservation.

[0011] Preferably, the virus sample preservation solution comprises the following components: lithium dodecyl sulfate 55mM, anhydrous sodium dihydrogen phosphate 15mM, disodium phosphate dodecahydrate 15mM, ethylenediaminetetraacetic acid 1mM, thio geraniol 1mM, phenol red 0.01v / v%, and water.

[0012] Preferably, the virus includes common viruses such as influenza virus (e.g. influenza A virus, etc.), novel coronavirus COVID-19, etc.

[0013] Preferably, the sample suitable for the preservation solution includes oropharyngeal swab, nasopharyngeal swab, and anal swab.

[0014] Further, the present application also provides a preparation method of the virus sample preservation solution, which comprises the following steps: uniformly mixing the components, and adjusting the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide to obtain the virus sample preservation solution.

[0015] Compared with the prior art, the present application has the following beneficial effects:

[0016] The present application provides an inactivated virus sample preservation solution, which inactivates the virus after sampling and effectively prevents the risk of secondary infection. The use of the preservation solution of the present application can effectively avoid the degradation and mutation of viral nucleic acid, which is beneficial to subsequent detection and analysis. The sample can be stored for more than 72h at 37℃, more than 7 days at room temperature, and long-term storage at-20℃ or below, without affecting the detection results.

[0017] The preservation solution product of the present application has stable quality and can be stored for a long time, with a shelf life of 12 months.

[0018] The preparation method of the present application is simple and easy to operate, does not require special equipment, and is easy to implement in industry. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 : Ct value change of virus sample after preservation in each group of preservation solution. The abscissa represents the group, and the ordinate represents the Ct value. DETAILED DESCRIPTION

[0020] In order to better understand the technical content of the present application for those skilled in the art, the present application will be further described in combination with specific examples.

[0021] Example 1 Virus sample preservation solution

[0022] Formulation:

[0023] No. Raw material Amount 1 Lithium dodecyl sulfate 55 mM 2 Sodium dihydrogen phosphate anhydrous 15 mM 3 Disodium hydrogen phosphate dodecahydrate 15 mM 4 Ethylene diamine tetraacetic acid (EDTA) 1 mM 5 Phenol red 0.01% (V / V) 6 Purified water Balance

[0024] Preparation method: mix the components evenly, adjust the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide, and obtain the product.

[0025] Example 2: Virus sample preservative

[0026] Formulation:

[0027] No. Raw material Amount 1 Lithium dodecyl sulfate 55 mM 2 Sodium dihydrogen phosphate anhydrous 15 mM 3 Disodium hydrogen phosphate dodecahydrate 15 mM 4 Ethylene diamine tetraacetic acid (EDTA) 1 mM 5 Thio geraniol 0.5 mM 6 Phenol red 0.01% (V / V) 7 Purified water Balance

[0028] Preparation method: mix the components evenly, adjust the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide, and obtain the product.

[0029] Example 3: Virus sample preservative

[0030] Formulation:

[0031] No. Raw material Amount 1 Lithium dodecyl sulfate 55 mM 2 Sodium dihydrogen phosphate anhydrous 15 mM 3 Disodium hydrogen phosphate dodecahydrate 15 mM 4 Ethylene diamine tetraacetic acid (EDTA) 1 mM 5 Thio geraniol 1 mM 6 Phenol red 0.01% (V / V) 7 Purified water Balance

[0032] Preparation method: mix the components evenly, adjust the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide, and obtain the product.

[0033] Example 4: Virus sample preservative

[0034] Formulation:

[0035]

[0036]

[0037] Preparation method: mix the components evenly, adjust the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide, and obtain the product.

[0038] Example 5: Virus sample preservative

[0039] Formulation:

[0040] No. Raw material Amount 1 Lithium dodecyl sulfate 55 mM 2 Sodium dihydrogen phosphate anhydrous 9 mM 3 Disodium hydrogen phosphate dodecahydrate 20 mM 4 Ethylene diamine tetraacetic acid (EDTA) 1 mM 5 Thio geraniol 1 mM 6 Phenol red 0.01% (V / V) 7 Purified water Balance

[0041] Preparation method: mix the components evenly, adjust the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide, and obtain the product.

[0042] Example 6: Virus sample preservative

[0043] Formulation:

[0044] No. Raw material Amount 1 Lithium dodecyl sulfate 60 mM 2 Sodium dihydrogen phosphate anhydrous 20 mM 3 Disodium hydrogen phosphate dodecahydrate 9 mM 4 Ethylene diamine tetraacetic acid (EDTA) 1 mM 5 Thio geraniol 1 mM 6 Phenol red 0.01% (V / V) 7 Purified water Balance

[0045] Preparation method: mix the components evenly, adjust the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide, and obtain the product.

[0046] Example 7: Virus sample preservative

[0047] Formula:

[0048]

[0049]

[0050] Preparation method: mix the components evenly, adjust the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide, and obtain the product.

[0051] Example 8: Virus sample preservative

[0052] Formula:

[0053] No. Raw material Amount 1 Lithium dodecyl sulfate 55 mM 2 Sodium dihydrogen phosphate anhydrous 15 mM 3 Disodium hydrogen phosphate dodecahydrate 15 mM 4 Ethylene diamine tetraacetic acid (EDTA) 0.5 mM 5 Thio geraniol 1 mM 6 Phenol red 0.01% (V / V) 7 Purified water Balance No. Raw material Amount Lithium dodecyl sulfate 55 mM Sodium dihydrogen phosphate anhydrous 15 mM Disodium hydrogen phosphate dodecahydrate 15 mM Ethylene diamine tetraacetic acid (EDTA) 0.5 mM Thio geraniol 1 mM Phenol red 0.01% (V / V) Purified water Balance

[0054] Preparation method: mix the components evenly, adjust the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide, and obtain the product.

[0055] Experiment 1:

[0056] Each group of preservatives was used to dilute the influenza A virus stock solution (titer 1×10 6 IU / mL) by 100 times, and the virus sample preservative was taken after 0h and 72h of 37℃ storage for RT-qPCR detection, recording the Ct value, and the average value of three repetitions. The detection process was carried out according to the instructions of the detection kit. The results are shown in the table below. Variance analysis was performed using SPSS software.

[0057] Table 1 Average Ct value of samples after 0h and 72h of 37℃ storage (average value±standard deviation)

[0058]

[0059] The results show that in Examples 2-8, the Ct value after 72h of 37℃ storage has no significant difference from 0h, and the difference in Example 3 is the smallest. The 72h Ct value of Example 1 is significantly improved, and there is a significant difference from 0h.

[0060] Experiment 2:

[0061] The preservatives of each group of examples were placed in the room at room temperature 25±2℃ without sealing, and the color change of the solution was observed after 7 days using test paper. The results show that except for Example 1, the other examples have no obvious color change, and the preservative after 7 days of storage is used to store virus samples, and the Ct value is not significantly changed after 72 hours of detection.

[0062] The above merely provides part of the embodiments of the present application but does not limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall fall into the protection scope of the present application.

Claims

1. A virus sample preservation solution, characterized in that, It includes the following components: lithium dodecyl sulfate 50-60 mM, anhydrous sodium dihydrogen phosphate 9-20 mM, disodium hydrogen phosphate dodecahydrate 9-20 mM, ethylenediaminetetraacetic acid 0.5-1.5 mM, thiogeraniol 0.5-3 mM, phenol red 0.005 v / v%-0.03 v / v% and water, and the viruses include influenza virus and novel coronavirus COVID-19.

2. The virus sample preservation solution according to claim 1, characterized in that, It includes the following ingredients: lithium dodecyl sulfate 55 mM, anhydrous sodium dihydrogen phosphate 15 mM, disodium hydrogen phosphate dodecahydrate 15 mM, ethylenediaminetetraacetic acid 1 mM, thiogeraniol 1 mM, phenol red 0.01 v / v% and water.

3. The virus sample preservation solution according to claim 1, characterized in that, The samples for which the preservation solution is applicable include oropharyngeal swab samples, nasopharyngeal swab samples, or anal swab samples.

4. The method for preparing the virus sample preservation solution according to claim 1, characterized in that, The process includes the following steps: mixing all components thoroughly, and adjusting the pH value to 7.2±0.2 using hydrochloric acid or sodium hydroxide.

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