Virus sample direct amplification type preservation liquid and application method

Abstract

The application belongs to the field of biological sample preservation. The application discloses a virus sample direct amplification type sample preservation liquid and an application method. The preservation liquid comprises a surfactant, a buffer, a salt ion, a reducing agent, alcohol and a nuclease inhibitor. The preservation liquid provided by the application can quickly inactivate viruses, realize direct sample amplification detection, and avoid a complicated nucleic acid extraction process.

Description

Technical Field

[0001] This invention relates to the field of biological sample preservation. In particular, it relates to a direct amplification-type sample preservation solution for viral samples and its application method. Background Technology

[0002] The description of the background art in this invention pertains to related technologies and is used merely for illustration and to facilitate understanding of the invention. It should not be construed as the applicant explicitly believing or presuming that the invention was prior art on the filing date of the first application.

[0003] Viruses are special organisms without cellular structure, consisting of a protein coat and internal genetic material. Viruses are tiny, structurally simple, and contain only one type of genetic material (DNA or RNA). Viral infections are extremely dangerous; for example, SARS or SARS-CoV-2. Initial symptoms often include fever, fatigue, and dry cough, followed by difficulty breathing. In severe cases, acute respiratory distress syndrome or septic shock can occur, even leading to death.

[0004] Viral nucleic acid detection is suitable for early diagnosis of diseases; therefore, preserving intact viral nucleic acid is of great significance for downstream gene analysis. Viral nucleic acids are easily degraded, especially RNA viruses, which are very easily degraded by widely distributed endogenous or exogenous RNases. Existing conventional methods for preserving viral nucleic acids are (1) conventional viral preservation solutions using universal transport media (UTM) or viral transport media (VTM) with buffers such as Hank's balanced salts and PBS as the main components; and (2) inactivation-type preservation solutions with guanidine salts as the main component, which can be stored at room temperature for a short period of time.

[0005] Existing UTM or VTM technologies require conventional ultra-low temperature storage at -80℃, necessitating cold chain equipment and inconvenient operation. However, inactivating preservation solutions based on guanidine salts are already available on the market, capable of inactivating viruses and protecting the safety of testing personnel. For example, Chinese patent CN111718908A discloses a virus sample preservation solution, its preparation method, and its application. The main components of Example 1, which shows good results, are: 50 mmol / L Tris-HCl, 5% (w / v) guanidine isothiocyanate, 5% (w / v) polyethylene glycol p-isooctylphenyl ether, 5% (w / v) sodium lauroyl sarcosinate, and 0.1 mmol / L EDTA-2Na. Another example is Chinese patent CN111172239A, which discloses a virus sample preservation solution containing the following components: guanidine thiocyanate, Tween-20, podocarboxylic acid, ethanol, trisodium citrate, citric acid, and dithiothreitol. The reagents disclosed in the two patents mentioned above can inactivate the virus, stabilize the RNA in the virus for a short period of time, and then perform qualitative and quantitative RT-PCR detection of the RNA after conventional nucleic acid extraction steps.

[0006] In summary, while guanidine salt-based inactivation preservation solutions can solve the technical problems of virus inactivation and enable short-term room temperature preservation and transport of viral RNA, their components often contain common RT-PCR inhibitors such as guanidine isothiocyanate, chelating agents, ethanol, or high concentrations of surfactants. Therefore, the subsequent RNA extraction step is still necessary. Currently, there is a need for a faster testing procedure and protocol for SARS-CoV-2 detection to improve the convenience of existing RT-PCR testing or clinical diagnosis. Summary of the Invention

[0007] The purpose of this invention is to provide a direct amplification preservation solution for virus samples and its application method. The preservation solution provided by this invention can rapidly inactivate viruses, enabling direct sample loading, amplification, and detection, avoiding the cumbersome nucleic acid extraction process.

[0008] The objective of this invention is achieved through the following technical solutions:

[0009] An embodiment of the first aspect of the present invention provides a direct amplification preservation solution for virus samples, comprising a surfactant, a buffer, salt ions, a reducing agent, an alcohol, and a nuclease inhibitor; wherein the surfactant has a mass-volume percentage of 0.01-5%; the buffer has a final concentration of 2-250 mM; the salt ion concentration is 10-500 mM; the reducing agent concentration is 0.5-50 mM; the alcohol has a mass-volume percentage of 0.1-15%; the nuclease inhibitor has a mass-volume percentage of 0.01-1%; and the preservation solution has a pH of 5.5-9.5.

[0010] Furthermore, the preservation solution may also include a gene-releasing agent, which is bovine serum albumin (BSA), 1,2-dioleoyl lecithin, 1,2-dioleoyl lecithin derivatives, and / or combinations thereof.

[0011] The structural formula of 1,2-dioleoyl lecithin is as follows:

[0012]

[0013] Furthermore, the gene-releasing agent is bovine serum albumin at a weight-volume percentage of 0.05-0.5%, 1,2-dioleoyl lecithin at a weight-volume percentage of 0.01-0.5%, 1,2-dioleoyl lecithin derivatives at a weight-volume percentage of 0.01-0.35%, and / or combinations thereof.

[0014] Furthermore, the surfactant is triethanolamine dodecyl sulfate, triethanolamine dodecylbenzene, triethanolamine dodecyl sulfate, Brij 35, Brij 56, Brij 58, sodium dodecyl sulfate, NP40, polyethylene glycol octylphenyl ether, Span-80, Span-20, lithium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium dodecyl sulfonate, sodium dodecyl sarcosinate, octadecylamine polyoxyethylene ether bisquaternary ammonium salt, hexadecyl alcohol polyoxyethylene ether dimethyl octyl ammonium chloride, octyl polyoxyethylene tetradecyl ammonium chloride, octyl polyoxyethylene dodecyl ammonium chloride and / or combinations thereof.

[0015] Furthermore, the buffer is trihydroxyaminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium ethanesulfonate, sodium acetate, potassium hydrogen phthalate, boric acid, potassium dihydrogen phosphate, diethanolamine, triethanolamine, propanesulfonic acid, sodium dihydrogen phosphate, ammonium acetate, ammonium sulfate, ammonium chloride, N,N-dihydroxyethylglycine, 3-(cyclohexylamine)-1-propanesulfonic acid, 3-morpholine propanesulfonic acid and / or combinations thereof.

[0016] Furthermore, the salt ion is sodium chloride, ammonium chloride, sodium sulfate, ammonium sulfate, potassium chloride, lithium chloride, sodium acetate, and / or a combination thereof.

[0017] Furthermore, the reducing agent is DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine), TCEP-HCl (tris(2-carboxyethyl)phosphine hydrochloride), uric acid, mercaptoethanol, cysteine, vitamin C, dithionite, mercaptoacetic acid, and / or combinations thereof.

[0018] Furthermore, the alcohol is methanol, ethanol, isopropanol, ethylene glycol, 1-propanol, glycerol, 2-butanol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 800, polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000, polyethylene glycol 8000 and / or combinations thereof.

[0019] Furthermore, the nuclease inhibitor is diethyl pyrophosphate (DEPC), guanidine isothiocyanate, vanadate ribonucleoside complex, RNase protein inhibitor (RNasin), urea, theaflavins, anthocyanins, leucosin, flavonoids, lignans, N-acetylglucosamine, hexuronic acid, and / or combinations thereof.

[0020] A second aspect of the present invention provides a method for applying a direct amplification preservation solution for virus samples, comprising the following steps:

[0021] Obtain samples that may contain the virus;

[0022] Add any potentially virus-containing samples to a tube containing the preservation solution;

[0023] After mixing by inverting the top and bottom, let it stand at room temperature for 1-30 minutes before storing and transporting at ambient temperature.

[0024] Directly add samples to the corresponding RT-PCR amplification kit for detection; the sample volume should account for 2.5-50% (v / v) of the total amplification system.

[0025] The embodiments of the present invention have the following beneficial effects:

[0026] It enables the transport and preservation of viral RNA under environmentally stable conditions;

[0027] It can rapidly inactivate pathogenic microorganisms, inhibit the growth of various viruses or bacteria, prevent secondary infection, and ensure the safe transportation of infectious samples.

[0028] The preservation solution of this invention can be used to modify the "sampling-inactivation-extraction-amplification detection" process to "sampling-amplification detection", which shortens the overall process time. Attached Figure Description

[0029] Figure 1 This is an RT-PCR amplification curve of different droplets of the virus added to preservation solution V5 in Example 12 of this invention. From left to right, they are 10* 5 10* 4 and 10* 3 The curve of direct amplification of pseudoviruses added to the preservation solution. Detailed Implementation

[0030] The present application will be further described below with reference to the embodiments.

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, in the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Different embodiments can be substituted or combined, and for those skilled in the art, other implementation methods can be obtained based on these embodiments without creative effort.

[0032] definition

[0033] Before describing this teaching in detail, it should be understood that this disclosure is not limited to specific compositions or process steps, as these can vary. It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly indicates otherwise.

[0034] As used herein, the term "sample" or "sample" will be understood to mean any such sample that may contain the target nucleic acid material. The term "sample" or "sample" may also include solutions such as aqueous solutions, cells, tissues, biopsies, powders, or combinations thereof. Samples can be biological samples such as saliva, sputum, oral swab samples, serum, plasma, blood, erythrocyte sedimentation rate (ESR), pharyngeal, nasal / nasopharyngeal, or sinus swabs or secretions, laryngeal swabs or scrapings, urine, mucus, fecal excretions, rectal swabs, vomit, gastric juice, gastrointestinal fluid, semen, sperm, urethral swabs and secretions, cerebrospinal fluid, lactational or menstrual products, egg yolk, amniotic fluid, aqueous humor, vitreous humor, cervical secretions, vaginal discharge, etc. Fluids, secretions, swabs or scrapes, bone marrow samples and extracts, pleural fluid and exudate, sweat, pus, tears, lymph, bronchial or lung lavage fluid or extracts, cell cultures and cell suspensions, connective tissue, epithelium, epithelial swabs and smears, mucous membranes, muscle tissue, placental tissue, biopsies, exudates, organ tissues, nerve tissues, hair, skin, or nails, wherein the aforementioned samples may be obtained from, for example, vertebrates, including mammals.

[0035] It should be understood that the terms "about" are implied before the temperature, mass, weight, volume ratio, concentration, time, etc., discussed in this disclosure, so that minor and non-substantial deviations are within the scope of the teachings herein. Generally, the term "about" indicates a non-substantial change in the amount of a component in the composition that has no significant effect on the effect or stability of the composition. Furthermore, the use of "comprising," "containing," and "including" is not intended to be limiting. It should be understood that the foregoing general and detailed descriptions are exemplary and explanatory only and are not intended to limit the teachings. In the event of any inconsistency between any material incorporated by reference and the statements of this disclosure, the statements shall prevail.

[0036] Unless otherwise specified, embodiments described in the specification as “comprising” various components are also considered to be “composed of” or “substantially composed of” said components; embodiments described in the specification as “composed of various components” are also considered to be “comprising” or “substantially composed of” said components.

[0037] "Nucleic acid" refers to a polymeric compound containing two or more covalently bonded nucleosides or nucleoside analogs having nitrogen-containing heterocyclic bases or base analogs, wherein the nucleosides are linked together by phosphodiester bonds or other bonds to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides and their analogs. The nucleic acid "backbone" can be composed of a variety of linkages, including one or more sugar-phosphodiester bonds and peptide-nucleic acid bonds. Nucleic acids may include modified bases to alter their function or behavior, such as adding a 3'-terminal dideoxynucleotide to prevent the addition of additional nucleotides to the nucleic acid. Synthetic methods for the in vitro preparation of nucleic acids are well known in the art, although nucleic acids can be purified from natural sources using conventional techniques.

[0038] In this application, "extraction," "separation," or "purification" refers to the removal or separation of one or more components of a sample from other sample components. The sample components contain target nucleic acids typically present in an aqueous solution phase, and may also contain cellular fragments, proteins, carbohydrates, lipids, salt ions, metal ions, and other nucleic acids. "Extraction," "separation," or "purification" does not imply any degree of purification. Typically, separation or purification removes at least 70%, at least 80%, or at least 90% of the target nucleic acids from other sample components.

[0039] Rapidly inactivate the virus, enabling direct sample loading, amplification, and detection, avoiding the cumbersome nucleic acid extraction process.

[0040] A direct amplification preservation solution for virus samples includes surfactants, buffers, salt ions, reducing agents, alcohols, and nuclease inhibitors.

[0041] Furthermore, the preservation solution may also include a gene-releasing agent.

[0042] In other embodiments of the present invention, the gene-releasing agent may be bovine serum albumin (BSA), 1,2-dioleoyl lecithin (DOPC), 1,2-dioleoyl lecithin derivative (DOPC-R), and / or a combination thereof.

[0043] The structural formula of 1,2-dioleoyl lecithin is as follows:

[0044]

[0045] The structure of the 1,2-dioleoyl lecithin derivative is as follows:

[0046]

[0047] The chemical name of the derivative of DOPC is (R)-2-methyl-2,3-di(oleoyl)propyl(2-(trimethylamino)ethyl)phosphate, abbreviated as DOPC-R.

[0048] In other embodiments of the present invention, the gene-releasing agent may be 0.05-0.5% bovine serum albumin, 0.01-0.5% DOPC, 0.01-0.35% DOPC-R and / or combinations thereof by weight / volume percentage.

[0049] Furthermore, the preservation solution comprises the following components: 0.01-5% by weight / volume of surfactant, 2-250mM of buffer, 10-500mM of salt ions, 0.5-50mM of reducing agent, 0.1-15% by weight / volume of alcohol, and 0.01-1% by weight / volume of nuclease inhibitor.

[0050] In some embodiments of the present invention, the pH of the preservation solution is 5.5-9.5.

[0051] In other embodiments of the present invention, the pH of the preservation solution is 6.5-8.5.

[0052] In some embodiments of the present invention, the surfactant is a nonionic surfactant, anionic surfactant, cationic surfactant, or amphoteric surfactant and / or a combination thereof.

[0053] In other embodiments of the present invention, the surfactant is triethanolamine dodecyl sulfate, triethanolamine dodecylbenzene, triethanolamine dodecyl sulfate, Brij 35, Brij 56, Brij 58, sodium dodecyl sulfate, NP40, polyethylene glycol octylphenyl ether, Span-80, Span-20, lithium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium dodecyl sulfonate, sodium dodecyl sarcosinate, octadecylamine polyoxyethylene ether bisquaternary ammonium salt, hexadecyl alcohol polyoxyethylene ether dimethyl octyl ammonium chloride, octyl polyoxyethylene tetradecyl ammonium chloride, octyl polyoxyethylene dodecyl ammonium chloride and / or combinations thereof.

[0054] In other embodiments of the present invention, the surfactant is triethanolamine dodecyl sulfate, triethanolamine dodecylbenzene, triethanolamine dodecyl sulfate, Brij 35, Brij 56, Brij 58, sodium dodecyl sulfate, NP40, polyethylene glycol octylphenyl ether, lithium dodecyl sulfate, cetyl alcohol polyoxyethylene ether dimethyl octyl ammonium chloride, octyl polyoxyethylene tetradecyl ammonium chloride, octyl polyoxyethylene dodecyl ammonium chloride and / or combinations thereof.

[0055] In other embodiments of the present invention, the surfactant is dodecyl triethanolamine, dodecylbenzene triethanolamine, Brij 56, Brij 58, sodium dodecyl sulfate, NP40, polyethylene glycol octylphenyl ether, lithium dodecyl sulfate, octyl polyoxyethylene tetradecyl ammonium chloride and / or combinations thereof.

[0056] In other embodiments of the invention, the surfactant is 0.02-0.1% by weight / volume of dodecyl triethanolamine, 0.02-0.2% of dodecylbenzene triethanolamine, 0.5-2% of Brij 56, 0.5-2% of Brij 58, 0.01-0.2% of sodium dodecyl sulfate, 0.2-2% of NP40, 0.2-5% of polyethylene glycol octylphenyl ether, 0.02-0.4% of lithium dodecyl sulfate, 0.1-2% of octyl polyoxyethylene tetradecyl ammonium chloride, and / or combinations thereof.

[0057] In some embodiments of the present invention, the buffer is trihydroxyaminomethane, 4-hydroxyethylpiperazine ethanesulfonic acid, sodium ethanesulfonate, sodium acetate, potassium hydrogen phthalate, boric acid, potassium dihydrogen phosphate, diethanolamine, triethanolamine, propanesulfonic acid, sodium dihydrogen phosphate, ammonium acetate, ammonium sulfate, ammonium chloride, N,N-dihydroxyethylglycine, 3-(cyclohexylamine)-1-propanesulfonic acid, 3-morpholine propanesulfonic acid and / or combinations thereof.

[0058] In other embodiments of the invention, the buffer solution is trihydroxyaminomethane, boric acid, diethanolamine, ammonium sulfate, ammonium chloride, N,N-dihydroxyethylglycine, 3-(cyclohexylamine)-1-propanesulfonic acid and / or combinations thereof.

[0059] In other embodiments of the invention, the buffer is 5-50 mM trihydroxyaminomethane, 2-50 mM ammonium sulfate, 2-40 mM ammonium chloride, 2-100 mM N,N-dihydroxyethylglycine, 5-250 mM 3-(cyclohexylamine)-1-propanesulfonic acid and / or combinations thereof.

[0060] In some embodiments of the present invention, the salt ion is sodium chloride, ammonium chloride, sodium sulfate, ammonium sulfate, potassium chloride, lithium chloride, sodium acetate, and / or a combination thereof.

[0061] In other embodiments of the invention, the salt ion is sodium chloride, ammonium chloride, ammonium sulfate, potassium chloride, and / or a combination thereof.

[0062] In other embodiments of the invention, the salt ions are 20-200 mM sodium chloride, 10-200 mM ammonium chloride, 20-300 mM ammonium sulfate, 30-500 mM potassium chloride, and / or combinations thereof.

[0063] In some embodiments of the present invention, the reducing agent is DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine), TCEP-HCl (tris(2-carboxyethyl)phosphine hydrochloride), uric acid, mercaptoethanol, cysteine, vitamin C, dithionite, mercaptoacetic acid, and / or combinations thereof.

[0064] In other embodiments of the present invention, the reducing agent is DTT, TCEP, vitamin C and / or a combination thereof.

[0065] In other embodiments of the present invention, the reducing agent is 0.5-25 mM DTT, 0.5-10 mM TCEP, 2-50 mM vitamin C and / or a combination thereof.

[0066] In some embodiments of the present invention, the alcohol is methanol, ethanol, isopropanol, ethylene glycol, 1-propanol, glycerol, 2-butanol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 800, polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000, polyethylene glycol 8000 and / or combinations thereof.

[0067] In other embodiments of the invention, the alcohol is ethanol, isopropanol, ethylene glycol, glycerol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 2000 and / or combinations thereof.

[0068] In other embodiments of the invention, the alcohol is 0.1-2% by weight / volume of ethanol, 0.2-2.5% of isopropanol, 0.5-5% of ethylene glycol, 1-10% of glycerol, 1-15% of polyethylene glycol 200, 1-10% of polyethylene glycol 600, 2-10% of polyethylene glycol 2000 and / or combinations thereof.

[0069] In some embodiments of the present invention, the nuclease inhibitor is diethyl pyrophosphate (DEPC), guanidine isothiocyanate, vanadate ribonucleoside complex, RNase protein inhibitor (RNasin), urea, theaflavins, anthocyanins, leucogens, flavonoids, lignans, N-acetylglucosamine, hexuronic acid and / or combinations thereof.

[0070] In other embodiments of the present invention, the nuclease inhibitor is theaflavins, anthocyanins, lignans, N-acetylglucosamine, and / or combinations thereof.

[0071] In other embodiments of the invention, the nuclease inhibitor is 0.01-0.5% by weight / volume of theaflavins, 0.01-1% of anthocyanins, 0.01-0.25% of lignans and / or combinations thereof.

[0072] In other embodiments of the present invention, the nuclease inhibitor is 0.01-0.2% by weight / volume of theaflavins, 0.01-0.5% of anthocyanins, 0.01-0.1% of lignans and / or combinations thereof.

[0073] In one embodiment, the preservation solution comprises the following components: 0.75% by weight / volume of Brij58, 0.05% by weight / volume of lithium dodecyl sulfate, 20 mM of trihydroxyaminomethane, 150 mM of potassium chloride, 1 mM of TCEP, 0.5% by weight / volume of ethanol, 0.05% by weight / volume of anthocyanins, and pH 7.4.

[0074] In another embodiment, the preservation solution comprises the following components: 0.1% by weight (w / v) dodecylbenzene triethanolamine, 0.5% Brij 56, 0.1% octyl polyoxyethylene tetradecyl ammonium chloride, 20 mM ammonium sulfate, 10 mM vitamin C, 5% by weight (w / v) polyethylene glycol 600, 0.05% by weight (w / v) anthocyanins, and pH 8.0.

[0075] In another embodiment, the preservation solution comprises the following components: 0.75% (w / v) Brij 58, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 1 mM TCEP, 0.5% (w / v) ethanol, 0.05% (w / v) anthocyanins, 0.1% (w / v) BSA, and pH 7.4.

[0076] In another embodiment, the preservation solution comprises the following components: 0.75% (w / v) Brij 58, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 1 mM TCEP, 0.5% (w / v) ethanol, 0.05% (w / v) anthocyanins, 0.1% (w / v) DOPC, and pH 7.4.

[0077] In another embodiment, the preservation solution comprises the following components: 0.75% (w / v) Brij 58, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 1 mM TCEP, 0.5% (w / v) ethanol, 0.05% (w / v) anthocyanins, 0.05% (w / v) DOPC-R, and pH 7.4.

[0078] According to a first aspect of the present invention, the preservation solution can preserve viral RNA for at least 2 days under environmentally stable conditions.

[0079] In other embodiments of the invention, the preservation solution can preserve viral RNA stably at ambient temperature for at least 3 days, or more than 4 days, or more than 5 days, or more than 7 days, or up to 14 days.

[0080] The term "preserving viral RNA" refers to the fact that, as detected by RT-PCR, the Ct value is no more than two times the initial preservation point.

[0081] The second aspect of the present invention provides a method for using a direct amplification preservation solution for virus samples, comprising the following steps: a) obtaining a sample that may contain a virus; b) adding the potentially virus sample to a tube containing the preservation solution; c) mixing by inverting the tube and allowing it to stand at room temperature for a period of time before storage and transportation at ambient temperature; d) directly adding the sample to a corresponding RT-PCR amplification kit for detection.

[0082] In some embodiments of the present invention, a) "obtaining samples that may contain viruses" means obtaining samples that may contain viruses by means of sampling tools such as nasal swabs and throat swabs;

[0083] In some embodiments of the present invention, the tube for "adding the preservation solution" in b) can be 1 mL, 1.5 mL, 2 mL, 3 mL, 5 mL, 10 mL, 12 mL, and 15 mL. The volume of the preservation solution added can be 0.5 mL, 1 mL, 1.5 mL, 2 mL, 3 mL, 40 mL, 5 mL, and 6 mL.

[0084] In some embodiments of the present invention, the time for c) "standing at room temperature for a period of time" is 1-30 minutes.

[0085] In some other embodiments of the present invention, the time for c) "standing at room temperature for a period of time" is 5-15 minutes.

[0086] In some embodiments of the present invention, the sampling volume of d) "directly sampling and adding to the corresponding RT-PCR amplification kit" accounts for 2.5-50% (v / v) of the entire amplification system.

[0087] In other embodiments of the present invention, the sampling volume may account for 2-40% (v / v) of the entire amplification system.

[0088] In other embodiments of the present invention, the sampling volume may account for 7.5-30% (v / v) of the entire amplification system.

[0089] In other embodiments of the present invention, the sampling volume may account for 10-25% (v / v) of the entire amplification system.

[0090] Example

[0091] To further understand the present invention, preferred embodiments are described below with reference to examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims. Unless otherwise specified, the experimental methods described in the following examples are conventional methods; and the materials described, unless otherwise specified, are commercially available.

[0092] Example 1: Preservative solution V1

[0093] This embodiment provides a preservation solution comprising 0.75% Brij 58 by mass / volume, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 1 mM TCEP, 0.5% ethanol by mass / volume, 0.05% anthocyanins by mass / volume, and pH 7.4.

[0094] Comparative Example 1: Preservative Solution W1

[0095] This embodiment provides a preservation solution comprising 0.75% Brij 58 by mass / volume, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 1 mM TCEP, 0.5% ethanol by mass / volume, and pH 7.4.

[0096] Comparative Example 2: Preservative Solution W2

[0097] This embodiment provides a preservation solution comprising 0.75% Brij 58 by mass / volume, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, 150 mM potassium chloride, and 0.5% ethanol by mass / volume, with a pH of 7.4.

[0098] Comparative Example 3: Preservative Solution W3

[0099] An example provides a preservation solution comprising 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 1 mM TCEP, 0.5% ethanol by weight / volume, 0.05% anthocyanins by weight / volume, and pH 7.4.

[0100] Example 5 uses the preservation solution V1 prepared in Example 1, the comparative preservation solutions W1-W3, UTM preservation solution, deionized water, and physiological saline for preservation and direct amplification detection.

[0101] Referring to the second aspect of the present invention, a method for applying a direct amplification preservation solution for virus samples is provided, wherein 10* is added to 1 mL of different preservation solutions. 5 The pseudovirus (COVID-19-pseudovirus) was mixed by inverting it 10 times and allowed to stand at room temperature for 10 minutes. Then, using the NEB Luna universal probe one-step RT-qPCR kit (Cat#E3006S) (following the kit instructions), 2 μL of the preservation solution containing the pseudovirus was added to a 20 μL system. The OFR1ab gene fragment of the pseudovirus was detected. Primer and probe sequences were obtained from the Chinese CDC. The direct amplification effect of the RNA virus and the preservation performance of viral RNA in the preservation solution were analyzed using an ABI 7500fast real-time PCR instrument. The Ct values ​​of the pseudovirus samples were measured at room temperature (25℃) on days 0, 3, and 5 in different preservation solutions. Table 1 shows that the addition of anthocyanins maintained the preservation performance of preservation solution V, while W1, without anthocyanins, could not preserve viral RNA at room temperature. Comparing the initial Ct values ​​of preservation solutions V1 and W2, we found that the reducing agent also played a significant role in the lysis of the viral sample and the release of RNA. A comparison of preservation solutions V1 and W3 reveals that without the addition of surfactants, viral samples cannot release their nucleic acid content, making them difficult to detect. In contrast, the control solutions UTM, deionized water, and physiological saline also fail to release nucleic acid.

[0102] The qPCR instrument conditions are as follows:

[0103]

[0104] Table 1: Viral RNA preservation at room temperature and direct amplification detection using different sample preservation solutions

[0105] V1 22.1 22.4 22.6 <![CDATA[ 0.5 ]]> W1 22.5 25.6 27.7 5.2 W2 24.4 26.7 28.9 4.5 W3 34.4 35.4 NT / UTM NT* NT NT / Deionized water NT NT NT / physiological saline NT 37.6 38.6 /

[0106] *△Ct=Day 0Ct-Day 5Ct

[0107] *NT indicates undetected.

[0108] Example 6: Verification of the effect of different anthocyanin concentrations on the preservation of viral RNA

[0109] In the formulation of preservation solution V1 prepared in Example 1, we prepared a series of preservation solutions containing different concentrations of anthocyanins, and will conduct experiments according to the scheme of Example 5. 10* 5 The pseudovirus (COVID-19-pseudovirus) was mixed by inverting 10 times and allowed to stand at room temperature for 10 minutes. Then, using the NEB Lunauniversalprobe one-step RT-qPCR kit (Cat#E3006S), following the kit instructions, 2 μL of the preservation solution containing the pseudovirus was added to a 20 μL system. The Ct values ​​of the pseudovirus samples stored at room temperature (25°C) for 0, 3, 5, and 7 days were analyzed by detecting the OFR1ab gene fragment of the pseudovirus. Surprisingly, we found that adding excessive anthocyanins led to a delay in Ct values ​​or even no detection signal. This indicates that excessive anthocyanins inhibit the RT-PCR reaction, while insufficient anthocyanins (e.g., 0.01%) are ineffective in preserving RNA. Therefore, in the following examples, we used 0.05% anthocyanins as an additive in the preservation solution formulation.

[0110] Table 2: Viral RNA preservation at room temperature and direct amplification detection using different sample preservation solutions

[0111]

[0112]

[0113] Example 7: Preservative Solution V2

[0114] The present invention provides a preservation solution comprising the following components: 0.1% by weight (w / v) of dodecylbenzene triethanolamine, 0.5% of Brij 56, 0.1% of octyl polyoxyethylene tetradecyl ammonium chloride, 20 mM of ammonium sulfate, 10 mM of vitamin C, 5% by weight (w / v) of polyethylene glycol 600, and 0.05% by weight (w / v) of anthocyanins, with a pH of 8.0.

[0115] Example 8: Preservative Solution V3

[0116] This invention provides a preservation solution comprising the following components: 0.75% (w / v) Brij 58, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 1 mM TCEP, 0.5% (w / v) ethanol, 0.05% (w / v) anthocyanins, 0.1% (w / v) BSA, and pH 7.4.

[0117] Example 9: Preservative Solution V4

[0118] This invention provides a preservation solution comprising the following components: 0.75% (w / v) Brij 58, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 1 mM TCEP, 0.5% (w / v) ethanol, 0.05% (w / v) anthocyanins, 0.1% (w / v) DOPC, and pH 7.4.

[0119] Example 10: Preservative Solution V5

[0120] This invention provides a preservation solution comprising the following components: 0.75% (w / v) Brij 58, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 1 mM TCEP, 0.5% (w / v) ethanol, 0.05% (w / v) anthocyanins, 0.05% (w / v) DOPC-R, and pH 7.4.

[0121] Comparative Example 4 Preservation Solution B1

[0122] 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 0.1% BSA, pH 7.4.

[0123] Comparative Example 5 Preservative Solution B2

[0124] 20 mM trihydroxyaminomethane, 150 mM potassium chloride, 0.75% Brij 58, 0.05% lithium dodecyl sulfate, 20 mM trihydroxyaminomethane, pH 7.4.

[0125] Example 11: Preservation and direct amplification detection were performed using the preservation solutions and deionized water prepared in Examples 7-10, as well as Comparative Examples 4 and 5.

[0126] Referring to the second aspect of the present invention, a method for applying a direct amplification preservation solution for virus samples is provided, wherein 10* is added to 1 mL of different preservation solutions. 5The pseudovirus (COVID-19-pseudovirus) was mixed by inverting 10 times and allowed to stand at room temperature for 10 minutes. Then, using the NEB Luna universal probe one-step RT-qPCR kit (Cat#E3006S), following the kit instructions, 2 μL of the preservation solution containing the pseudovirus was added to a 20 μL system. The Ct values ​​of the pseudovirus samples stored at room temperature (25°C) for 0, 3, and 5 days were analyzed by detecting the OFR1ab gene fragment. Data in Table 3 shows that the initial Ct values ​​were lower after adding different gene release agents, indicating that the addition of gene release agents is more conducive to the release of nucleic acids from the viral samples. Surprisingly, BSA, as a component of the formulation for preserving the virus and cell integrity in UTM and VTM preservation solutions, played a role in protecting cells. However, in our formulation tests, preservation solution B2 alone also provided a method for releasing viral nucleic acids. Furthermore, DOPC-R and DOPC showed superior gene release effects compared to BSA as gene release agents.

[0127] Table 3: Viral RNA preservation at room temperature and direct amplification detection using different sample preservation solutions

[0128]

[0129]

[0130] Example 12: Testing direct amplification of different concentrations of virus using preservation solution V5

[0131] Add 10* to 1 mL of the preservation solution V5 prepared in Example 10. 5 10* 4 10* 3 Mix the COVID-19 pseudovirus with the sample by inverting it 10 times until well combined. After standing at room temperature for 10 minutes, use the NEB Luna universalprobe one-step RT-qPCR kit (Cat#E3006S) to add 2 μL of the preservation solution containing the pseudovirus to a 20 μL system. Analyze the detection results of different drops of the pseudovirus sample in the preservation solution by detecting the OFR1ab gene fragment of the pseudovirus. The results are as follows: Figure 1 As shown, sample preservation solution V5 has a good release effect on viral RNA in pseudoviruses of different titers.

[0132] The above description of the embodiments is merely for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of the present invention. The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

[0133] It should be noted that the above embodiments can be freely combined as needed. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A direct amplification preservation solution for virus samples, comprising a surfactant, a buffer, salt ions, a reducing agent, an alcohol, and a nuclease inhibitor; wherein, The surfactant has a mass-volume percentage of 0.01-5%; the buffer has a final concentration of 2-250 mM; the salt ion concentration is 10-500 mM; the reducing agent concentration is 0.5-50 mM; the alcohol has a mass-volume percentage of 0.1-15%; the nuclease inhibitor has a mass-volume percentage of 0.02-0.2%; and the preservation solution has a pH of 5.5-9.

5. in: The surfactant is triethanolamine dodecyl sulfate, triethanolamine dodecylbenzene, triethanolamine dodecylbenzene, Brij 35, Brij 56, Brij 58, sodium dodecyl sulfate, NP40, lithium dodecyl sulfate, sodium dodecylbenzene sulfonate, sodium dodecyl sulfonate, octyl polyoxyethylene tetradecyl ammonium chloride, octyl polyoxyethylene dodecyl ammonium chloride and / or combinations thereof; The buffer is trihydroxyaminomethane and / or ammonium sulfate; The salt ion is sodium chloride, ammonium sulfate, potassium chloride, lithium chloride, and / or a combination thereof; The reducing agent is tris(2-carboxyethyl)phosphine, tris(2-carboxyethyl)phosphine hydrochloride, vitamin C, and / or a combination thereof; The alcohol is methanol, ethanol, isopropanol, ethylene glycol, 1-propanol, glycerol, 2-butanol, polyethylene glycol 200, polyethylene glycol 400, polyethylene glycol 600, polyethylene glycol 800, polyethylene glycol 1000, polyethylene glycol 2000, polyethylene glycol 6000, polyethylene glycol 8000 and / or combinations thereof; The nuclease inhibitor is anthocyanin.

2. The virus sample direct amplification preservation solution according to claim 1, characterized in that, The preservation solution may also include a gene-releasing agent, which is bovine serum albumin, 1,2-dioleoyl lecithin, 1,2-dioleoyl lecithin derivatives and / or combinations thereof; The structural formula of 1,2-dioleoyl lecithin is as follows: The structure of the 1,2-dioleoyl lecithin derivative is as follows: 。 3. The virus sample direct amplification preservation solution according to claim 2, characterized in that, The gene-releasing agent is bovine serum albumin at a weight-volume percentage of 0.05-0.5%, 1,2-dioleoyl lecithin at a weight-volume percentage of 0.01-0.5%, 1,2-dioleoyl lecithin derivatives at a weight-volume percentage of 0.01-0.35%, and / or combinations thereof.

4. The virus sample direct amplification preservation solution according to claim 1, characterized in that, The surfactant is dodecylbenzene triethanolamine, Brij 56, Brij 58, lithium dodecyl sulfate, octyl polyoxyethylene tetradecyl ammonium chloride and / or combinations thereof.

5. The virus sample direct amplification preservation solution according to claim 1, characterized in that, The salt ions are ammonium sulfate and / or potassium chloride.

6. The virus sample direct amplification preservation solution according to claim 1, characterized in that, The reducing agent is tris(2-carboxyethyl)phosphine and / or vitamin C.

7. A method for using a direct amplification preservation solution for virus samples for non-diagnostic purposes, characterized in that, The preservation solution is the preservation solution according to any one of claims 1-6; the application method includes the following steps: Obtain samples that may contain the virus; Add any potentially virus-containing samples to a tube containing the preservation solution; After mixing by inverting the top and bottom, let it stand at room temperature for 1-30 minutes before storing and transporting at ambient temperature. Directly add samples to the corresponding RT-PCR amplification kit for detection; the sample volume should account for 2.5-50% (v / v) of the total amplification system.

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