A body fluid sample virus preservation solution, a preparation method and application thereof

Abstract

The application discloses a kind of body fluid sample virus preservative and its preparation method and application, it is related to virus nucleic acid detection technical field.The component includes: sodium chloride 12.8-19.2g / L, sodium hydrogen phosphate 2.28-3.41g / L, potassium chloride 0.32-0.48g / L, potassium dihydrogen phosphate 0.43-0.65g / L, liquid biological preservative Proclin300 0.16-0.24g / L, trans-1,2-cyclohexanediaminetetraacetic acid 0.58-0.87g / L, polyethylene glycol 128-192g / L according to final concentration.It can save virus nucleic acid integrity in body fluid, solve the problem of nucleic acid degradation possibly caused by body fluid virus sample sampling and transportation, improve the sensitivity and accuracy of virus detection using fluorescent quantitative PCR method, ensure the effective amplification of downstream PCR reaction, maintain the virus nucleic acid concentration to be detected, improve detection rate.

Description

Technical Field

[0001] This invention relates to the field of biomolecular technology, and more specifically, to a viral preservation solution for bodily fluid samples, its preparation method, and its application. Background Technology

[0002] Viruses are special organisms composed of a protein coat and internal genetic material, lacking cellular structure. Due to their very simple structure, they cannot perform metabolism for survival and require substances and energy from host cells to produce new generations of viruses, thus achieving replicative proliferation.

[0003] Viral nucleic acid detection is applicable to many important fields, including early disease diagnosis, auxiliary diagnosis of difficult samples, and disease progression monitoring and prediction. Currently, the most effective and sensitive method for viral nucleic acid detection in clinical practice is real-time quantitative PCR. However, this method requires the viral nucleic acid to have a certain degree of integrity to ensure the sensitivity and accuracy of the detection. Therefore, ensuring the integrity of viral nucleic acid is of great significance for downstream gene analysis.

[0004] Animal clinical samples containing viral bodily fluids typically include blood (serum, plasma), oral fluid, semen, castration fluid, and amniotic fluid. These samples have complex compositions, contain numerous endogenous enzymes, and are prone to bacterial and fungal growth during transport. These factors can decompose and damage the viral protein coat, preventing the virus from maintaining its original integrity. Viral nucleic acids, once exposed, are easily degraded by widely distributed endogenous or exogenous RNases or DNases.

[0005] In view of this, the present invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to provide a virus preservation solution for bodily fluid samples, its preparation method, and its application. This solution can preserve the integrity of viral nucleic acid in bodily fluids, specifically addressing the problem of nucleic acid degradation that may occur during the sampling and transportation of viral samples from bodily fluids. It improves the sensitivity and accuracy of virus detection using quantitative real-time PCR, ensures effective amplification of downstream PCR reactions, maintains the concentration of viral nucleic acid to be detected, and increases the detection rate.

[0007] This invention is implemented as follows:

[0008] In a first aspect, the present invention provides a viral preservation solution for bodily fluid samples, the components of which, based on final concentration, include: sodium chloride 12.8-19.2 g / L, disodium hydrogen phosphate 2.28-3.41 g / L, potassium chloride 0.32-0.48 g / L, potassium dihydrogen phosphate 0.43-0.65 g / L, liquid biological preservative Proclin 300 0.16-0.24 g / L, trans-1,2-cyclohexanediaminetetraacetic acid 0.58-0.87 g / L, and polyethylene glycol 128-192 g / L.

[0009] In an optional embodiment, the components, based on the final concentration, include: sodium chloride 14-18 g / L, disodium hydrogen phosphate 2.6-3.1 g / L, potassium chloride 0.35-0.45 g / L, potassium dihydrogen phosphate 0.5-0.6 g / L, liquid biological preservative Proclin 300 0.18-0.22 g / L, trans-1,2-cyclohexanediaminetetraacetic acid 0.6-0.8 g / L, and polyethylene glycol 150-170 g / L.

[0010] In an optional embodiment, the components, based on final concentration, include: 16.0 g / L sodium chloride, 2.84 g / L disodium hydrogen phosphate, 0.4 g / L potassium chloride, 0.54 g / L potassium dihydrogen phosphate, 0.2 g / L liquid biological preservative Proclin 300, 0.72 g / L trans-1,2-cyclohexanediaminetetraacetic acid, and 160 g / L polyethylene glycol.

[0011] In an optional embodiment, the polyethylene glycol is PEG 6000.

[0012] In an optional embodiment, the pH of the viral preservation solution for the bodily fluid sample is 7.2-7.5.

[0013] In an optional embodiment, the component further includes the remainder water.

[0014] Secondly, the present invention provides a method for preparing a viral preservation solution for bodily fluid samples, which includes mixing the components of the viral preservation solution for bodily fluid samples as described in any of the foregoing embodiments with water and preparing it to the aforementioned final concentration.

[0015] Thirdly, the present invention provides the application of the viral preservation solution for bodily fluid samples as described in any of the foregoing embodiments in the preparation of viral nucleic acid detection.

[0016] Fourthly, the present invention provides a method for detecting viral nucleic acid, wherein a viral sample is placed in a viral preservation solution for bodily fluid samples as described in any of the foregoing embodiments and mixed evenly. Before detection, the mixture is centrifuged, the precipitate is resuspended and mixed evenly, and then the viral nucleic acid is purified and detected.

[0017] Fifthly, the present invention provides a viral nucleic acid detection kit, which includes a viral preservation solution for body fluid samples, a purification reagent, and a detection reagent as described in any of the foregoing embodiments.

[0018] The present invention has the following beneficial effects:

[0019] The viral preservation solution for bodily fluid samples provided in this application is formulated with sodium chloride, disodium hydrogen phosphate, potassium chloride, and potassium dihydrogen phosphate as a buffer solution. This buffer solution is isotonic, maintaining the morphology of viral particles while maintaining a relative pH, ensuring that each component functions optimally in the optimal pH environment. The liquid biological preservative Proclin 300 is a highly effective sterilizing agent for controlling microbial content. With its broad-spectrum activity, excellent compatibility and stability, and low toxicity at its usage concentration, it can eradicate bacteria and fungi for a longer period, inhibit cell growth and metabolism, and promote apoptosis. Trans-1,2-cyclohexanediaminetetraacetic acid (trans-1,2-cyclohexanediaminetetraacetic acid) is a potent metal ion chelating agent that chelates divalent metal cations in the sample, inhibiting the activity of nucleases and some endogenous enzymes, thus preventing the degradation of viral particles and free nucleic acids. In this application, the combined use of the liquid biological preservative Proclin 300 and trans-1,2-cyclohexanediaminetetraacetic acid maximizes the maintenance of the effective nucleic acid concentration of the virus to be tested, improving the detection rate of PCR detection. Compared to previous sample preservation solutions, this application adds polyethylene glycol as a virus settling agent. Centrifugation and precipitation concentrate the virus, increasing its concentration and reducing the likelihood of false negatives due to low-concentration samples failing to reach the detection limit of the kit. In terms of usage, simply invert and shake the virus preservation solution thoroughly, then mix it with the body fluid virus sample at a 1:1 volume ratio. Upon receiving the sample, the laboratory should centrifuge it at 12,000 rpm for 10 minutes during sample pretreatment, and then use the precipitate for nucleic acid purification. The virus preservation solution of this invention is simple to prepare, uses readily available raw materials, is stable, has a long shelf life, and is generally convenient to use, making it highly valuable. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0021] This invention provides a virus preservation solution for bodily fluid samples, the components of which, based on final concentration, include: sodium chloride 12.8-19.2 g / L, disodium hydrogen phosphate 2.28-3.41 g / L, potassium chloride 0.32-0.48 g / L, potassium dihydrogen phosphate 0.43-0.65 g / L, liquid biological preservative Proclin 300 0.16-0.24 g / L, trans-1,2-cyclohexanediaminetetraacetic acid 0.58-0.87 g / L, and polyethylene glycol 128-192 g / L.

[0022] The components also include the remainder water, which is used as a solvent in this application to prepare the above components to a specific concentration. In this application, the pH of the viral preservation solution for bodily fluid samples is 7.2-7.5. It should be understood that the components may also include a pH adjuster to adjust the pH of the viral preservation solution for bodily fluid samples.

[0023] Sodium chloride, disodium hydrogen phosphate, potassium chloride, and potassium dihydrogen phosphate are formulated as a solution buffer, providing salt balance and pH buffering effects to maintain viral cell morphology and relative pH. Proclin 300, a liquid biological preservative, is a highly effective sterilizing agent for controlling microbial content. With its broad-spectrum activity, superior compatibility and stability, and low toxicity at its application concentration, it can eradicate bacteria and fungi over a longer period, inhibit cell growth and metabolism, and promote apoptosis. Trans-1,2-cyclohexanediaminetetraacetic acid is a potent metal ion chelating agent that chelates divalent metal cations in the sample, inhibiting the activity of nucleases and some endogenous enzymes, thus preventing the degradation of viral particles and free nucleic acids. Polyethylene glycol (PEG) is a water-soluble nonionic polymer. In this application, the PEG used is PEG 6000. Using PEG with a molecular weight of 6000 allows for co-precipitation with viral particles. It operates under mild conditions and is less likely to cause denaturation of the viral protein coat. It has high precipitation efficiency; a small amount of PEG can precipitate a significant number of viral particles, thus increasing the viral load in the sample. Compared to previous sample preservation solutions, this application, by adding PEG as a viral settling agent, allows for the concentration of the virus and increases viral concentration through centrifugation and precipitation. This reduces the likelihood of false negatives due to low-concentration samples failing to reach the detection limit of the kit.

[0024] In this application, by using the biological preservative Proclin300 in combination with trans-1,2-cyclohexanediaminetetraacetic acid, the concentration of effective nucleic acid of the virus to be tested can be maintained to the greatest extent, thereby improving the detection rate of PCR detection.

[0025] Preferably, the components, based on the final concentration, include: sodium chloride 14-18 g / L, disodium hydrogen phosphate 2.6-3.1 g / L, potassium chloride 0.35-0.45 g / L, potassium dihydrogen phosphate 0.5-0.6 g / L, liquid biological preservative Proclin 300 0.18-0.22 g / L, trans-1,2-cyclohexanediaminetetraacetic acid 0.6-0.8 g / L, and polyethylene glycol 150-170 g / L.

[0026] More preferably, the components, based on final concentration, include: sodium chloride 16.0 g / L, disodium hydrogen phosphate 2.84 g / L, potassium chloride 0.4 g / L, potassium dihydrogen phosphate 0.54 g / L, liquid biological preservative Proclin 300 0.2 g / L, trans-1,2-cyclohexanediaminetetraacetic acid 0.72 g / L, and polyethylene glycol 160 g / L.

[0027] The preparation method of the above-mentioned viral preservation solution for bodily fluid samples is simple. It only requires mixing the components of the above-mentioned viral preservation solution with water and preparing it to the above-mentioned final concentration. The operation is simple and easy to implement.

[0028] The prepared viral preservation solution for bodily fluid samples can be widely used as a viral preservation solution in viral nucleic acid detection.

[0029] In addition, this application also provides a method for detecting viral nucleic acid, wherein the viral sample is placed in the above-mentioned viral preservation solution of body fluid sample and mixed evenly. Before detection, the mixture is centrifuged, the precipitate is taken, resuspended and mixed evenly, and then the viral nucleic acid is purified and detected.

[0030] Correspondingly, this invention provides a viral nucleic acid detection kit, which includes a viral preservation solution for body fluid samples, purification reagents, and detection reagents. The purification reagent is Hangzhou Bori Technology's MagaBio Virus DNA / RNA Purification Kit II (catalog number BSC71S1E). The detection reagents used are Century Yuanheng's Porcine Reproductive and Respiratory Syndrome Virus Universal Fluorescent RT-PCR Kit (batch number PRRSV202210923P) and Century Yuanheng's High-Sensitivity African Swine Fever Fluorescent PCR Kit (batch number ASFV202110630HS). During detection, each experimental sample was tested three times using a GeneRotex96 real-time PCR instrument, and the average value was taken. The program settings were based on the kit instructions.

[0031] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0032] Example 1

[0033] This embodiment provides a virus preservation solution for body fluid samples, the preparation method of which includes: weighing 16.0g of sodium chloride; 2.84g of disodium hydrogen phosphate; 0.4g of potassium chloride; 0.54g of potassium dihydrogen phosphate; and 0.2g of Proclin 300; dissolving them in 1L of ultrapure water; adjusting the pH to 7.2-7.5; and then adding 160g of polyethylene glycol (PEG 6000) and 0.72g of trans-1,2-cyclohexanediaminetetraacetic acid.

[0034] Example 2

[0035] This embodiment provides a virus preservation solution for body fluid samples, the preparation method of which includes: weighing 18.0g of sodium chloride; 3g of disodium hydrogen phosphate; 0.5g of potassium chloride; 0.6g of potassium dihydrogen phosphate; and 0.22g of Proclin 300; dissolving them in 1L of ultrapure water; adjusting the pH to 7.2-7.5; and then adding 170g of polyethylene glycol (PEG 6000) and 0.8g of trans-1,2-cyclohexanediaminetetraacetic acid.

[0036] Example 3

[0037] This embodiment provides a viral preservation solution for bodily fluid samples, the preparation method of which includes: weighing 14.0g of sodium chloride; 2.6g of disodium hydrogen phosphate; 0.35g of potassium chloride; 0.5g of potassium dihydrogen phosphate; and 0.18g of Proclin 300; dissolving them in 1L of ultrapure water; adjusting the pH to 7.2-7.5; and then adding 150g of polyethylene glycol (PEG 6000) and 0.6g of trans-1,2-cyclohexanediaminetetraacetic acid.

[0038] Example 4

[0039] This embodiment provides a virus preservation solution for body fluid samples, the preparation method of which includes: weighing 19.0g of sodium chloride; 3.2g of disodium hydrogen phosphate; 0.48g of potassium chloride; 0.62g of potassium dihydrogen phosphate; and 0.24g of Proclin 300; dissolving them in 1L of ultrapure water; adjusting the pH to 7.2-7.5; and then adding 180g of polyethylene glycol (PEG 6000) and 0.82g of trans-1,2-cyclohexanediaminetetraacetic acid.

[0040] Comparative Example 1

[0041] This comparative example provides a virus preservation solution for bodily fluid samples, the preparation method of which includes: weighing 16.0g of sodium chloride; 2.84g of disodium hydrogen phosphate; 0.4g of potassium chloride; 0.54g of potassium dihydrogen phosphate; and 0.2g of Proclin 300; dissolving them in 1L of ultrapure water, adjusting the pH to 7.2-7.5, and then adding 0.72g of trans-1,2-cyclohexanediaminetetraacetic acid.

[0042] Comparative Example 2

[0043] This comparative example provides a virus preservation solution for body fluid samples, the preparation method of which includes: weighing 16.0g of sodium chloride; 2.84g of disodium hydrogen phosphate; 0.4g of potassium chloride; 0.54g of potassium dihydrogen phosphate; and 0.2g of Proclin 300; dissolving them in 1L of ultrapure water, adjusting the pH to 7.2-7.5, and then adding 160g of polyethylene glycol (PEG).

[0044] Comparative Example 3

[0045] This comparative example provides a viral preservation solution for bodily fluid samples, the preparation method of which includes: weighing 20.0g of sodium chloride; 4g of disodium hydrogen phosphate; 0.5g of potassium chloride; 0.7g of potassium dihydrogen phosphate; and 0.3g of Proclin 300; dissolving them in 1L of ultrapure water; adjusting the pH to 7.2-7.5; and then adding 200g of polyethylene glycol (PEG 6000) and 0.9g of trans-1,2-cyclohexanediaminetetraacetic acid.

[0046] Comparative Example 4

[0047] This comparative example provides a viral preservation solution for bodily fluid samples, the preparation method of which includes: weighing 12g of sodium chloride; 2g of disodium hydrogen phosphate; 0.3g of potassium chloride; 0.4g of potassium dihydrogen phosphate; and 0.1g of Proclin 300; dissolving them in 1L of ultrapure water; adjusting the pH to 7.2-7.5; and then adding 100g of polyethylene glycol (PEG 6000) and 0.5g of trans-1,2-cyclohexanediaminetetraacetic acid.

[0048] Experimental Example 1

[0049] The viral preservation solutions for bodily fluid samples provided in Examples 1-4 and Comparative Examples 1-4 were tested using the following methods:

[0050] Two fresh positive saliva or serum samples with different viral concentrations were selected for each experimental group. Each sample was shaken well and divided into 10 portions, each containing 0.5 mL. 0.5 mL of virus preservation solution was added to each sample in the experimental group, and 0.5 mL of phosphate-buffered saline (PBS) was added to each sample in the control group. The samples were thoroughly shaken and placed at room temperature. Samples were collected on days 0, 1, 3, 5, and 7 and centrifuged at 12,000 rpm for 10 min. Add 300 μL to resuspend the precipitate and purify the viral nucleic acid using Hangzhou Bori Technology MagaBio Virus DNA / RNA Purification Kit II (catalog number BSC71S1E). Detection reagents used were Century Yuanheng's Porcine Reproductive and Respiratory Syndrome Virus Universal Fluorescent RT-PCR Kit (batch number PRRSV202210923P) and Century Yuanheng's High-Sensitivity African Swine Fever Fluorescent PCR Kit (batch number ASFV202110630HS). Each experimental sample was tested three times using a GeneRotex96 real-time PCR instrument, and the average value was taken. The program settings were based on the kit instructions. The degree of sample degradation was compared based on changes in Ct values.

[0051] Please refer to Tables 1, 2, 3 and 4 for the test results.

[0052] Table 1. Effects on positive saliva samples of porcine reproductive and respiratory syndrome virus (PRRSV)

[0053]

[0054] Table 2. Effects on positive serum samples of porcine reproductive and respiratory syndrome virus (PRRSV)

[0055]

[0056] Table 3. Effects on African swine fever virus positive saliva samples

[0057]

[0058] Table 4. Effects on African swine fever virus positive serum samples

[0059]

[0060] As shown in Tables 1 and 2, the experimental results indicate that after adding the sample preservation solutions prepared in Example 1 and Comparative Examples 1-2 to saliva and serum samples positive for porcine reproductive and respiratory syndrome virus (PRRSV) and storing them at room temperature for 0-7 days, the CT values ​​detected by fluorescent RT-PCR consistently showed lower changes than the Ct values ​​of the control group; and the change range was greater in Comparative Example 1 than in Comparative Example 2, which in turn was greater in Example 1. In contrast, the Ct values ​​of the control group samples containing PBS increased significantly after 7 days, indicating severe degradation of the target nucleic acid. The experimental results demonstrate that the combined use of Proclin 300 with trans-1,2-cyclohexanediaminetetraacetic acid (trans-1,2-cyclohexanediaminetetraacetic acid) and polyethylene glycol is more effective than Proclin 300 alone with either trans-1,2-cyclohexanediaminetetraacetic acid or polyethylene glycol. In other words, the addition of the virus sample preservation solution from Example 1 of this invention provides good preservation of body fluid samples containing RNA viruses and significantly increases the concentration of extracted viral nucleic acid. Furthermore, comparing the data from Examples 1-4 and Comparative Examples 3-4, it can be seen that the selection of component concentrations in this application has a significant impact on the preservation effect of the samples. When the concentration range of Comparative Examples 3-4 is not within the range of this application, the preservation solution obtained has a poor preservation effect on the virus, and the virus samples have the problem of nucleic acid degradation.

[0061] As shown in Tables 3 and 4, after adding the sample preservation solution of this invention to saliva and serum samples positive for African swine fever virus and storing them at room temperature for 0-7 days, the change in CT values ​​using fluorescent PCR was consistently lower than that of the control group; and the change was greater in Comparative Example 1 than in Comparative Example 2, which in turn was greater in Example 1. In contrast, the control group samples with added PBS showed a significant increase in Ct values ​​after 7 days, indicating severe degradation of the target nucleic acid. African swine fever virus is relatively stable in blood samples, resulting in a smaller increase in Ct values. The experimental results demonstrate that the combined use of Proclin 300 with trans-1,2-cyclohexanediaminetetraacetic acid (TGA) and polyethylene glycol is more effective than using Proclin 300 alone with either TGA or polyethylene glycol. Therefore, the virus sample preservation solution from Example 1 of this invention has a good preservation effect on body fluid samples containing DNA viruses and can significantly improve the concentration of purified viral nucleic acids. Furthermore, comparing the data from Examples 1-4 and Comparative Examples 3-4, it can be seen that the selection of component concentrations in this application has a significant impact on the preservation effect of the samples. When the concentration range of Comparative Examples 3-4 is not within the range of this application, the preservation solution obtained has a poor preservation effect on the virus, and the virus samples have the problem of nucleic acid degradation.

[0062] In summary, the viral preservation solution for bodily fluid samples provided in this application is formulated with sodium chloride, disodium hydrogen phosphate, potassium chloride, and potassium dihydrogen phosphate as a buffer solution. This buffer solution is isotonic, maintaining the morphology of viral particles while maintaining a relative pH, ensuring that each component functions optimally in the optimal pH environment. The liquid biological preservative Proclin 300 is a highly effective sterilizing agent for controlling microbial content. With its broad-spectrum activity, superior compatibility and stability, and low toxicity at its usage concentration, it can eradicate bacteria and fungi for a longer period, inhibit cell growth and metabolism, and promote apoptosis. Trans-1,2-cyclohexanediaminetetraacetic acid (trans-1,2-cyclohexanediaminetetraacetic acid) is a potent metal ion chelating agent that chelates divalent metal cations in the sample, inhibiting the activity of nucleases and some endogenous enzymes, preventing the degradation of viral particles and free nucleic acids. In this application, the combined use of the liquid biological preservative Proclin 300 and trans-1,2-cyclohexanediaminetetraacetic acid maximizes the maintenance of the effective nucleic acid concentration of the virus to be tested, improving the detection rate of PCR detection and facilitating early disease diagnosis. Compared to previous sample preservation solutions, this application adds polyethylene glycol as a virus settling agent. Centrifugation and precipitation concentrate the virus, increasing its concentration and reducing the likelihood of false negatives due to low-concentration samples failing to reach the detection limit of the kit. In terms of usage, simply invert and shake the virus preservation solution thoroughly, then mix it with the body fluid virus sample at a 1:1 volume ratio. Upon receiving the sample, the laboratory should centrifuge it at 12,000 rpm for 10 minutes during sample pretreatment, and then use the precipitate for nucleic acid purification. The virus preservation solution of this invention is simple to prepare, uses readily available raw materials, is stable, has a long shelf life, and is generally convenient to use, making it highly valuable.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A virus preservation solution for bodily fluid samples, characterized in that, The components, based on final concentration, include: sodium chloride 16.0 g / L, disodium hydrogen phosphate 2.84 g / L, potassium chloride 0.4 g / L, potassium dihydrogen phosphate 0.54 g / L, liquid biological preservative Proclin 300 0.2 g / L, trans-1,2-cyclohexanediaminetetraacetic acid 0.72 g / L, and polyethylene glycol 160 g / L, wherein the polyethylene glycol is PEG6000, and the pH of the viral preservation solution for the body fluid sample is 7.2-7.

5.

2. The viral preservation solution for bodily fluid samples according to claim 1, characterized in that, The components also include the remainder water.

3. A method for preparing a viral preservation solution for bodily fluid samples, characterized in that, It includes mixing the components of the viral preservation solution for bodily fluid samples as described in any one of claims 1-2 with water and preparing it to the aforementioned final concentration.

4. The application of the viral preservation solution for bodily fluid samples as described in any one of claims 1-2 in the preparation of viral nucleic acid detection.

5. A viral nucleic acid detection kit, characterized in that, It includes the viral preservation solution, purification reagent and detection reagent for bodily fluid samples as described in any one of claims 1-2.

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