Preservative for room temperature storage of samples of various types of rna viruses

Abstract

This invention provides a preservation solution for preserving various types of RNA virus samples at room temperature, belonging to the field of molecular biology technology. The preservation solution is based on a low-concentration guanidine salt, and incorporates three stabilizers—single-stranded binding protein, trehalose, and BSA—along with a Tris-HCl buffer system, a nonionic surfactant, and EDTA. The low-concentration guanidine salt cleaves and releases viral RNA while inhibiting nucleases, and the single-stranded binding protein actively protects the released viral RNA. Trehalose and BSA provide physical and competitive protection, respectively. This solution not only overcomes the technical bottleneck of 18-day stable preservation of RNA virus samples at room temperature but also is compatible with complex samples such as blood, feces, and tissues, addressing the compatibility issues for various clinical samples and downstream RT-qPCR detection. The preservation solution of this invention provides stable preservation of various RNA virus samples at room temperature for up to 18 days (ΔCt≤1.5), significantly superior to existing high-concentration guanidine salt-based preservation solutions (preservation period only 7 days, 14-day ΔCt>3), making it suitable for clinical nucleic acid testing and large-scale screening applications where high accuracy is required.

Description

Technical Field

[0001] This invention relates to the field of molecular biology technology, and mainly to a preservation solution for long-term preservation of RNA viruses at room temperature, which is particularly suitable for different types of RNA virus samples. In particular, RNA viruses preserved in this preservation solution can be directly used for subsequent nucleic acid detection. Background Technology

[0002] In vitro preservation of RNA viruses is a key step in virological research, disease diagnosis, and epidemiological surveillance. Currently, preserving RNA viruses at room temperature still faces significant challenges. The main difficulty lies in the poor stability of RNA molecules themselves, and the difficulty of simultaneously meeting the requirements of biosafety, nucleic acid integrity, and long-term stable preservation using existing preservation methods. Although various preservation solutions have been developed to extend the in vitro preservation time of RNA viruses, the effective preservation time of samples at room temperature (18–25°C) is generally still short. For example, after SARS-CoV-2 nucleic acid detection samples are placed at 20–22°C for only 1 day, the RT-qPCR detection results may be affected by nucleic acid degradation and become biased (Reference [1]); in saliva samples, SARS-CoV-2 RNA can remain stable for about 24 hours at 25°C (Reference [2]). The preservation time can be extended by drying. For example, inactivated SARS-CoV-2 RNA can be fixed on a silk fibroin matrix and can be stably preserved at room temperature for more than 21 weeks (about 6 months) (Reference [3]); dengue virus RNA can be extracted and dried on a glass fiber membrane using the RNAES method and can be preserved at ambient temperature for 35 days (Reference [4]). However, the operation process of drying preservation method is complicated and the application scenario is different from that of conventional liquid preservation solution, so its practical application is limited.

[0003] Currently, virus sample preservation techniques are divided into cryopreservation and room temperature preservation based on their dependence on the cold chain. 1) Cryopreservation is the most mature and widely used method. By placing the sample directly in an environment of -20℃, -70℃ or even liquid nitrogen (-196℃), the rate of viral metabolism and nucleic acid degradation can be significantly reduced, effectively maintaining the integrity and infectivity of the virus (reference [5]). In particular, ultra-low temperature conditions of -70℃ and below can achieve long-term preservation for months or even years, which is suitable for scientific research, vaccine development and virus strain bank construction with high requirements for viral infectivity. However, cryopreservation is highly dependent on professional equipment and strict cold chain management. It is costly and complicated to operate, and it is difficult to promote and popularize in areas with limited resources or remote areas. Furthermore, repeated freezing and thawing may damage virus particles and affect sample quality. 2) Room temperature preservation, which is further divided into inactivated preservation and non-inactivated preservation according to the mechanism of action: ① Inactivated preservation can inactivate the virus and inhibit nuclease activity through chemical reagents, thereby protecting the integrity of viral nucleic acid at room temperature (Reference [6]); ② Non-inactivated preservation can achieve room temperature transportation without relying on the cold chain by optimizing the freeze-drying scheme of the freeze-drying protectant components, reducing logistics costs and biosafety risks, and is particularly suitable for the collection and transportation of highly pathogenic virus samples, such as SARS-CoV-2 (Reference [7]). However, the process threshold for freeze-drying and thawing is high and the popularity is low. The chemical reagents used in room temperature inactivation preservation include high concentrations of guanidine salts, alcohols, etc.; guanidine salts protect nucleic acids by destroying viral structures and proteins and inhibiting RNase activity. For example, rotavirus RNA can still be successfully amplified 100% after being stored in guanidine salt buffer for 180 days (reference [8]); alcohols protect nucleic acids through dehydration and denaturation, but they are volatile and not only have poor RNA preservation effect, but their buffer system also has weak resistance to environmental interference; although room temperature inactivation claims to be able to stably preserve samples for 3-7 days at room temperature (20-25℃), which is the mainstream preservation method and is suitable for RT-qPCR detection, the room temperature preservation period is usually only a few days to a week, which is difficult to meet the needs of long-term transportation or storage. In addition, there are some non-inactivation preservation methods that emphasize maintaining viral activity and structural integrity without inactivating the virus, and inhibiting microbial contamination and nucleic acid degradation. However, due to the limited room temperature storage time, they still cannot get rid of the strong dependence on the cold chain and are only suitable for specific scenarios such as clinical diagnosis and virus isolation and culture. Although the new biopolymer technology fixes biological materials by replacing water molecules in the sample, it can achieve the purpose of storing samples at room temperature without refrigeration, but it is limited to use in professional laboratories and has a lower application popularity (reference [9]). It can be seen that virus sample preservation technologies, whether dependent on the cold chain or requiring inactivation, each have specific application scenarios and different technical advantages, and non-inactivation preservation schemes also have biosafety risks.

[0004] Current mainstream methods for inactivating and preserving RNA viruses at room temperature have significant performance limitations. They rely on components such as guanidine salts, alcohols, and buffer salts, making it difficult to simultaneously meet the requirements of biosafety, low cost, ease of operation, and long-term preservation at room temperature for more than one week. Among these, high-concentration (3 mol / L~6 mol / L) guanidine salt preservation products have the largest market share. However, high-concentration guanidine salts are classified as hazardous materials, exhibiting strong irritant and corrosive properties, high storage and transportation costs, strict control, and stringent operational protection requirements. Residual guanidine salt components can reduce detection sensitivity by inhibiting enzyme activity in downstream RT-qPCR reactions. Furthermore, guanidine salts cannot prevent the spontaneous hydrolysis of RNA single strands, resulting in a stable preservation time of only 3–7 days at room temperature, and high-temperature environments can accelerate RNA degradation. While low-concentration (0.5 mol / L~1.5 mol / L) guanidine salt preservation methods have emerged, reducing toxicity, corrosivity, and storage and transportation costs while retaining basic virus inactivation and antibacterial capabilities, they cannot inhibit non-enzymatic autohydrolysis or stabilize RNA single-stranded structures. RNA still degrades rapidly at room temperature. Furthermore, the reduced guanidine salt concentration leads to poor persistence of inactivation and antibacterial effects, enabling only very short-term sample preservation. This falls short of the requirement for long-term room temperature preservation of 14 days or more, and cannot meet the needs of sampling and transportation in remote areas or without cold chain conditions. Therefore, at room temperature, none of the above preservation methods can simultaneously achieve the goals of safe, efficient, stable, and long-term preservation.

[0005] Different types of RNA virus biological samples, such as blood, feces, and tissues, are difficult to preserve using a uniform method or solution. Typically, anti-interference or anti-degradation components need to be designed specifically based on the differences in the core components of each sample. Specifically: for blood samples, the focus is on anticoagulation and interference removal; for fecal samples, the key is sterilization and de-inhibition; and for tissue samples, the emphasis is on rapid penetration and deep RNase inhibition. In actual research and development, guanidine salts, which are inactivation / stabilization base systems, are usually used, and specific functional modules are added according to the sample type to achieve targeted optimization of preservation. Currently, there is no universal "one-size-fits-all" preservation solution or method. Therefore, there is an urgent need to develop a universal preservation solution suitable for room temperature conditions and compatible with multiple RNA virus sample types. This preservation solution should not only be easy to operate but also achieve three core objectives: first, to fully guarantee safety and stability, ensuring the biosafety of samples during transportation and storage; second, to significantly extend the preservation period, overcoming the technical bottleneck of stable preservation at room temperature for more than 14 days; and third, to be highly compatible with different sample types and adaptable to downstream RT-PCR nucleic acid detection, ensuring high-sensitivity detection stability.

[0006] References: [1] Effects of Storage Temperature and Media / Buffer for SARS-CoV-2 Nucleic Acid Detection. Namhee Kim et al., *American Journal of Clinical Pathology*, 2021(2), pp. 280-285, July 23, 2021; doi:10.1093 / ajcp / aqaa207; [2] Meals and Room Temperature Storage do not Significantly Affect Feasibility of Direct RT-PCR Tests for SARS-CoV-2 Using Saliva. Koji Tsuchiya et al., *Clinical Laboratory*, 2022; 68(6); doi:10.7754 / Clin.Lab.2021.210451; [3] Long-Term Preservation of SARS-CoV-2 RNA in Silk for Downstream RT-PCR Tests. Raphael Nyaruaba et al., *Analytical Chemistry*, 2022, 94 (10), pp. 4522-4530, 2022; doi:10.1021 / acs.analchem.2c00169; [4] Simple and Economical Extraction of Viral RNA and Storage at Ambient Temperature. Sarah Hernandez et al., *Microbiology Spectrum*, 2022, 10(3), pp. 2165-0497, June 1, 2022; doi:10.1128 / spectrum.00859-22; [5] Crucial aspects for maintaining rAAV stability. Johannes Lengler et al., *Scientific Reports*, 2022, 14(1):27685, November 12, 2024; doi:10.1038 / s41598-024-79369-0; [6] Standardized preservation, extraction and quantificationtechniques for detection of fecal SARS-CoV-2 RNA. Aravind Natarajan et al., "Nature Communications" (2021) 12:5753, October 1, 2021; doi:10.1038 / s41467-021-25576-6; [7] Development and evaluation of a lyophilization protocol for colorimetric RT-LAMP diagnostic assay for COVID-19.‌ Nayra Oliveira Prado et al., "Scientific Reports" (2024) 14:10612, May 9, 2024; doi:10.1038 / s41598-024-61163-7; [8] Comparison of storage methods for reverse-transcriptase PCRamplification of rotavirus RNA from gorilla (Gorilla g. gorilla) fecalsamples. Christopher A Whittier et al., Journal of Virological Methods (2004) 116: pp. 11–17, 2004; doi:10.1016 / j.jviromet.2003.10.003; [9] Biopolymers for sample collection, protection, and preservation. Iryna Sorokulova et al., Applied Microbiology and Biotechnology, 2015, 99(13): 5397–5406, May 19, 2015; doi:10.1007 / s00253-015-6681-3. Summary of the Invention

[0007] In order to overcome the above problems, the present invention provides a preservation solution that can break through the technical bottleneck of stably preserving RNA viruses at room temperature for more than 14 days.

[0008] Another objective of this invention is to provide a preservation solution that can overcome the differences in the sources of preserved RNA virus samples.

[0009] The present invention also aims to provide a preservation solution that is compatible with and can be directly applied to subsequent nucleic acid testing of preserved RNA virus samples.

[0010] To achieve the above-mentioned objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a preservation solution for preserving RNA virus samples at room temperature, comprising a denaturant, a stabilizer, a pH buffer, a surfactant, and a chelating agent; wherein the denaturant is a low-concentration guanidine salt; the stabilizer comprises 0.1-1.0 μmol / L of single-chain binding protein, 1%-10% (m / v) of trehalose, and 0.1%-0.5% (m / v) of BSA; the pH buffer is a buffer solution with a pH value stable within the physiological pH range of 7.4±0.1, and a concentration range of 20-50 mmol / L; and the surfactant is a nonionic surfactant with a volume percentage (v / v) concentration of 0.1%-1%.

[0011] According to a preferred embodiment of the present invention, the RNA virus sample is blood, feces, or tissue.

[0012] According to a preferred embodiment of the present invention, the remaining components in the preservation solution, except for denaturant, stabilizer, pH buffer, surfactant, and chelating agent, are enzyme-free sterile water.

[0013] According to a preferred embodiment of the present invention, in the preservation solution, the low-concentration guanidine salt is selected from either guanidine isothiocyanate or guanidine hydrochloride, and the added concentration is controlled in the range of 0.5-1 mol / L.

[0014] According to a preferred embodiment of the present invention, the pH buffer in the preservation solution is a Tris-HCl buffer solution, wherein the chemical name of Tris-HCl is tris(hydroxymethyl)aminomethane hydrochloride.

[0015] According to a preferred embodiment of the present invention, the chelating agent in the preservation solution is 1 mmol / L EDTA, wherein the chemical name of EDTA is ethylenediaminetetraacetic acid.

[0016] According to a preferred embodiment of the present invention, the nonionic surfactant in the preservation solution is Triton X-100, Tween 20, or ethyl phenyl polyethylene glycol.

[0017] Preferably, the denaturant in the preservation solution is 0.75 mol / L guanidine isothiocyanate.

[0018] Preferably, the stabilizer in the preservation solution is 0.3 μmol / L single-chain binding protein, 5% trehalose by mass and volume, and 0.2% BSA by mass and volume.

[0019] Preferably, the pH buffer in the preservation solution is a 35 mmol / L Tris-HCl buffer solution.

[0020] Preferably, in the preservation solution, the surfactant is Triton X-100 at a volume percentage of 0.1%-1%, where Triton X-100 is abbreviated as Triton X-100.

[0021] In a second aspect, the present invention provides an apparatus for preserving RNA virus samples, comprising the preservation solution for preserving RNA virus samples at room temperature as described in the first aspect.

[0022] According to a preferred embodiment of the present invention, the device for preserving RNA virus samples is a disposable virus sampling tube.

[0023] Thirdly, the present invention provides a method for preparing the preservation solution for preserving RNA virus samples at room temperature as described in the first aspect. The preparation steps are as follows: 1) In a pH buffer, denaturant, surfactant, chelating agent, SSB, trehalose, and BSA are added sequentially and stirred until completely dissolved and mixed; 2) The pH is finely adjusted to 7.4±0.1 with the pH buffer, and then diluted to volume with sterile water and mixed evenly; 3) After filtration and sterilization using a 0.22μm aqueous filter membrane, the solution is dispensed and stored.

[0024] According to a preferred embodiment of the present invention, the denaturant, surfactant, chelating agent, SSB, trehalose, BSA and sterile water are all selected as enzyme-free reagents.

[0025] Fourthly, the present invention provides a method for using the preservation solution for preserving RNA virus samples at room temperature as described in the first aspect, wherein the preservation solution for preserving RNA virus samples at room temperature is mixed evenly with the RNA virus sample that needs to be preserved at room temperature according to a volume ratio.

[0026] According to a preferred embodiment of the present invention, the RNA virus sample that needs to be stored at room temperature is a pretreated sample working solution.

[0027] Preferably, when the RNA virus sample is a blood sample, it can be used directly as the working solution without pretreatment.

[0028] Preferably, when the RNA virus sample is a fecal sample, pretreatment is required. The pretreatment steps are as follows: use a sterile sampling spoon to pick up about 5 grams (pea-sized) of fresh fecal sample, add PBS or physiological saline, and then vortex to make a uniform suspension as the sample working solution.

[0029] Preferably, when the RNA virus sample is a tissue sample, pretreatment is required. The pretreatment steps are as follows: first, wash with sterile saline, then cut the tissue block into the smallest possible pieces with a sterile scalpel, then add the tissue pieces to PBS or saline and grind them into a homogenate under sterile conditions, and use the homogenate as the working solution for the sample.

[0030] According to a preferred embodiment of the present invention, the volume ratio is greater than 4:1.

[0031] Fifthly, the present invention also claims protection for the preservation solution for preserving RNA virus samples at room temperature as described in the first aspect, and the application of the apparatus for preserving RNA virus samples as described in the second aspect in the room temperature preservation and nucleic acid detection of RNA virus samples.

[0032] The beneficial effects of this invention are: This invention provides a preservation solution for storing various RNA virus samples at room temperature. This solution is based on a low-concentration guanidine salt, and incorporates three stabilizers—single-stranded binding protein, trehalose, and BSA—along with a Tris-HCl buffer system, a nonionic surfactant, and EDTA. The components do not contain high-concentration, highly corrosive, or inhibitory components for subsequent nucleic acid detection. Verification has shown that the preservation solution optimized by this invention overcomes the technical bottleneck of stabilizing RNA virus samples at room temperature for more than 14 days, achieving an actual stable preservation period of at least 18 days. The preservation solution releases viral RNA through cleavage with a low-concentration guanidine salt and inhibits nucleases. The single-stranded binding protein actively protects the released viral RNA, while trehalose and BSA provide physical and competitive protection, respectively. This makes the preservation solution compatible with complex samples such as blood, feces, and tissues, specifically reducing matrix interference from different types of clinical samples. It synergistically overcomes and solves the compatibility issues between various types of clinical samples and downstream RT-qPCR detection. The performance of the preservation solution of this invention (ΔCt≤1.5) is significantly superior to conventional high-concentration solutions (3 mol / L~6 mol / L). A guanidine salt-type preservation solution (mol / L) is suitable for clinical nucleic acid testing and large-scale screening applications.

[0033] The preservation solution formulation provided by this invention includes a denaturant, a stabilizer, a pH buffer, a surfactant, and a chelating agent. In a preferred formulation, the denaturant is a guanidine salt, preferably guanidine isothiocyanate, and secondarily guanidine hydrochloride. Verification has shown that the minimum concentration range for balancing guanidine salt cleavage and RNA stability is 0.5-1 mol / L, with the optimal equilibrium concentration of guanidine isothiocyanate preferably being 0.75 mol / L. The stabilizers include SSB, trehalose, and BSA. The minimum effective and optimal working concentration of SSB for protecting RNA integrity is preferably 0.1-1.0 μmol / L; trehalose acts as both a nucleic acid protectant and an osmotic pressure regulator, with a preferred mass-volume percentage concentration ((m / v)%) ranging from 1% to 10%; and the preferred mass-volume percentage concentration ((m / v)%) of BSA added to reduce adsorption and provide protein protection ranges from 0.1% to 0.5%. The preferred pH buffer is Tris-HCl buffer (pH 7.4). The key buffer concentration for maintaining a stable pH of 7.4 is 20-50 mmol / L, with an optimal concentration of 35 mmol / L. The preferred surfactant is a nonionic surfactant. Validated components include Triton X-100, Tween-20, and NP-40, all of which can assist in the cleavage of guanidine salts to disrupt lipid membranes and are compatible with subsequent purification and nucleic acid detection steps. Triton X-100 is the preferred component, with a preferred volume percentage (v / v) concentration range of 0.1%-1.0%. Considering both economy and synergistic effect, the optimal concentration is 0.1%. The preferred chelating agent is 1 mmol / L EDTA, used to bind magnesium, calcium, and other metal ions in blood samples. EDTA can also bind metal ions released after tissue sample homogenization, further inhibiting the activity of nucleases that may be present in the sample by blocking metal ions.

[0034] The preferred preservation solution formulation of this invention, while still based on guanidine salts, uses ultra-low concentrations of guanidine salts to avoid inhibiting subsequent PCR amplification, making it compatible with room temperature preservation of completely different types of RNA viruses. The main function of guanidine salts as denaturants is to achieve virus inactivation, nuclease inactivation, and nucleic acid release. On the one hand, guanidine salts are suitable for different types of biological samples (blood, feces, tissues, etc.), all possessing the most basic ability to lyse viruses and release RNA, and acting as RNase inhibitors to strongly inactivate various RNases in the sample, completely blocking the enzymatic degradation pathway of RNA and inhibiting RNases from degrading RNA. On the other hand, low concentrations of guanidine salts can achieve the most basic purpose of virus inactivation by gently lysing RNA virus particles to release naked single-stranded RNA and denaturing viral capsid proteins, without causing SSB protein denaturation and inactivation, thus providing favorable conditions for SSB protein to target and bind to nucleic acids.

[0035] This invention's preservation solution utilizes the organic combination and synergistic effect of four key components: guanidine salt, SSB, trehalose, and BSA, to construct a multi-level, multi-target virus inactivation and RNA protection system. Guanidine salt, as a strong protein denaturant, can rapidly lyse the viral envelope and capsid, penetrate tissue cells, and release nucleic acids. Simultaneously, it broadly inhibits endogenous and exogenous ribonucleases and proteases in the sample, forming the core basis for efficient inactivation and preliminary viral RNA preservation. SSB, as a single-stranded nucleic acid-binding protein, is introduced into the preservation solution system for the first time. It can actively and reversibly bind to exposed viral single-stranded RNA, masking potential enzyme cleavage sites and inhibiting secondary structure formation, directly maintaining RNA integrity and linear length at the molecular level—a functional active protection targeting RNA. Trehalose, as a non-reducing disaccharide, possesses both chemical stability and osmotic pressure regulation functions. It can form a stable glassy hydration layer around RNA and protein molecules, reducing the degrees of freedom of molecular motion through a water substitution mechanism. This prevents degradation or aggregation during dehydration, temperature fluctuations, or long-term storage. Simultaneously, it maintains an isotonic environment in the preservation solution to avoid the release of endogenous nucleases from ruptured sampling cells, thus providing broad-spectrum physical passive protection. BSA, on the other hand, utilizes surface adsorption blocking and competitive binding mechanisms. It preferentially occupies non-specific binding sites on the sampling tube wall and consumable surfaces to reduce the physical loss of viral particles or nucleic acids. It also competitively binds to any residual trace amounts of proteases, impurities, and other interfering substances in the system, causing these substances to preferentially act on BSA rather than the target viral RNA or viral proteins. This represents indirect protection through a dual mechanism of sacrificial and competitive action. The four key components—guanidine salt, SSB, trehalose, and BSA—work synergistically from four dimensions: chemical denaturation and lysis, targeted binding protection, physical layer stabilization, and competitive clearance of interfering substances. This forms a complete protection chain of "lysis and inhibition—active binding—passive isolation—competitive clearance," ensuring efficient viral denaturation and lysis, long-term stable preservation of viral RNA at room temperature, and compatibility and detection sensitivity with downstream RT-PCR detection. This overcomes the technical difficulties of achieving compatibility and coordination between inactivation efficiency, nucleic acid stability, and detection sensitivity in single-component or traditional high-concentration guanidine salt-based preservation solutions.

[0036] Furthermore, the stabilizers preferably added to the preservation solution of this invention include a specific combination of three: single-chain binding protein (SSB), trehalose, and BSA. In particular, this invention innovatively combines SSB with a low-concentration guanidine salt inactivated virus preservation solution to address the short room temperature preservation period of RNA viruses in the prior art, aiming to achieve a breakthrough in room temperature preservation time of more than 14 days. However, the negative control verification experiment found that without any of the stabilizers SSB, trehalose, or BSA, the preservation solution could only stably preserve RNA viruses at room temperature for 14 days (ΔCt≤1.5) for subsequent stable nucleic acid detection. Only when SSB, trehalose, and BSA were all present could the preservation solution of this invention effectively preserve RNA virus samples at room temperature for more than 14 days, extending to at least 18 days. This shows that there is an irreplaceable synergistic effect among the three stabilizers SSB, trehalose, and BSA. It is speculated that SSB mainly protects single-stranded RNA specifically at the molecular level, trehalose mainly stabilizes the physical and chemical environment, and BSA mainly reduces non-specific loss. Therefore, SSB, trehalose, and BSA work synergistically from three different dimensions, thereby comprehensively improving the preservation stability of guanidine salt-cleaved viral RNA.

[0037] This invention innovatively introduces a single-stranded DNA-binding protein (SSB) as a core stabilizer in the preservation solution. SSBs are proteins that specifically bind to single-stranded nucleic acids, known for their high affinity for single-stranded DNA. However, research has shown that they can also bind to and protect single-stranded RNA. In routine molecular detection applications, SSBs are mainly used for amplification reactions such as PCR, qPCR, RT-PCR, and RT-qPCR to improve amplification efficiency and detection sensitivity by reducing non-specific amplification. Their conventional addition concentration is typically 0.1–1.0 μg / μL (equivalent to 5.6–55.6 μmol / L). This invention breaks through the traditional use of SSBs solely as additives for amplification reactions, applying them for the first time at a much lower concentration (0.1–1 μmol / L) in RNA virus preservation solutions, achieving specific coating and protection of viral single-stranded RNA. The mechanism of action of SSB in the preservation solution of this invention is as follows: SSB selectively binds to exposed viral single-stranded RNA, blocking the 2'-hydroxyl active site in the RNA backbone that is prone to nucleophilic attack, effectively inhibiting the inherent non-enzymatic autohydrolysis reaction of RNA from the source. This blocking protection mechanism makes up for the problem that the preservation solution of low concentration guanidine salt only relies on its chemical denaturation to inhibit RNase, but cannot block the rapid degradation caused by the chemical instability of RNA molecules themselves. It provides comprehensive protection for viral RNA from enzymatic degradation to chemical autohydrolysis, and finally, together with other components, achieves the technical breakthrough of stable preservation of viral RNA for more than 14 days at room temperature.

[0038] In this invention, the denaturing agents, stabilizers (SSB, trehalose, BSA), pH buffers, surfactants, and chelating agents in the preservation solution collectively construct a highly efficient synergistic network suitable for different types of clinical samples: low-concentration guanidine salts act as denaturing agents, rapidly lysing the viral envelope and capsid while broadly inhibiting RNase activity, providing basic preservation for various samples. Among the stabilizers, SSB provides active targeted protection against naked single-stranded RNA on the basis of guanidine salts, effectively preventing RNA auto-hydrolysis, especially suitable for complex samples with a large amount of exposed RNA after cell lysis (such as blood, feces, and tissue homogenates); trehalose provides physical isolation protection for RNA and proteins by forming a glassy hydration layer, while maintaining the isotonic physical and chemical environment of the preservation solution, avoiding the release of endogenous enzymes from blood cell rupture in blood samples, and also reducing additional damage to cell debris in fecal and tissue samples; BSA preferentially adsorbs onto the tube wall and consumable surface through competitive binding, reducing non-specific loss of viral particles and neutralizing interference from residual trace amounts of proteases in the sample. Tris-HCl buffer, as a preferred pH buffer adapted to physiological conditions, can maintain pH stability for different samples in the preservation solution environment. EDTA, as a preferred chelating agent, can inhibit metal-dependent nucleases by integrating metal ions. Triton X-100, as a preferred nonionic surfactant, can weaken the lipid bilayer structure of cell membranes and viral envelopes, facilitating the full release of viral nucleic acids from cells; at the same time, by selecting an appropriate concentration, it avoids excessive disruption of the spatial conformation of protein protective components (SSB, BSA), ensuring that the nucleic acid stability system is not affected. Thus, the multi-component synergistic network of the preservation solution of this invention achieves efficient removal of anticoagulants and PCR interferences for blood samples (containing anticoagulant interference), achieves strong microbial inactivation and neutralization of inhibitors such as humic acid for fecal samples (containing a large number of PCR inhibitors and microbial flora), and can rapidly penetrate and deeply inhibit RNases for tissue samples (with dense structures requiring full lysis), perfectly adapting to the differentiated preservation needs of three different types of clinical samples.

[0039] The preservation solution provided by this invention for preserving RNA virus samples at room temperature is based on a low-concentration guanidine salt and incorporates a low-concentration saline solution (SSB). This overcomes the limitations of traditional technologies that rely on high-concentration guanidine salts or only inhibit enzymatic degradation of RNA. By targeting and blocking the non-enzymatic self-hydrolysis pathway of RNA, it achieves the core objective of stable preservation of RNA virus samples at room temperature for more than 14 days. Simultaneously, it avoids the industry pain points associated with high-concentration guanidine salts, such as strong corrosivity, strict storage and transportation control, and inhibition of downstream PCR reactions. The preservation solution of this invention achieves stable preservation of different types of RNA virus samples for more than 18 days (ΔCt≤1.5) at room temperature through a multi-component synergistic network, significantly outperforming the 7-day preservation period of existing commercial products. The preservation solution of this invention has broad application prospects in clinical nucleic acid testing: in primary healthcare and resource-limited areas, it can significantly reduce the dependence on cold chain transportation for samples, lower the logistics costs of long-distance sample transportation, and improve the accessibility of cross-regional transfer testing; in scenarios of sudden outbreaks and large-scale screening, it can effectively alleviate the pressure on low-temperature storage equipment and transportation capacity, and improve the organizational efficiency of large-scale testing; in the construction of biobanks, it can provide a reliable technical solution for the long-term room temperature preservation of RNA biological samples; in addition, given the good preservation effect of the preservation solution on RNA viruses with poor stability such as HIV-1, it can also provide strong technical support for infectious disease monitoring, epidemiological investigation, and entry-exit inspection and quarantine. In summary, this invention has significant practical value and broad market prospects in improving the convenience of nucleic acid testing, reducing testing costs, ensuring testing quality, and expanding testing coverage. Specifically, for virus types, it provides a novel technical solution for the clinical testing and epidemiological monitoring of various common RNA viruses such as respiratory viruses and enteroviruses, possessing extremely high clinical promotion value and public health application prospects. Detailed Implementation

[0040] Exemplary embodiments of the present invention are described in detail below, wherein the same or similar terms represent the same concepts, such as single-chain binding protein = SSB, bovine serum albumin = BSA, hepatitis E virus = HEV, novel coronavirus = SARS-CoV-2, human immunodeficiency virus = HIV, and tris(hydroxymethyl)aminomethane hydrochloride buffer = Tris-HCl buffer. In the following detailed description, numerous specific details are set forth to provide a full understanding of the embodiments disclosed herein for ease of explanation. The following examples are for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available. Specifically, the third-party reagents used to compare and verify the preservation solution formulation of this invention were: disposable virus sampling tubes (Luwei Medical Device Registration No. 20200172) (guanidine salt-containing, inactivated, 3mL) purchased from Haiweigao Biotechnology Co., Ltd.; Hepatitis E Virus RNA Detection Kit (PCR Fluorescence Method) (National Medical Device Registration No. 20193401917) purchased from Aikon Biotechnology (Hangzhou) Co., Ltd.; Novel Coronavirus Nucleic Acid Detection Kit (PCR-Fluorescent Probe Method) (National Medical Device Registration No. 20233400645) purchased from Beijing Wantai Biological Pharmacy Co., Ltd.; Human Immunodeficiency Virus Type 1 (HIV-1) Nucleic Acid Detection Kit (National Medical Device Registration No. 20263400710) (PCR-Fluorescent Probe Method) purchased from Beijing Wantai Biological Pharmacy Co., Ltd.; and a real-time fluorescence quantitative PCR instrument (model ABI7500) purchased from Thermo Fisher Scientific. However, it is obvious that one or more embodiments can be implemented without these specific details, and conditions not specifically specified in the embodiments should be performed under standard conditions or conditions recommended by the manufacturer. Molecular biology experimental methods not specifically described in the following embodiments are performed according to the specific methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition), or according to the kit and product instructions.

[0042] The application objects measured in this invention refer to biological samples of bodily fluids collected from human or animal bodies. After the sample collection is completed, the biological sample has been separated from the living human or animal body. For example, blood samples (whole blood / serum / plasma), excrement (feces), tissues (pig liver), bodily fluids, etc., are all non-living biological samples separated from the body. The measurement process is completed in vitro. The direct purpose of the measurement is to determine whether viral RNA (such as HEV RNA, SARS-CoV-2 RNA, HIV-1 RNA) or fragments derived from the partial degradation of the target nucleic acid are present in the sample. The detection described in this invention is only used to assist in determining whether the living organism from which the sample originates has RNA viruses. However, the entire detection process does not involve any process that can directly obtain a disease diagnosis result or health status. Therefore, this invention does not belong to the diagnostic method for diseases and meets the basic requirements of the Patent Law for the subject matter of patent protection. The specific embodiments of this invention are described below.

[0043] Example 1: Formulation design of the preservation solution.

[0044] The formulation design aims to minimize the adverse effects of the preservation solution on subsequent nucleic acid detection. The preferred denaturant in this invention is a low-concentration (0.5-2 mol / L) guanidine salt. This is because guanidine salts can lyse blood cells, fecal microorganisms, and viruses to release viral RNA. They can rapidly penetrate the internal cells of tissue blocks, immediately denature and fix proteins, and inhibit RNases in different samples or inactivate RNase / proteases to prevent enzymatic degradation of RNA. Therefore, they can be broadly used as a denaturant, inactivator, and RNase inhibitor in blood, fecal, and tissue samples. In addition to using a low-concentration guanidine salt denaturant, to compensate for the insufficient RNase inhibition effect of low concentrations, 1 mmol / L EDTA (ethylenediaminetetraacetic acid) is added as a chelating agent. EDTA can integrate metal ions in the system, thereby assisting in the inhibition of ion-dependent nucleases. Furthermore, EDTA can replace heparin as an anticoagulant for blood samples, avoiding the influence of heparin on subsequent PCR amplification reactions. To accommodate diverse clinical samples such as blood, feces, and tissue, the preferred pH buffer is Tris-HCl buffer covering the physiological pH range (pH 7.4 ± 0.1). This buffer overcomes the complexity of different clinical samples, maintains a stable, weakly alkaline physiological environment over a long period, prevents non-enzymatic autohydrolysis of RNA due to drastic pH fluctuations, and does not inhibit Taq enzyme activity in downstream RT-PCR, perfectly meeting the end-to-end buffering requirements of virus preservation solutions. Nonionic surfactants, such as Triton, are preferred. X-100, Tween-20, NP-40, etc., do not contain charged groups and can further assist in the gentle destruction of viral envelopes and cell lysis at low concentrations, while dissolving and inactivating RNase, further compensating for the insufficient inhibitory effect of low-concentration guanidine salts on RNase. In addition, nonionic surfactants can also be adapted to different clinical samples such as blood, feces, and tissues: For blood samples, they can gently lyse blood cells and efficiently release viral nucleic acids encapsulated in cells, without releasing large amounts of hemoglobin like strong denaturants, significantly reducing pigment interference in subsequent PCR; For fecal samples, they can destroy the cell membranes of microorganisms in feces, assisting guanidine salts to achieve rapid sterilization, while dissolving lipid impurities in feces, reducing lipid encapsulation of nucleic acids, and allowing nucleic acids to be fully released; For tissue samples, they can significantly reduce the surface tension of the preservation solution, greatly increasing the penetration rate of the liquid into the interior of dense tissue blocks, assisting in the deep inactivation of highly active RNases in tissues, and further avoiding enzymatic degradation of RNA; Moreover, low concentrations (≤1%) of nonionic surfactants will not adversely affect the subsequent nucleic acid detection reaction and sensitivity.

[0045] To overcome the technical bottleneck of stable preservation at room temperature for more than 14 days, this invention explores in depth how to maintain the stability of viral RNA released from different types of clinical samples by adding stabilizers. Through preliminary screening, the added components are mainly focused on optimizing the addition of three components: single strand DNA-binding protein (SSB), trehalose, and bovine serum albumin (BSA). The concentration of the formula is optimized and adjusted according to the effect. The design concept will be analyzed in detail later.

[0046] The preliminary preferred formulation of the preservation solution of this invention includes a denaturant, a pH buffer, a surfactant, a chelating agent, and a stabilizer. The denaturant is preferably a low concentration of guanidine salt, at a concentration of 0.5-1 mol / L, and the guanidine salt is preferably guanidine isothiocyanate or guanidine hydrochloride. The pH buffer is preferably a Tris-HCl buffer solution, with a pH buffering range preferably weakly alkaline and covering the physiological pH range of pH 7.4 ± 0.1. The preferred concentration range of the pH buffer is 20-50 mmol / L, where 20 mmol / L is close to the lower limit of the physiological pH buffer, and 50 mmol / L provides a stronger buffering capacity to resist pH fluctuations caused by the sample itself or guanidine salt cleavage, and also ensures that RNA remains stable under optimal pH conditions. The surfactant is preferably a low-concentration nonionic surfactant with a volume percentage ((v / v)%) of 0.1%-1%, preferably Triton X-100 (polyethylene glycol octylphenyl ether), Tween-20 (polysorbate-20), or ethylphenyl polyethylene glycol (NP-40). The chelating agent is preferably 1 mmol / L ethylenediaminetetraacetic acid (EDTA). The stabilizer preferably comprises three components: 0.1-1.0 μmol / L single-chain binding protein (SSB), 1%-10% trehalose ((m / v)%), and 0.1%-0.5% BSA ((m / v)%).

[0047] Based on the above-mentioned preferred formulations confirmed by preliminary experiments, the following 37 specific formulations were designed, and subsequent specific indicators and optimization verifications were carried out based on these 37 formulations.

[0048] The preservation solution of Formula 1 contains the following components: guanidine isothiocyanate 0.5 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0049] Formula 2's preservation solution contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0050] Formula 3's preservation solution contains the following components: guanidine isothiocyanate 1 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0051] Formula 4's preservation solution contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.1 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0052] Formula 5's preservation solution contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.5 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0053] Formula 6's preservation solution contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 1.0 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0054] The preservation solution of Formula 7 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 1% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0055] Formula 8's preservation solution contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 3% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0056] Formula 9's preservation solution contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 10% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0057] The preservation solution of Formula 10 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.1% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0058] The preservation solution of Formula 11 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.3% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0059] The preservation solution of Formula 12 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0060] The preservation solution of Formula 13 contains the following components: guanidine isothiocyanate 0.5 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.1 μmol / L, trehalose 1% (m / v), bovine serum albumin (BSA) 0.1% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0061] The preservation solution of Formula 14 contains the following components: guanidine isothiocyanate 0.5 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.1 μmol / L, trehalose 1% (m / v), bovine serum albumin (BSA) 0.5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0062] Formula 15 contains the following components: guanidine isothiocyanate 0.5 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.1 μmol / L, trehalose 10% (m / v), bovine serum albumin (BSA) 0.1% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0063] The preservation solution of Formula 16 contains the following components: guanidine isothiocyanate 0.5 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.1 μmol / L, trehalose 10% (m / v), bovine serum albumin (BSA) 0.5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0064] The preservation solution of Formula 17 contains the following components: guanidine isothiocyanate 0.5 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 1.0 μmol / L, trehalose 1% (m / v), bovine serum albumin (BSA) 0.1% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0065] The preservation solution of Formula 18 contains the following components: guanidine isothiocyanate 0.5 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 1.0 μmol / L, trehalose 1% (m / v), bovine serum albumin (BSA) 0.5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0066] The preservation solution of Formula 19 contains the following components: guanidine isothiocyanate 0.5 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 1.0 μmol / L, trehalose 10% (m / v), bovine serum albumin (BSA) 0.1% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0067] The preservation solution of Formula 20 contains the following components: guanidine isothiocyanate 0.5 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 1.0 μmol / L, trehalose 10% (m / v), bovine serum albumin (BSA) 0.5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0068] The preservation solution of Formula 21 contains the following components: guanidine isothiocyanate 1.0 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.1 μmol / L, trehalose 1% (m / v), bovine serum albumin (BSA) 0.1% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0069] The preservation solution of Formula 22 contains the following components: guanidine isothiocyanate 1.0 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.1 μmol / L, trehalose 1% (m / v), bovine serum albumin (BSA) 0.5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0070] The preservation solution of Formula 23 contains the following components: guanidine isothiocyanate 1.0 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.1 μmol / L, trehalose 10% (m / v), bovine serum albumin (BSA) 0.1% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0071] The preservation solution of Formula 24 contains the following components: guanidine isothiocyanate 1.0 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.1 μmol / L, trehalose 10% (m / v), bovine serum albumin (BSA) 0.5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0072] Formula 25's preservation solution contains the following components: guanidine isothiocyanate 1.0 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 1.0 μmol / L, trehalose 1% (m / v), bovine serum albumin (BSA) 0.1% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0073] The preservation solution of Formula 26 contains the following components: guanidine isothiocyanate 1.0 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 1.0 μmol / L, trehalose 1% (m / v), bovine serum albumin (BSA) 0.5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0074] The preservation solution of Formula 27 contains the following components: guanidine isothiocyanate 1.0 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 1.0 μmol / L, trehalose 10% (m / v), bovine serum albumin (BSA) 0.1% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0075] The preservation solution of Formula 28 contains the following components: guanidine isothiocyanate 1.0 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 1.0 μmol / L, trehalose 10% (m / v), bovine serum albumin (BSA) 0.5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0076] The preservation solution of Formula 29 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 20 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0077] The preservation solution of Formula 30 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 50 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0078] The preservation solution of Formula 31 contains the following components: guanidine hydrochloride 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 1.0% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0079] The preservation solution of Formula 32 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Tween-20 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0080] The preservation solution of Formula 33 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, ethylphenyl polyethylene glycol (NP-40) 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0081] The preservation solution of Formula 34 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0082] The preservation solution of Formula 35 contains the following components: ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0083] The preservation solution of Formula 36 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, bovine serum albumin (BSA) 0.2% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0084] The preservation solution of Formula 37 contains the following components: guanidine isothiocyanate 0.75 mol / L, ethylenediaminetetraacetic acid (EDTA) 1 mmol / L, tris(hydroxymethyl)aminomethane-hydrochloric acid (Tris-HCl) buffer (pH 7.4) 35 mmol / L, Triton X-100 0.1% (v / v), single-stranded DNA binding protein (SSB) 0.3 μmol / L, trehalose 5% (m / v), with the balance made up to 100% with enzyme-free sterile water.

[0085] This invention employs 37 formulations for systematic formulation screening and validation. Formulations 1-12 constitute a single-factor concentration gradient screening experiment, systematically designing concentration gradients for guanidine salt concentration (formulations 1-3), SSB concentration (formulations 4-6), trehalose concentration (formulations 7-9), and BSA concentration (formulations 10-12), exploring the independent action thresholds and effective concentration ranges of each key component. Formulations 13-28 are multi-factor full-endpoint combination gradient validations, covering combinations of the highest and lowest full-endpoint values ​​of the four key components (guanidine salt, SSB, trehalose, and BSA) within their respective screening ranges, aiming to evaluate the synergistic adaptation and interaction effects between components with different endpoint concentration values. Formulations 29-30 explore the potential impact of buffer (Tris-HCl) concentration on the pH stability and nucleic acid preservation effect of the system when the deformable agent (guanidine salt), SSB, trehalose, and BSA are intermediate concentration parameters, broadening and connecting the endpoint concentrations of the buffer system in the experimental components before and after validation. Formulas 31 to 33, with denaturant (guanidine salt), SSB, trehalose, and BSA as intermediate concentration parameters, explored the potential substitution and adjustment possibilities of different guanidine salts (guanidine hydrochloride) / surfactant components (Tween-20, NP-40), broadening and validating the optimal cleavage and solubilization conditions for denaturant and surfactant. Formulas 34 to 37 served as negative control formulas for key components, respectively lacking key components SSB (Formula 34), denaturant (Formula 35), trehalose (Formula 36), and BSA (Formula 37), verifying the irreplaceable role of each of the four key components in virus inactivation and nucleic acid stable preservation.

[0086] Example 2: Preparation, Use and Validation Methods of Preservative Solution 1. Preparation of preservation solution: 1) According to the formulation in Example 1, measure the reagents of each component in proportion and prepare them as follows: In the pre-prepared Tris-HCl (pH 7.4) stock solution, add denaturant, surfactant, chelating agent, SSB, trehalose and BSA in sequence, stir until completely dissolved and mixed. Note that all components should be enzyme-free (DNase / RNase-free) reagents; 2) Fine-tune the pH to 7.4±0.1 with pH buffer, then make up to volume with enzyme-free sterile water and mix evenly; 3) Filter sterilize with a 0.22μm aqueous filter membrane to obtain the preservation solution optimized by this invention for storing RNA virus samples at room temperature. Dispense the preservation solution into enzyme-free centrifuge tubes, seal and store for later use.

[0087] 2. Preparation of virus preservation solution: 1) Sample collection and pretreatment: Collect clinical samples (blood, feces, tissue) suspected of being positive for RNA virus. Blood samples do not require pretreatment. Feces and tissue samples are pretreated and aliquoted to prepare working solutions. It is recommended to keep at least one tube of working solution directly at -80℃ as a control to avoid repeated freeze-thaw cycles during cryopreservation.

[0088] For blood samples, no pretreatment is required; the sample can be directly aliquoted and used to prepare the working solution after adding preservation solution. For fecal samples, about 5 grams (pea-sized) of fresh fecal sample is taken with a sterile sampling spoon, PBS or physiological saline is added, and the sample is vortexed to form a homogeneous suspension for use as the working solution. For tissue samples, the tissue is first washed with sterile physiological saline, then cut into the smallest possible pieces (no more than 5 mm on any side) with a sterile scalpel. The tissue pieces are then added to PBS or physiological saline and ground into a homogenate under sterile conditions. The working solution is prepared based on the homogenate.

[0089] If the virus preservation solution is to be prepared for verification, RNA viruses (HEV, SARS-CoV-2, HIV, preferably HEV) can be collected from sensitive cell cultures. After extracting viral RNA, the RNA concentration is quantitatively determined. The solution is quantitatively diluted with enzyme-free sterile ddH2O to an initial Ct value of 21.43±0.5. This quantitatively diluted RNA solution is used as the stock solution for the working solution of the verification sample. It is aliquoted into 200μL / tube. It is recommended to keep at least one tube frozen at -80℃ as a control for later use.

[0090] 2) Preparation of virus preservation solution: Mix the virus preservation solution and sample working solution in a volume ratio greater than 4:1, preferably 4:1 to 19:1, and the mixture is the virus preservation solution. In the above verification, the sample working solution used for virus preservation solution was extracted and purified viral RNA. After adding the preservation solution, the initial Ct value of the sample preservation solution was quantified to 24.15±0.5. At this time, the preferred volume ratio of preservation solution to sample working solution was 9:1.

[0091] 3) Aliquoting: Aliquot the above virus preservation solution into 200-1000μL / tube, which can be used as a virus preservation solution that can be stably stored at room temperature (15-25℃) and used for subsequent nucleic acid testing.

[0092] The method of using the preservation solution described in this invention is the same as the preparation process of the virus preservation solution above. It is evident that the operation of the preservation solution is simple. The core step is to directly mix the preservation solution and the sample working solution requiring room temperature storage at a certain volume ratio (4:1 to 19:1) until homogeneous. This mixture can then be directly used for storage or transportation at room temperature (15–25°C). During use, the room-temperature preserved virus solution can also be directly aspirated for subsequent nucleic acid testing. Note: For different clinical samples (blood, feces, tissue), it is recommended to complete the pretreatment and prepare the virus preservation solution as soon as possible within 30 minutes of removal from the body, following the steps above.

[0093] 3. Screening of preservation solution components and stability experiments The following standardized operating procedures were adopted for the stability test of the preservation solution at room temperature to ensure that the experimental results are reproducible and traceable. The specific operating steps are as follows: Take an appropriate amount of virus preservation solution in aliquots and store it at room temperature (15-25℃). A third-party preservation solution can be set up as a positive control at the same time. After taking it out at the preset time points (3 days to 18 days), immediately transfer it to the freezing condition (-16℃ to -20℃) for continued storage. After all the samples at the preset time points have been collected, the subsequent nucleic acid test is carried out in a unified manner to eliminate the error caused by the testing process.

[0094] 4. Nucleic acid testing and stability assessment Nucleic acid detection validation experiments, constructed based on the screening and optimization of preservation solution formulations, were conducted using commercially available kits according to the kit instructions. For example, HEV was detected using a hepatitis E virus RNA detection kit (PCR fluorescence method), SARS-CoV-2 using a novel coronavirus nucleic acid detection kit (PCR-fluorescent probe method), and HIV using a human immunodeficiency virus type 1 (HIV-1) nucleic acid detection kit (PCR-fluorescent probe method). Taking HEV RNA detection as an example, the specific conditions and evaluation criteria for nucleic acid detection are as follows: RT-PCR detection used a 40 μL reaction system containing: 18 μL RT-PCR buffer, 0.6 μL hepatitis E virus fluorescent probe, 1.4 μL enzyme mixture, and 20 μL sample. The sample was taken from the virus preservation solution that had been stored at room temperature at preset time points. The amplification reaction was set as follows: reverse transcription at 50℃ for 20 minutes, pre-denaturation at 70℃ for 15 minutes, followed by 40 cycles of amplification, each cycle consisting of denaturation at 94℃ for 15 seconds and annealing and extension at 60℃ for 30 seconds, with simultaneous acquisition of the FAM channel fluorescence signal at 60℃. Each sample was tested in triplicate, and the average Ct value was used as the final detection result.

[0095] The stability of the preservation solution was evaluated using the difference between the Ct value after preservation and the initial Ct value (ΔCt) as the core indicator. The judgment criteria were as follows: when the absolute value of ΔCt did not exceed 1.5 (ΔCt≤1.5), the stability was deemed to meet the requirements: that is, the nucleic acid was stable and there was no significant degradation, meeting the performance requirements for nucleic acid detection of clinical samples; when the absolute value of ΔCt did not exceed 0.5 (ΔCt≤0.5), the preservation stability of RNA virus samples was deemed to be excellent, indicating that there was almost no loss of nucleic acid integrity in the sample.

[0096] 5. Stability results after long-term storage at room temperature The preservation solutions prepared according to formulations 1-33 in Example 1 were used to conduct stability experiments with the above-mentioned preferred HEV-corresponding verification virus preservation solutions. The preset time points for the stability experiments were set to 0 days, 7 days, 14 days and 18 days, respectively. The test results are shown in Table 1 below. The CV values ​​between the three replicates were all <2% (in all subsequent verification experiments, unless otherwise specified, the CV values ​​of nucleic acid detection are assumed to be <2%).

[0097] Table 1. Stability of HEV virus preservation solution ; Table 1 shows the stability results: the preservation solutions prepared according to formulations 1-33 all contain 0.5-1 mol / L denaturant (guanidine salt), 20-50 mmol / L pH buffer (Tris-HCl buffer) (pH 7.4±0.1), 0.1%-1% (v / v) surfactant (Triton X-100, Tween-20, NP-40), chelating agent (1 mmol / L EDTA), and stabilizer (0.1-1 μmol / L SSB, 1%-10% (v / v) trehalose, 0.1%-0.5% (v / v) BSA). When the HEV RNA virus preservation solution was placed at room temperature (15-25℃) for 18 days using these 33 preservation solutions, the preservation stability of all the preservation solution pairs except for formulation 21 was determined to meet the stability requirements (ΔCt≤1.5).

[0098] In the stability results, formulations consistently rated as having excellent stability up to 18 days of storage at room temperature included formulations 2, 5, 11, 12, and 30. These four formulations shared the common characteristic that the concentrations of guanidine salt, SSB, trehalose, and BSA in their components were all at intermediate values. The only formulation that failed to meet stability requirements after 18 days of storage at room temperature (ΔCt=1.57>1.5) was formulation 21. In formulation 21, with the denaturant guanidine salt concentration at the high end (1 mol / L), the concentrations of the stabilizers SSB, trehalose, and BSA were all at the low end. Further analysis of these four formulations with excellent stability and the one that failed to meet stability requirements suggests that the optimal formulations leading to good stability of the storage solution might be: ① The 0.75 mol / L guanidine salt concentration is an effective concentration at the midpoint between sufficient lysis and RNase inhibition. The 0.75 mol / L concentration, being lower than the highest end value of 1 mol / L, may have less potential interference with subsequent nucleic acid amplification experiments. ② The concentrations of SSB, trehalose, and BSA in the stable system were all at the intermediate optimal values ​​(SSB 0.3 μmol / L, trehalose 5%, BSA 0.2%), and the expected synergistic effect was relatively optimal.

[0099] When the concentrations of stabilizers SSB, trehalose, and BSA are all at their lowest endpoints, the stability of formulation 21 is unsatisfactory when the guanidine salt concentration is at its highest endpoint (1 mol / L). However, when the relative guanidine salt concentration is at its lowest endpoint (0.5 mol / L), the stability of formulation 13 (ΔCt = 0.34, 0.83, 0.6, all less than 1.5) meets the requirements. This shows that for the endpoint concentrations of the corresponding guanidine salt, lower concentrations are better than higher concentrations, i.e., 0.5 mol / L is relatively better than 1 mol / L. The stability results shown in Table 1 preliminarily show that the core key components of the preservation solution formulation prepared in this invention include four types: 0.5-1 mol / L guanidine salts (guanidine isothiocyanate, guanidine hydrochloride), 0.1-1 μmol / L single-chain binding protein (SSB), 1%-10% (m / v) trehalose, and 0.1%-0.5% (m / v) BSA.

[0100] Stability verification of key components: Further stability comparison experiments were conducted using the four negative control formulations (formulations 34-37) in Example 1, the most preferred formulation 2 among the four formulations with excellent stability, and commercial third-party reagents. The preset time points for the stability experiments were refined to 0 days, 3 days, 5 days, 7 days, 14 days, and 18 days, respectively. The test results are shown in Table 2 below.

[0101] Table 2. Stability of the negative control formulation of key components (Ct value of HEV RNA qPCR detection) ; Table 2 shows the stability results of the negative control formulations for the key components. It is evident that only the preservation solution prepared according to formulation 2, which contains all four key components, can maintain the stability of HEV RNA up to day 18. Conversely, when the preservation solutions prepared according to the four negative control formulations and the third-party reagents were stored at room temperature (15-25°C), the longest preservation time for HEV RNA meeting the stability requirements was only 7 days (ΔCt≤1.5). When the room temperature storage time was extended to day 14 and day 18, the stability of the four negative control formulations and the third-party reagents without the four key components of this invention for HEV RNA preservation was comparable, and neither met the stability requirements (ΔCt>1.5). Therefore, the four key components (guanidine salt, SSB, trehalose, and BSA) in the preservation solution formulation of this invention have a crucial impact on prolonging the long-term room temperature storage time of RNA viruses.

[0102] In summary, the preservation solutions prepared using formulations 1-37 of this invention have confirmed the key components and their optimal concentrations for extending the long-term storage time of RNA viruses at room temperature: 0.5-1 mol / L guanidine salts (guanidine isothiocyanate, guanidine hydrochloride), 0.1-1 μmol / L single-chain binding protein (SSB), 1%-10% (m / v) trehalose, and 0.1%-0.5% (m / v) BSA. The preferred components of the guanidine salt are guanidine isothiocyanate and guanidine hydrochloride, with a preferred low concentration range of 0.5-1 mol / L. Guanidine salt acts as a strong protein denaturant in the preservation solution and is a core component of the formulation that combines virus inactivation and nucleic acid protection. On the one hand, guanidine salt can rapidly penetrate tissue cells, lyse virus particles, and effectively lyse blood cells and microorganisms in blood and fecal samples, releasing viral RNA. On the other hand, guanidine salt can broadly inhibit endogenous and exogenous ribonucleases and proteases in the sample, thus providing long-term preservation for RNA. Therefore, guanidine salt can be adapted to different sample types. The stability verification experiment results show that 0.75 mol / L > 0.5 mol / L > 1.0 mol / L, indicating that the guanidine salt concentration has a non-linear effect on the preservation effect. The lower limit of concentration (0.5 mol / L) may not be sufficient for viral lysis and RNase inhibition; while the upper limit of concentration (1.0 mol / L) ensures efficient viral inactivation, it has a significant inhibitory effect on subsequent RT-PCR reactions, leading to a decrease in detection sensitivity. The optimal intermediate concentration (0.75 mol / L) achieves the best balance between sufficient viral lysis and minimal PCR inhibition, exhibiting the best nucleic acid stability in room temperature storage experiments. Therefore, screening and obtaining guanidine salts and their optimal concentrations that combine inactivation efficiency and detection compatibility is one of the key inventive points of this invention.

[0103] Among the stabilizers, SSB (single-strand DNA-binding protein) is a class of proteins that can specifically bind to single-stranded DNA (ssDNA) and single-stranded RNA; therefore, it is referred to as single-strand binding protein in this invention. SSB is routinely used in PCR, qPCR, RT-PCR, RT-qPCR, or other DNA / RNA modifying enzyme amplification reactions. Its main function is to reduce non-specific amplification and improve amplification efficiency and detection sensitivity. Its conventional addition concentration is usually 0.1–1.0 μg / μL (equivalent to approximately 5.6 μmol / L to 55.6 μmol / L based on the molecular weight of the SSB tetramer being approximately 75 kDa). This invention introduces SSB into the preservation solution components of RNA viruses for the first time, aiming to solve the stability problem of viral RNA in different sample types. After screening and verification, adding a low concentration (0.1–1.0 μg / μL) to the preservation solution... SSB at a concentration of 0.1–1.0 μmol / L can rapidly, efficiently, and reversibly bind to exposed viral single-stranded RNA, thereby achieving active protection of viral RNA at the molecular level. On the one hand, SSB binding prevents non-enzymatic self-hydrolysis of RNA due to thermal motion; on the other hand, SSB can mask potential restriction enzyme sites on the RNA strand, avoiding enzymatic degradation or breakage caused by residual nucleases, while inhibiting the formation of RNA secondary structures and maximizing the maintenance of RNA integrity and length. Therefore, SSB is the most critical stabilizer in this invention for directly functionally protecting viral RNA targets. Furthermore, the SSB concentration used in this invention (0.1–1.0 μmol / L) is far lower than its commonly used concentration in routine amplification reactions (5.6–55.6 μmol / L). When directly applied to the preservation solution, it will not impose a cumulative burden on SSB already present in the subsequent RT-qPCR detection system, nor will it adversely affect the detection sensitivity and accuracy.

[0104] Trehalose is a non-reducing disaccharide that, in this invention, possesses both chemical stabilization and osmotic pressure regulation functions. As a chemical stabilizer, trehalose can form a stable "glassy" hydration layer around biological macromolecules (including RNA, viral proteins, etc.), reducing the degree of freedom of molecular motion through a water substitution mechanism, thereby effectively preventing physical breakage or aggregation of nucleic acids and proteins during dehydration, temperature fluctuations, or long-term storage at room temperature. As an osmotic pressure regulator, trehalose can maintain the physiological isotonic environment provided by the preservation solution, reducing osmotic pressure shocks to different types of samples (such as blood, feces, and tissues), and preventing the release of more endogenous substances (such as nucleases) due to drastic changes in osmotic pressure during the initial stage of preservation, thus indirectly and synergistically ensuring the stability of viral RNA.

[0105] In this invention, BSA (bovine serum albumin) acts as both a protein competitor and a surface adsorption blocker. Its mechanism of action can be categorized into two types: "sacrificial protection" and "competitive protection." On one hand, as a surface adsorption blocker, BSA preferentially adsorbs onto the sampling tube wall, nucleic acid surface, and other locations, blocking non-specific binding sites in the preservation environment. This effectively reduces the non-specific adsorption loss of the sample due to physical adsorption of target RNA virus particles or free RNA. On the other hand, as a protein competitor, BSA competitively binds to trace amounts of proteases, impurities, and other interfering substances that may remain in the preservation solution. This causes the interfering substances to preferentially act on BSA rather than viral proteins or viral RNA, thereby reducing the risk of degradation of target viral RNA and proteins. The above analysis suggests that the stabilizing effect of BSA does not stem from its direct protection as a stabilizing matrix, but rather from indirect protection achieved through "sacrificial consumption" and "competitive binding."

[0106] Example 3: Performance Indicators of Preservative Solution 1. Preservation effect on different RNA viruses To verify the preservation effect of the preservation solution of the present invention on different types of RNA virus samples, three clinical blood samples (approximately 2 mL) confirmed to be positive for HEV, SARS-CoV-2, and HIV-1 nucleic acid, respectively, were used as test samples. The above test samples were prepared according to the preservation solution preparation and virus preservation solution preparation methods described in Example 2. The preservation solution prepared according to Formula 2 of Example 1 was mixed with the test samples in a certain proportion to prepare virus preservation solutions corresponding to three different RNA viruses, and then aliquoted into at least six portions. These were stored at room temperature (15–25°C) for preset time points (0 days, 3 days, 5 days, 7 days, 14 days, and 18 days). Simultaneously, a commercially approved third-party preservation solution was used as a positive control. After all the virus preservation solution samples corresponding to the preset time points were collected, RT-PCR detection was performed using third-party reagents for three RNA viruses (Hepatitis E virus RNA detection kit (PCR fluorescence method), Novel Coronavirus nucleic acid detection kit (PCR-fluorescent probe method), and Human Immunodeficiency Virus Type 1 (HIV-1) nucleic acid detection kit (PCR-fluorescent probe method)). The Ct values ​​and ΔCt at different time points were recorded and calculated. The results of nucleic acid detection stability of clinical samples of the three RNA viruses after being stored at room temperature for different times are shown in Table 3 below.

[0107] Table 3: Room temperature storage time and nucleic acid detection stability of three RNA virus samples ; Table 3 shows that the preservation solution prepared by the preferred formulation 2 of this invention exhibits significantly better nucleic acid detection stability than the third-party control preservation solution when storing clinical blood samples of three different RNA viruses (HEV, SARS-CoV-2, and HIV-1) at room temperature. Specifically, the third-party control preservation solution only meets the detection requirements (ΔCt ≤ 1.5) within the first 7 days of storage; by day 14, ΔCt is generally greater than 3, and by day 18, ΔCt is generally greater than 5, indicating that it cannot support long-term storage of RNA virus samples at room temperature. In contrast, the preservation solution prepared by the preferred formulation 2 of this invention shows that the ΔCt values ​​for all three RNA virus samples are less than 1.5 by day 18, meeting the clinical detection stability standards, demonstrating the excellent performance of the preservation solution in providing ultra-long-term stable storage of viral RNA at room temperature and compatibility with nucleic acid detection.

[0108] As shown in Table 3, firstly, the preferred preservation solution of this invention exhibits stable preservation effects on different types of RNA viruses (such as HEV, SARS-CoV-2, and HIV-1), demonstrating good broad-spectrum applicability and meeting the preservation requirements for nucleic acid detection of various RNA virus samples. Secondly, the preferred preservation solution of this invention significantly extends the effective storage period of clinical RNA virus samples at room temperature from 7 days of existing products to 18 days or more, breaking through the technical bottleneck of short storage time at room temperature for current mainstream preservation solutions. Thirdly, the stability index of a ΔCt value of less than 1.5 for nucleic acid detection after 18 days of storage at room temperature proves that RNA virus samples preserved with the preferred preservation solution of this invention can still meet the accuracy requirements for nucleic acid detection, thus ensuring the reliability and accuracy of nucleic acid detection results while reducing the risk of false negatives due to nucleic acid degradation (especially the potential risk of missed detection for HIV).

[0109] Therefore, the preferred preservation solution of this invention has broad practical value. For example, in clinical testing scenarios, this preservation solution can significantly extend the time window from sample collection to testing, especially suitable for situations where samples collected from primary healthcare institutions need to be transported long distances to central laboratories for testing, effectively reducing cold chain transportation costs and logistical pressure. In scenarios of sudden outbreaks or large-scale screening, the long-term room temperature preservation characteristic can simplify sample management procedures, reduce reliance on low-temperature storage equipment, and improve the organizational efficiency of large-scale testing. The excellent preservation effect on RNA viruses such as HIV-1, which have highly variable genomes and poor stability, provides technical support for their widespread application in infectious disease surveillance, epidemiological investigations, and biobank construction, and can also significantly avoid the huge risk of false negatives in nucleic acid screening for highly infectious viruses such as HIV-1. In summary, the preservation solution of this invention has significant performance advantages in improving the convenience of room temperature preservation of different RNA virus samples, compatibility with subsequent nucleic acid testing, reducing testing and transportation costs, and ensuring the quality of nucleic acid testing.

[0110] 2. Preservation effect on different sample types To verify the preservation effect of the preservation solution of this invention on viral RNA in different types of clinical samples, based on Formula 2 of Example 1, and following the preparation methods of the preservation solution and virus preservation solution described in Example 2, the preservation solution prepared in Formula 2 was used to preserve 5 HEV nucleic acid positive blood samples, 5 positive fecal samples, and 5 positive pig liver tissue samples at room temperature (15-25°C) for a preset time point of 2 weeks (14 days). After the preset preservation time point, 15 different types of clinical samples were tested using a third-party reagent (Hepatitis E Virus RNA Detection Kit (PCR Fluorescence Method)) strictly following the instructions. The test results are shown in Table 4 below.

[0111] Table 4. Stability of HEV RNA detection for different sample types stored at room temperature ; Table 4 shows that among the 15 positive samples, the clinical sample "feces-05" was excluded because its initial viral RNA load was extremely low (0.19 copy / μL and 0.16 copy / μL at 0 and 14 days, respectively, as verified by digital PCR), which was below the detection limit of ordinary nucleic acid test kits. Therefore, the test results of the "feces-05" sample were not related to the preservation effect of the preservation solution of this invention. The remaining 14 positive samples, after being stored at room temperature (15-25℃) for 2 weeks using the preservation solution prepared by the preferred formula 2 of this invention, all met the stability requirements (ΔCt≤1.5) in nucleic acid testing. The above results indicate that the preferred preservation solution of this invention can effectively adapt to various types of clinical samples such as serum, feces, and liver tissue, and achieve stable preservation for at least 2 weeks at room temperature. Its preservation effect is significantly better than that of existing commercial preservation solutions (such as 7 days for third-party reagents).

[0112] Specific embodiments of the present invention have been described in detail to enable those skilled in the art to readily understand them. However, based on all the disclosed descriptions, different or similar modifications or substitutions may be made to certain specific details, and all such changes are within the scope of protection of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. A preservation solution for storing RNA virus samples at room temperature, characterized in that, It contains denaturants, stabilizers, pH buffers, surfactants, and chelating agents; among which, The denaturant is a low-concentration guanidine salt; The stabilizer comprises 0.1-1.0 μmol / L of single-chain binding protein, 1%-10% trehalose by weight / volume, and 0.1%-0.5% BSA by weight / volume. The pH buffer is a buffer solution with a stable pH value within the physiological pH range of 7.4 ± 0.1, and a concentration range of 20-50 mmol / L; The surfactant is a nonionic surfactant with a volume percentage concentration of 0.1%-1%.

2. The preservation solution for preserving RNA virus samples at room temperature according to claim 1, characterized in that, The low-concentration guanidine salt is selected from either guanidine isothiocyanate or guanidine hydrochloride, and the concentration is controlled within the range of 0.5-1 mol / L.

3. The preservation solution for preserving RNA virus samples at room temperature according to claim 1, characterized in that, The RNA virus sample is blood, feces, or tissue.

4. The preservation solution for preserving RNA virus samples at room temperature according to claim 1, characterized in that, The pH buffer is Tris-HCl buffer solution.

5. The preservation solution for preserving RNA virus samples at room temperature according to claim 1, characterized in that, The chelating agent is 1 mmol / L ethylenediaminetetraacetic acid.

6. The preservation solution for preserving RNA virus samples at room temperature according to claim 1, characterized in that, The nonionic surfactants are Triton X-100, Tween 20, and ethyl phenyl polyethylene glycol.

7. A device for preserving RNA virus samples, characterized in that, Includes the preservation solution for preserving RNA virus samples at room temperature as described in any one of claims 1-6.

8. The method for preparing the preservation solution for preserving RNA virus samples at room temperature as described in any one of claims 1-6, characterized in that, The preparation steps are as follows: 1) Add denaturant, surfactant, chelating agent, SSB, trehalose and BSA to the pH buffer in sequence, and stir until completely dissolved and mixed. 2) Fine-tune the pH to 7.4±0.1 with pH buffer, then bring the volume up with enzyme-free sterile water and mix thoroughly; 3) After sterilization by filtration through a 0.22μm aqueous filter membrane, the product is dispensed and stored.

9. The method of using the preservation solution for preserving RNA virus samples at room temperature as described in any one of claims 1-6, characterized in that, Simply mix the preservation solution for room temperature RNA virus samples with the RNA virus samples that need to be preserved at room temperature according to the volume ratio.

10. The preservation solution for preserving RNA virus samples at room temperature as described in any one of claims 1-6, and the application of the device for preserving RNA virus samples as described in claim 7 in the room temperature preservation and nucleic acid detection of RNA virus samples.