Low-temperature-resistant inactivated guanidine salt virus preserving fluid and preparation method thereof

Abstract

The invention provides a low-temperature-resistant inactivated guanidine salt virus preservation solution and a preparation method thereof, and relates to the field of virus preservation solutions. Comprising the following raw materials: NaCl, KCl, glucose, KH2PO4, Na2HPO4, guanidine hydrochloride, penicillin, streptomycin, amphotericin B and glycerol. The raw materials are mixed and uniformly stirred. The preserving fluid is suitable for being used in a low-temperature environment, can preserve a liquid state within several hours in the low-temperature environment, can significantly inactivate pathogens, can also maintain completeness of subsequent nucleic acid extraction, and has no significant influence on amplification and the like.

Description

Technical Field

[0001] The present invention relates to the field of virus preservation fluids, and in particular to a low-temperature-resistant inactivated guanidine virus preservation fluid and a preparation method thereof. Background Art

[0002] Nucleic acids (RNA and DNA) are commonly found in animals, plants, microorganisms, and some viruses and bacteriophages, and are carriers of genetic information. Fresh samples used to extract nucleic acids are generally frozen in a -80°C refrigerator and then tested in batches. However, ultra-low temperature refrigerators are often not popular in clinical departments, and the cost of preservation is relatively high. Therefore, it is of practical value to find a new method to preserve samples.

[0003] Clinical pathogens are the main pathogens that cause clinical diseases. Timely and accurate testing of patient specimens suspected of being infected with clinical pathogens is of great significance to clinical treatment and epidemic control. The quality control of nucleic acid testing involves many aspects, including specimen collection, storage and transportation, detection reagents, and result interpretation. Among them, specimen storage and transportation is the most easily overlooked link. Due to improper specimen storage, the pathogen nucleic acid is degraded and destroyed, which will directly lead to a "negative" test result. Real-time fluorescence reverse transcription polymerase chain reaction is one of the main methods for clinical diagnosis of clinical pathogens, but there may be a risk of biosafety exposure during nucleic acid extraction. Therefore, it is particularly important to inactivate pathogenic microorganisms in samples before testing.

[0004] Adding inactivation preservation solution to the sample preservation tube can inactivate pathogens immediately after sampling and prevent ribonuclease RNase from degrading nucleic acids. However, during the winter epidemic in northern regions, the sample preservation solution used for sample collection often solidifies due to low temperatures, resulting in the inability of the preservation solution to inactivate pathogens and also causing the pathogen structure to be destroyed, affecting the test results. Summary of the invention

[0005] The object of the present invention is to provide a low-temperature-resistant inactivated guanidine virus preservation solution, which is suitable for use in a low-temperature environment. The liquid state can be preserved for several hours in the low-temperature environment, and the pathogen can be significantly inactivated. The integrity of subsequent nucleic acid extraction can also be maintained, and amplification and the like have no significant effect.

[0006] Another object of the present invention is to provide a method for preparing a low-temperature-resistant inactivated guanidine virus preservation solution, which is simple and convenient.

[0007] The present invention solves the technical problem by adopting the following technical solutions.

[0008] On the one hand, an embodiment of the present invention provides a low-temperature-resistant inactivated guanidine virus preservation solution, comprising the following raw materials: NaCl, KCl, glucose, KH2PO4, Na2HPO4, guanidine hydrochloride, penicillin, streptomycin, amphotericin B and glycerol.

[0009] In some embodiments of the present invention, the concentration of each raw material in the preservation solution is: 7-8g / L NaCl, 0.3-0.6g / L KCl, 0.8-1.2g / L glucose, 40-60mg / L KH2PO4, 40-50mg / L Na2HPO4, 400-600g / L guanidine hydrochloride, 100000-200000U / L penicillin, 0.1-0.5g / L streptomycin, 0.25-0.5g / L amphotericin B, 10%-50% vol glycerol. The concentration of glycerol is a volume percentage.

[0010] In some embodiments of the present invention, the concentration of each raw material is: 8g / L NaCl, 0.4g / L KCl, 1g / L glucose, 60mg / L KH2PO4, 47.5mg / L Na2HPO4, 573g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 50% vol glycerol.

[0011] In some embodiments of the present invention, the concentration of each raw material is: 7g / L NaCl, 0.3g / L KCl, 0.8g / L glucose, 40mg / L KH2PO4, 40mg / L Na2HPO4, 400g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 10% vol glycerol.

[0012] On the other hand, an embodiment of the present invention provides a method for preparing a low-temperature-resistant inactivated guanidine virus preservation solution, which comprises: mixing various raw materials, adding deionized water and adjusting the volume to 1 L, and stirring evenly.

[0013] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects:

[0014] The preservation solution provided by the present invention includes NaCl, KCl, glucose, KH2PO4, Na2HPO4, guanidine hydrochloride, penicillin, streptomycin, amphotericin B and glycerol, wherein NaCl, KCl, glucose, KH2PO4 and Na2HPO4 provide an environment with a suitable pH value for preserving pathogenic microorganisms, guanidine hydrochloride can quickly inactivate pathogens and protect nucleic acid components from being easily degraded; penicillin, streptomycin and amphotericin B have the effect of inhibiting bacteria and fungi; glycerol is an antifreeze agent, which makes the preservation solution not easy to solidify under low temperature conditions. That is, the virus preservation solution provided by the present invention can quickly dissolve pathogens, make them lose their infectivity, and keep the released nucleic acids in a stable state, and is particularly suitable for outdoor sample collection in northern winter. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0016] Figure 1 This is the storage solution after being stored at 4°C for 4 hours;

[0017] Figure 2 This is the storage solution after being stored at -10°C for 4 hours;

[0018] Figure 3 This is the sterilization comparison of each preservation solution after being stored at 4°C for 2 hours;

[0019] Figure 4 The effect of each preservation solution on gene amplification;

[0020] Figure 5 The effects of various preservation solutions on nucleic acid extraction from N2A cells; DETAILED DESCRIPTION

[0021] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0022] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other. The present invention will be described in detail below with reference to specific embodiments.

[0023] On the one hand, the embodiment of the present invention provides a low-temperature resistant inactivated guanidine virus preservation solution, comprising the following raw materials, NaCl, KCl, glucose, KH2PO4, Na2HPO4, guanidine hydrochloride, penicillin, streptomycin, amphotericin B and glycerol. NaCl, KCl, glucose, KH2PO4, and Na2HPO4 provide an environment with a suitable pH value for preserving pathogenic microorganisms, wherein KH2PO4 and Na2HPO4 can form a pH buffer system to stabilize the pH value of the preservation solution; guanidine hydrochloride can quickly inactivate pathogens and protect nucleic acid components from being easily degraded; penicillin, streptomycin and amphotericin B have the effect of inhibiting bacteria and fungi, and glycerol makes the preservation solution less likely to solidify under low temperature conditions.

[0024] In some embodiments of the present invention, the concentration of each raw material is: 7-8 g / L NaCl, 0.3-0.6 g / L KCl, 0.8-1.2 g / L glucose, 40-60 mg / L KH2PO4, 40-50 mg / L Na2HPO4, 400-600 g / L guanidine hydrochloride, 100000-200000 U / L penicillin, 0.1-0.5 g / L streptomycin, 0.25-0.5 g / L amphotericin B, 10%-50% vol glycerol.

[0025] It should be noted that in the embodiments provided by the present invention, the concentration of the above raw materials refers to the concentration in the preservation solution, and the concentration of glycerol refers to the volume percentage of glycerol in the preservation solution. Among the above raw materials, except for glycerol which is liquid, the other raw materials are solid.

[0026] In some embodiments of the present invention, the concentration of each raw material is: 8g / L NaCl, 0.4g / L KCl, 1g / L glucose, 60mg / L KH2PO4, 47.5mg / L Na2HPO4, 573g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 50% vol glycerol.

[0027] In some embodiments of the present invention, the concentration of each raw material is: 7g / L NaCl, 0.3g / L KCl, 0.8g / L glucose, 40mg / L KH2PO4, 40mg / L Na2HPO4, 400g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 10% vol glycerol.

[0028] On the other hand, an embodiment of the present invention provides a method for preparing a low-temperature-resistant inactivated guanidine virus preservation solution, which comprises: mixing various raw materials, adding deionized water and adjusting the volume to 1 L, and stirring evenly.

[0029] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0030] Example 1

[0031] Prepare the raw materials and configure them according to the following concentrations:

[0032] 8g / L NaCl, 0.4g / L KCl, 1g / L glucose, 60mg / L KH2PO4, 47.5mg / L Na2HPO4, 573g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 5%vol glycerol;

[0033] The preparation method is:

[0034] According to the above concentration ratio, calculate the amount of each raw material, add the above raw materials into the container, add deionized water, make up to 1L, and stir evenly.

[0035] Example 2

[0036] Prepare the raw materials and configure them according to the following concentrations:

[0037] 8g / L NaCl, 0.4g / L KCl, 1g / L glucose, 60mg / L KH2PO4, 47.5mg / L Na2HPO4, 573g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 10% glycerol;

[0038] The preparation method is:

[0039] According to the above concentration ratio, calculate the amount of each raw material, add the above raw materials into the container, add deionized water, make up to 1L, and stir evenly.

[0040] Example 3

[0041] Prepare the raw materials and configure them according to the following concentrations:

[0042] 8g / L NaCl, 0.4g / L KCl, 1g / L glucose, 60mg / L KH2PO4, 47.5mg / L Na2HPO4, 573g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 20% glycerol;

[0043] The preparation method is:

[0044] According to the above concentration ratio, calculate the amount of each raw material, add the above raw materials into the container, add deionized water and make the volume to 1L, and stir evenly.

[0045] Example 4

[0046] Prepare the raw materials and configure them according to the following concentrations:

[0047] 8g / L NaCl, 0.4g / L KCl, 1g / L glucose, 60mg / L KH2PO4, 47.5mg / L Na2HPO4, 573g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 30% glycerol;

[0048] The preparation method is:

[0049] According to the above concentration ratio, calculate the amount of each raw material, add the above raw materials into the container, add deionized water, make up to 1L, and stir evenly.

[0050] Example 5

[0051] Prepare the raw materials and configure them according to the following concentrations:

[0052] 8g / L NaCl, 0.4g / L KCl, 1g / L glucose, 60mg / L KH2PO4, 47.5mg / L Na2HPO4, 573g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 50% glycerol by volume;

[0053] The preparation method is:

[0054] According to the above concentration ratio, calculate the amount of each raw material, add the above raw materials into the container, add deionized water and make the volume to 1L, and stir evenly.

[0055] Example 6

[0056] Prepare the raw materials and configure them according to the following concentrations:

[0057] 7g / L NaCl, 0.3g / L KCl, 0.8g / L glucose, 40mg / L KH2PO4, 40mg / L Na2HPO4, 400g / L guanidine hydrochloride, 100000U / L penicillin, 0.1g / L streptomycin, 0.25g / L amphotericin B, 10% glycerol;

[0058] The preparation method is:

[0059] According to the above concentration ratio, calculate the amount of each raw material, add the above raw materials into the container, add deionized water and make the volume to 1L, and stir evenly.

[0060] Experimental example

[0061] 1. Low temperature storage performance

[0062] The storage solutions of Examples 1-5 and those without glycerol were placed in sampling tubes and then stored at 4°C and -20°C for 4 hours. The physical states of the samples were observed. The results are as follows: Figure 1-2 The glycerol concentrations corresponding to Examples 1-5 are 5%, 10%, 20%, 30%, and 50%, and the storage solution without glycerol corresponds to 0%.

[0063] from Figure 1-2 It can be seen that Examples 1-5 added 5%-50% glycerol respectively, and they were all liquid after being stored at 4°C and -20°C for 4 hours, indicating that the storage solution did not solidify in a low temperature environment, and the storage solution at low temperature still had the performance of inactivating and preserving pathogens. However, the storage solution without glycerol (0%) solidified after being stored at -20°C for 4 hours, and ice crystals appeared in the storage solution.

[0064] 2. Bactericidal properties

[0065] The standard Escherichia coli 25922 strain was used as a sample and placed in the preservation solution of Example 1-5 without glycerol at -20°C. After 2 hours, the growth of the strain was observed. The results are as follows: Figure 3 As shown. Among them, Figure 3 A is a normal saline control group, i.e., normal saline is used instead of the preservation solution, B is a comparative example, and CG are Examples 1-5, respectively.

[0066] from Figure 3 It can be seen that the control group (Part A) has more colonies and a larger area, while adding 0% (B) and 50% glycerol (G) has a small amount of colonies, and the rest of the parts have no obvious colonies, indicating that the preservation solution provided in the embodiment of the present invention has good bactericidal and sterilizing properties.

[0067] 3. Effect of storage medium on gene amplification

[0068] COVID-19 quality control products were used to simulate clinical pathogens and extract and amplify them. Figure 4 shown.

[0069] from Figure 4 It can be seen that the Ct values ​​obtained by amplifying the N gene, ORF gene and internal reference gene using preservation solutions containing different concentrations of glycerol are very close and will not interfere with the interpretation of positive and negative results.

[0070] 4. Effect of storage solution on nucleic acid extraction from N2A cells

[0071] N2A cells were used as the research object. They were placed in the preservation solution at -20℃ for 2 hours and then the nucleic acid extraction experiment was performed. The results are as follows: Figure 5 As shown, from Figure 5 It can be concluded that the preservation solution provided by the present invention will not affect the extraction of nucleic acid.

[0072] In summary, the embodiment of the present invention provides a low-temperature resistant inactivated guanidine virus preservation solution, which is suitable for use in a low-temperature environment. It can be kept in a liquid state for several hours in a low-temperature environment, can significantly inactivate pathogens, and can also maintain the integrity of subsequent nucleic acid extraction without significant effect on amplification.

[0073] The embodiments described above are part of the embodiments of the present invention, rather than all of the embodiments. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A low-temperature-resistant inactivated guanidine virus preservation solution, characterized in that: Including the following raw materials, NaCl, KCl, glucose, KH2PO4, Na2HPO4, guanidine hydrochloride, penicillin, streptomycin, amphotericin B and glycerol.

2. The low-temperature-resistant inactivated guanidine virus preservation solution according to claim 1, characterized in that The concentrations of the raw materials are: 7-8 g / L NaCl, 0.3-0.6 g / L KCl, 0.8-1.2 g / L glucose, 40-60 mg / L KH2PO4, 40-50 mg / L Na2HPO4, 400-600 g / L guanidine hydrochloride, 100000-200000 U / L penicillin, 0.1-0.5 g / L streptomycin, 0.25-0.5 g / L amphotericin B, and 10%-50% vol glycerol.

3. The low-temperature-resistant inactivated guanidine virus preservation solution according to claim 2, characterized in that The concentrations of the raw materials are: 8 g / L NaCl, 0.4 g / L KCl, 1 g / L glucose, 60 mg / L KH2PO4, 47.5 mg / L Na2HPO4, 573 g / L guanidine hydrochloride, 100000 U / L penicillin, 0.1 g / L streptomycin, 0.25 g / L amphotericin B, and 50% vol glycerol.

4. The low-temperature-resistant inactivated guanidine virus preservation solution according to claim 1, characterized in that The concentrations of the raw materials are: 7 g / L NaCl, 0.3 g / L KCl, 0.8 g / L glucose, 40 mg / L KH2PO4, 40 mg / L Na2HPO4, 400 g / L guanidine hydrochloride, 100000 U / L penicillin, 0.1 g / L streptomycin, 0.25 g / L amphotericin B, and 10% vol glycerol.

5. A method for preparing a low-temperature-resistant inactivated guanidine virus preservation solution according to any one of claims 1 to 4, characterized in that: The following steps are included: Mix all the raw materials, add deionized water to make up to volume, and stir evenly.

Images