CN112680545A

CN112680545A - Virus sample direct amplification type preservation solution and application method thereof

Info

Publication number: CN112680545A
Application number: CN202011581588.6A
Authority: CN
Inventors: 张佳斌; 邹永龙; 曲峰; 何宗顺
Original assignee: Suzhou Cretaceous Biotechnology Co ltd
Current assignee: Suzhou Cretaceous Biotechnology Co ltd
Priority date: 2020-12-28
Filing date: 2020-12-28
Publication date: 2021-04-20

Abstract

The invention belongs to the field of biological sample preservation. The invention discloses a virus sample direct amplification type sample preserving fluid and an application method thereof. Wherein the preservation solution comprises a surfactant, a buffer, salt ions, a reducing agent, alcohol and a nuclease inhibitor. The preservation solution provided by the invention can quickly inactivate viruses, realize direct sample loading amplification detection and avoid a nucleic acid extraction process with complicated steps.

Description

Virus sample direct amplification type preservation solution and application method thereof

Technical Field

The invention relates to the field of biological sample preservation. In particular to a virus sample direct amplification type sample preserving fluid and an application method thereof.

Background

The description of the background of the invention pertaining to the related art to which this invention pertains is given for the purpose of illustration and understanding only of the summary of the invention and is not to be construed as an admission that the applicant is explicitly or implicitly admitted to be prior art to the date of filing this application as first filed with this invention.

A virus is a special organism without cellular structure, consisting of a protein coat and internal genetic material. The virus is small in individual and simple in structure, and only contains one genetic material (DNA or RNA). Viral infections, such as SARS or SARS-CoV-2, are extremely harmful, and the initial symptoms of the infected person are fever, fatigue and dry cough, and the subsequent breathing difficulty gradually appears, and the serious person can have acute respiratory distress syndrome or septic shock, and even die.

The detection of viral nucleic acids is suitable for early diagnosis of disease, and therefore, preservation of intact viral nucleic acids is of great significance for downstream genetic analysis. Viral nucleic acids, especially RNA viruses, are readily degraded by ubiquitous endogenous or exogenous rnases. The conventional preservation method of virus nucleic acid comprises (1) Universal Transport Medium (UTM) or Virus Transport Medium (VTM) conventional virus preservation solution using Hank's balanced salt, PBS and other buffer solution as main body; (2) the inactivated preserving fluid mainly containing guanidine salt can be preserved at room temperature for a short time.

UTM or VTM in the prior art needs to be preserved at the conventional ultralow temperature of-80 ℃, needs equipment such as a cold chain and the like, and is inconvenient to operate; and the inactivated preservation solution taking guanidine salt as a main body is already available in the market, and can inactivate viruses to protect the safety of inspectors. For example, chinese patent CN111718908A discloses a virus sample preservation solution, a preparation method and an application thereof, and the embodiment 1 with a better effect disclosed by the solution mainly comprises the following components: 50mmol/L Tris-HCl, 5% by mass of guanidinium isothiocyanate, 5% by volume of polyethylene glycol p-isooctylphenyl ether, 5% by volume of sodium lauroyl sarcosinate and a final concentration of EDTA-2Na of 0.1 mmol/L. For another example, chinese patent CN111172239A discloses a virus sample preservation solution comprising the following components: guanidine thiocyanate, tween-20, polidocanol, ethanol, trisodium citrate, citric acid and dithiothreitol. Both of the above patents disclose reagents that can inactivate viruses, preserve stable RNA in viruses for a short period of time, and perform qualitative and quantitative RT-PCR detection of RNA after conventional nucleic acid extraction procedures.

As described above, although the inactivation storage solution mainly containing guanidine salt can solve the technical problem of virus inactivation and can realize the storage and transportation of RNA in virus at room temperature for a short period of time, the composition thereof contains a common RT-PCR inhibitor such as guanidine isothiocyanate, a chelating agent, ethanol or a high concentration surfactant, and thus the subsequent RNA extraction step cannot be avoided. However, the SARS-CoV-2 assay requires a faster assay procedure and protocol to improve the convenience of the current RT-PCR assay or clinical diagnosis.

Disclosure of Invention

The embodiment of the invention aims to provide a virus sample direct amplification type preservation solution and an application method thereof. The preservation solution provided by the invention can quickly inactivate viruses, realize direct sample loading amplification detection and avoid a nucleic acid extraction process with complicated steps.

The purpose of the embodiment of the invention is realized by the following technical scheme:

the embodiment of the first aspect of the invention provides a virus sample direct amplification type preservation solution, which comprises a surfactant, a buffer, salt ions, a reducing agent, alcohol and a nuclease inhibitor; wherein, the mass volume percentage of the surfactant is 0.01-5%; the final concentration of the buffer is 2-250 mM; the concentration of salt ions is 10-500 mM; the concentration of the reducing agent is 0.5-50 mM; the mass volume percentage of the alcohol is 0.1-15%; the mass volume percentage of the nuclease inhibitor is 0.01-1%; the pH value of the preservation solution is 5.5-9.5.

Further, the preservation solution can also comprise a gene release agent, wherein the gene release agent is Bovine Serum Albumin (BSA), 1, 2-dioleoyl lecithin derivatives and/or combinations thereof.

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

further, the gene releasing agent is bovine serum albumin with the mass volume percentage of 0.05-0.5%, 1, 2-dioleoyl lecithin with the mass volume percentage of 0.01-0.5%, 1, 2-dioleoyl lecithin derivatives with the mass volume percentage of 0.01-0.35% and/or the combination thereof.

Further, the surfactant is lauryl triethanolamine sulfate, lauryl triethanolamine, dodecyl benzene triethanolamine, Brij 35, Brij 56, Brij 58, sodium dodecyl sulfate, NP40, polyethylene glycol octyl phenyl ether, Span-80, Span-20, lithium dodecyl sulfate, sodium dodecyl benzene sulfonate, sodium dodecyl sarcosinate, octadecyl amine polyoxyethylene ether biquaternary ammonium salt, cetyl alcohol polyoxyethylene ether dimethyl octane ammonium chloride, octyl polyoxyethylene tetradecyl ammonium chloride, octyl polyoxyethylene dodecyl ammonium chloride and/or a combination thereof.

Further, the buffer is trihydroxyaminomethane, 4-hydroxyethyl piperazine 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-dihydroxyethylglycine, 3- (cyclohexylamine) -1-propanesulfonic acid, 3-morpholine propanesulfonic acid, and/or combinations thereof.

Further, the salt ion is sodium chloride, ammonium chloride, sodium sulfate, ammonium sulfate, potassium chloride, lithium chloride, sodium acetate, and/or combinations thereof.

Further, 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, thioglycolic acid, and/or combinations thereof.

Further, 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.

Further, the nuclease inhibitor is diethyl pyrophosphate (DEPC), guanidinium isothiocyanate, vanadyl riboside complex, protein inhibitor of rnase (RNasin), urea, theaflavin, anthocyanidin, flavonoid, lignan, N-acetamido hexose, hexuronic acid and/or combinations thereof.

The embodiment of the second aspect of the invention provides an application method of a virus sample direct amplification type preservation solution, which comprises the following steps:

obtaining a sample that may contain a virus;

adding a virus sample possibly contained into a tube containing a preservation solution;

standing at room temperature for 1-30min after turning upside down and mixing, and storing and transporting at ambient temperature;

directly sampling and adding the sample into a corresponding RT-PCR amplification kit for detection; the proportion of the sampling volume in the whole amplification system is 2.5-50% (v/v).

The embodiment of the invention has the following beneficial effects:

the virus RNA can be transported and stored under stable environment;

quickly inactivates pathogenic microorganisms, inhibits the growth of various viruses or bacteria, avoids secondary infection and realizes the safe transportation of infectious samples.

The preservation solution can change the process of sampling-inactivation-extraction-amplification detection into the process of sampling-amplification detection, thereby shortening the time of the whole process.

Drawings

FIG. 1 is a diagram showing the amplification of RT-PCR curves for direct amplification of pseudoviruses of different titers in example 12 of the present invention after addition of preservation solution V5. From left to right are respectively 10^*5、10^*4And 10^*3Adding pseudovirus into preservation solution to directly amplify.

Detailed Description

The present application is further described below with reference to examples.

In the following description, different "one embodiment" or "an embodiment" may not necessarily refer to the same embodiment, in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art. Various embodiments may be replaced or combined, and other embodiments may be obtained according to the embodiments without creative efforts for those skilled in the art.

Definition of

Before the present teachings are described in detail, it is to be understood that this disclosure is not limited to particular compositions or process steps, as these may vary. It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

The term "sample" or "specimen" as used herein will be understood to mean any such sample: the term "sample" or "specimen" may include a sample that may contain the nucleic acid species of interest, or may include a solution, such as an aqueous solution, cells, tissue, biopsy, powder, or a combination of one or more thereof. The sample may be a biological sample such as saliva, sputum, buccal swab sample, serum, plasma, blood, buffy coat, pharynx, nasal/nasopharyngeal or sinus swab or secretion, laryngeal swab or scrapings, urine, mucus, fecal excrement, rectal swab, vomit, gastric juice, gastrointestinal fluids, semen, sperm, urethral swab and secretion, cerebrospinal fluid, lactation or menstrual products, egg yolk, amniotic fluid, aqueous humor, vitreous humor, cervical secretion, vaginal fluid, secretion, swab or scrapings, bone marrow samples and aspirates, pleural fluid and exudates, sweat, pus, tears, lymph, bronchial or pulmonary lavage fluids or aspirates, cell culture and cell suspensions, connective tissue, epithelium, epithelial swab and smear, mucosa, muscle tissue, placental tissue, biopsy, exudate, organ tissue, nervous tissue, Hair, skin, or nails, wherein the foregoing samples may be obtained from, for example, vertebrates, including mammals.

It is understood that there is an implied "about" before the temperatures, masses, weights, volume ratios, concentrations, times, etc. discussed in this disclosure such that slight and insubstantial deviations are within the scope of the teachings herein. Generally, the term "about" refers to insubstantial changes in the amounts of the components of the composition, which do not have any significant effect on the effectiveness or stability of the composition. Also, the use of "including," "containing," and "including" is not intended to be limiting. It is to be understood that both the foregoing general description and the detailed description are exemplary and explanatory only and are not restrictive of the present teachings. To the extent that any material incorporated by reference does not conform to the teachings of the present disclosure, that description controls.

Unless specifically stated otherwise, embodiments in the specification that are described as "comprising" various components are also considered to "consist of" or "consist essentially of" the components; embodiments in the specification that are described as "consisting of" various components are also considered to be "comprising" or "consisting essentially of" the components.

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

In this application, "extraction," "isolation," or "purification" refers to the removal of one or more components of a sample or the separation from other sample components. The sample components comprise target nucleic acids, often in a generally aqueous solution phase, which may also comprise cell fragments, proteins, carbohydrates, lipids, salt ions, metal ions, and other nucleic acids. "extraction", "isolation" or "purification" does not imply any degree of purification. Typically, the isolation or purification removes at least 70% or at least 80% or at least 90% of the target nucleic acid from other sample components.

The virus is quickly inactivated, direct sample loading amplification detection is realized, and the nucleic acid extraction process with complicated steps is avoided.

A virus sample direct amplification type preservation solution comprises a surfactant, a buffer, salt ions, a reducing agent, alcohol and a nuclease inhibitor.

Further, the preservation solution may further include a gene releasing agent.

In other embodiments of the present invention, the gene delivery agent may be Bovine Serum Albumin (BSA), 1, 2-dioleoyl lecithin (DOPC), 1, 2-dioleoyl lecithin derivatives (DOPC-R), and/or combinations thereof.

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

the 1, 2-dioleoyl lecithin derivative has the following structure:

the chemical name of the DOPC derivative is (R) -2-methyl-2, 3-di (oleoyl) propyl (2- (trimethylamino) ethyl) phosphate, which is called DOPC-R for short.

In other embodiments of the present invention, the gene delivery agent may be bovine serum albumin in an amount of 0.05-0.5% by weight, 0.01-0.5% by weight, DOPC in an amount of 0.01-0.35% by weight, DOPC-R in an amount of 0.01-0.35% by weight, and/or a combination thereof.

Further, the preservation solution comprises the following components: 0.01-5% of surfactant by mass volume, 2-250mM of buffer agent at final concentration, 10-500mM of salt ion, 0.5-50mM of reducing agent, 0.1-15% of alcohol by mass volume and 0.01-1% of nuclease inhibitor by mass volume.

In some embodiments of the invention, the preservation solution has a pH of 5.5 to 9.5.

In other embodiments of the invention, the preservation solution has a pH of 6.5 to 8.5.

In some embodiments of the invention, the surfactant is a nonionic, anionic, cationic or amphoteric surfactant and/or combinations thereof.

In other embodiments of the present invention, the surfactant is triethanolamine lauryl sulfate, triethanolamine lauryl, triethanolamine dodecylbenzene, Brij 35, Brij 56, Brij 58, sodium lauryl sulfate, NP40, polyethylene glycol octylphenyl ether, Span-80, Span-20, lithium lauryl sulfate, sodium dodecylbenzene sulfonate, sodium dodecylsulfonate, sodium dodecylsarcosinate, octadecyl amine polyoxyethylene ether diquaternary ammonium salt, cetyl polyoxyethylene ether dimethyloctane ammonium chloride, octyl polyoxyethylene tetradecyl ammonium chloride, octyl polyoxyethylene dodecyl ammonium chloride, and/or combinations thereof.

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

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

In other embodiments of the present invention, the surfactant is 0.02-0.1% by weight volume of dodecyltriethanolamine, 0.02-0.2% by weight of dodecylbenzenetriethanolamine, 0.5-2% by weight of Brij 56, 0.5-2% by weight of Brij 58, 0.01-0.2% by weight of sodium lauryl sulfate, 0.2-2% by weight of NP40, 0.2-5% by weight of polyethylene glycol octylphenyl ether, 0.02-0.4% by weight of lithium lauryl sulfate, 0.1-2% by weight of octylpolyoxyethylenetetradecylammonium chloride and/or combinations thereof.

In some embodiments of the invention, the buffer is tris, 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, bicine, 3- (cyclohexylamine) -1-propanesulfonic acid, 3-morpholinopropanesulfonic acid, and/or combinations thereof.

In other embodiments of the invention, the buffer is tris, boric acid, diethanolamine, ammonium sulfate, ammonium chloride, bicine, 3- (cyclohexylamine) -1-propanesulfonic acid, and/or combinations thereof.

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

In some embodiments of the invention, the salt ion is sodium chloride, ammonium chloride, sodium sulfate, ammonium sulfate, potassium chloride, lithium chloride, sodium acetate, and/or combinations thereof.

In other embodiments of the present invention, the salt ion is sodium chloride, ammonium sulfate, potassium chloride, and/or combinations thereof.

In other embodiments of the invention, the salt ion is 20-200mM sodium chloride, 10-200mM ammonium chloride, 20-300mM ammonium sulfate, 30-500mM potassium chloride, and/or combinations thereof.

In some embodiments of the 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, thioglycolic acid, and/or combinations thereof.

In other embodiments of the present invention, the reducing agent is DTT, TCEP, vitamin C, and/or combinations thereof.

In other embodiments of the invention, the reducing agent is DTT in the range of 0.5 to 25mM, TCEP in the range of 0.5 to 10mM, vitamin C in the range of 2 to 50mM, and/or combinations thereof.

In some embodiments of the 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.

In other embodiments of the present 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.

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

In some embodiments of the invention, the nuclease inhibitor is diethyl pyrophosphate (DEPC), guanidinium isothiocyanate, vanadyl riboside complex, a protein inhibitor of rnase (RNasin), urea, theaflavin, anthocyanidin, flavonoid, lignan, N-acetamido hexose, hexuronic acid and/or combinations thereof.

In other embodiments of the invention, the nuclease inhibitor is theaflavin, anthocyanidin, lignan, N-acetamidohexose and/or combinations thereof.

In other embodiments of the invention, the nuclease inhibitor is 0.01-0.5% by weight of theaflavin, 0.01-1% by weight of anthocyanin, 0.01-0.25% by weight of lignan and/or a combination thereof.

In other embodiments of the invention, the nuclease inhibitor is 0.01-0.2% by weight of theaflavin, 0.01-0.5% by weight of anthocyanin, 0.01-0.1% by weight of lignan and/or combination thereof.

In one embodiment, the preservation solution comprises the following composition: brij 58 with the mass percent by volume of 0.75%, lithium dodecyl sulfate with the mass percent by volume of 0.05%, trihydroxyaminomethane with the mass percent by volume of 20mM, potassium chloride with the mass percent of 150mM, TCEP with the mass percent by volume of 1mM, ethanol with the mass percent by volume of 0.5%, anthocyanin with the mass percent by volume of 0.05%, and the pH value is 7.4.

In another embodiment, the preservation solution comprises the following composition: 0.1 percent of dodecyl benzene triethanolamine, 0.5 percent of Brij 56, 0.1 percent of octyl polyoxyethylene tetradecyl ammonium chloride, 20mM of ammonium sulfate, 10mM of vitamin C, 5 percent of polyethylene glycol 600 and 0.05 percent of anthocyanin by mass, and the pH value is 8.0.

In another embodiment, the preservation solution comprises the following composition: brij 58 with the mass percent by volume of 0.75%, lithium dodecyl sulfate with the mass percent by volume of 0.05%, trihydroxy aminomethane with the mass percent by volume of 20mM, potassium chloride with the mass percent of 150mM, TCEP with the mass percent of 1mM, ethanol with the mass percent by volume of 0.5%, anthocyanin with the mass percent by volume of 0.05%, BSA with the mass percent by volume of 0.1% and pH value of 7.4.

In another embodiment, the preservation solution comprises the following composition: brij 58 with the mass percent by volume of 0.75%, lithium dodecyl sulfate with the mass percent by volume of 0.05%, trihydroxyaminomethane with the mass percent by volume of 20mM, potassium chloride with the mass percent by volume of 150mM, TCEP with the mass percent by volume of 1mM, ethanol with the mass percent by volume of 0.5%, anthocyanin with the mass percent by volume of 0.05%, DOPC with the mass percent by volume of 0.1% and pH7.4.

In another embodiment, the preservation solution comprises the following composition: brij 58 with the mass percent by volume of 0.75%, lithium dodecyl sulfate with the mass percent by volume of 0.05%, trihydroxyaminomethane with the mass percent by volume of 20mM, potassium chloride with the mass percent of 150mM, TCEP with the mass percent of 1mM, ethanol with the mass percent by volume of 0.5%, anthocyanin with the mass percent by volume of 0.05%, DOPC-R with the mass percent by volume of 0.05% and pH 7.4.

According to the first aspect of the present invention, the preservation solution can preserve the viral RNA for at least 2 days under ambient stability.

In other embodiments of the invention, the preservation solution may preserve viral RNA stable at ambient temperature for at least 3 days, or more to 4 days, or more to 5 days, or more to 7 days, or up to 14 days.

By "preserving viral RNA" is meant that no more than 2 Cts are detected by RT-PCR as compared to the initial preservation point.

The second aspect of the present invention provides a method for applying a virus sample direct amplification type preservation solution, comprising the steps of: a) obtaining a sample that may contain a virus; b) adding a virus sample possibly contained into a tube containing a preservation solution; c) after being turned upside down and mixed evenly, the mixture is kept stand for a period of time at room temperature and then can be stored and transported at the ambient temperature; d) directly sampling and adding into a corresponding RT-PCR amplification kit for detection.

In some embodiments of the present invention, the step a) "obtaining a sample that may contain a virus" refers to obtaining a sample that may contain a virus by a nasal swab, pharyngeal swab, or other sampling tool;

in some embodiments of the invention, the b) "add to tube containing preservation solution" tubes may be 1mL, 1.5mL, 2mL, 3mL, 5mL, 10mL, 12mL and 15 mL. The volume of preservative solution added may be 0.5mL, 1mL, 1.5mL, 2mL, 3mL, 40mL, 5mL, and 6 mL.

In some embodiments of the invention, the time for c) "standing at room temperature for a certain period" is 1 to 30 min.

In other embodiments of the present invention, the time for c) "standing at room temperature for a certain period" is 5-15 min.

In some embodiments of the invention, the d) "direct sampling to the corresponding RT-PCR amplification kit" sample volume is 2.5-50% (v/v) of the whole amplification system.

In other embodiments of the invention, the sampling volume may be 2-40% (v/v) of the total amplification system.

In other embodiments of the invention, the ratio of the sample volume to the total amplification system may be 7.5-30% (v/v).

In other embodiments of the invention, the sampling volume may be 10-25% (v/v) of the total amplification system.

Examples

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the materials are commercially available, unless otherwise specified.

Example 1: preservation solution V1

This example provides a preservation solution, which comprises Brij 58 in an amount of 0.75% by volume, lithium lauryl sulfate in an amount of 0.05% by volume, trishydroxyaminomethane in an amount of 20mM, potassium chloride in an amount of 150mM, TCEP in an amount of 1mM, ethanol in an amount of 0.5% by volume, anthocyanins in an amount of 0.05% by volume, and pH 7.4.

Comparative example 1: preservation solution W1

This example provides a preservation solution comprising, by mass, 0.75% Brij 58, 0.05% lithium lauryl sulfate, 20mM Tris, 150mM KCl, 1mM TCEP, 0.5% ethanol, pH 7.4.

Comparative example 2: preservation solution W2

This example provides a preservation solution, which comprises Brij 58 in an amount of 0.75% by volume, lithium lauryl sulfate in an amount of 0.05% by volume, 20mM of trishydroxyaminomethane, 150mM of potassium chloride, and 0.5% by volume of ethanol, and which has a pH of 7.4.

Comparative example 3: preservation solution W3

The embodiment provides a preservation solution, which comprises 20mM of trihydroxyaminomethane, 150mM of potassium chloride, 1mM of TCEP, 0.5% of ethanol by mass and volume, 0.05% of anthocyanin by mass and volume, and has a pH value of 7.4.

Example 5 preservation and direct amplification assays Using preservation solution V1 prepared in example 1 and preservation solutions W1-W3, UTM preservation solution, deionized water, physiological saline of comparative example

Referring to the second aspect of the present invention, there is provided a method of using a virus specimen directly amplified preservation solution, comprising adding 10 to 1mL of different preservation solutions^*5Pseudovirus (COVID-19-pseudovirus), reversing the virus upside down 10 times to mix well, standing at room temperature for 10min, using Luna univarial probe step RT-qPCRkit (Cat # E3006S) of NEB company, and using 2 uL containing pseudovirus (see kit instruction)A virus preservation solution sample is added into a 20 mu L system to detect a pseudovirus OFR1ab gene fragment, a primer probe sequence is derived from Chinese CDC, and the direct amplification effect of RNA virus and the preservation performance of virus RNA in a preservation solution are detected and analyzed by an ABI fluorescent quantitative PCR instrument 7500 fast. Detection of Ct values of pseudovirus samples stored at room temperature (25 ℃) for days 0, 3 and 5 in different storage solutions. As can be seen from table 1, the addition of anthocyanins maintained the preservation properties of preservation solution V, whereas viral RNA could not be preserved at room temperature without the addition of anthocyanins to W1. Comparison of initial Ct values with preservation solutions V1 and W2 we found that reducing agents also had a significant effect on the lysis of viral samples and the release of RNA. Comparison of the preservation solution V1 with W3 shows that the virus sample can not release nucleic acid substances in the virus sample and is difficult to detect if no surfactant is added, and the UTM preservation solution, deionized water and physiological saline of the control can not release nucleic acid.

The qPCR instrument conditions were as follows

Table 1: preservation of viral RNA in different sample preserving solutions at room temperature and direct amplification detection

Specimen preservation solution	Day0(Ct)	Day2(Ct)	Day5(Ct)	△Ct*
					V1	22.1	22.4	22.6	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	/

Δ Ct ═ Ct on day 0-Ct on day 5

NT is not detected

Example 6: verification of different contents of anthocyanins for preservation of viral RNA

In the formulation of the preservation solution V1 prepared in example 1, we prepared a series of preservation solutions containing anthocyanins at different concentrations, and experiments were carried out in the protocol of reference example 5. Adding 10 into 1mL of different storage solutions^*5Pseudovirus (COVID-19-pseudovirus), upside down for 10 times until mixing, after standing for 10min at room temperature, using the Luna univarial probe one step RT-qPCR kit (Cat # E3006S) of NEB company, taking 2 muL of a preservation solution sample containing the pseudovirus and adding the sample into a 20 muL system by using a method referring to the kit instruction, and analyzing the Ct values of the pseudovirus sample at the 0 th, the 3 th, the 5 th and the 7 th days in different preservation solutions at room temperature (25 ℃) by detecting the pseudovirus OFR1ab gene fragment. Surprisingly, we found that the addition of excess anthocyanins resulted in a delay in Ct values and even no detectable signal, indicating that excess anthocyanins inhibited RT-PCR reactions, while too little anthocyanins (e.g., 0.01%) did not function well for RNA preservation. Therefore in the formulation of the preservation solution in the following examples we used 0.05% anthocyanin as additive.

Table 2: preservation of viral RNA in different sample preserving solutions at room temperature and direct amplification detection

Example 7: preservation solution V2

The invention provides a preservation solution, which comprises the following components: 0.1 percent of dodecyl benzene triethanolamine, 0.5 percent of Brij 56, 0.1 percent of octyl polyoxyethylene tetradecyl ammonium chloride, 20mM of ammonium sulfate, 10mM of vitamin C, 5 percent of polyethylene glycol 600 and 0.05 percent of anthocyanin by mass, and the pH value is 8.0.

Example 8: preservation solution V3

The invention provides a preservation solution, which comprises the following components: brij 58 with the mass percent by volume of 0.75%, lithium dodecyl sulfate with the mass percent by volume of 0.05%, trihydroxy aminomethane with the mass percent by volume of 20mM, potassium chloride with the mass percent of 150mM, TCEP with the mass percent of 1mM, ethanol with the mass percent by volume of 0.5%, anthocyanin with the mass percent by volume of 0.05%, BSA with the mass percent by volume of 0.1% and pH value of 7.4.

Example 9: preservation solution V4

The invention provides a preservation solution, which comprises the following components: brij 58 with the mass percent by volume of 0.75%, lithium dodecyl sulfate with the mass percent by volume of 0.05%, trihydroxyaminomethane with the mass percent by volume of 20mM, potassium chloride with the mass percent by volume of 150mM, TCEP with the mass percent by volume of 1mM, ethanol with the mass percent by volume of 0.5%, anthocyanin with the mass percent by volume of 0.05%, DOPC with the mass percent by volume of 0.1% and pH7.4.

Example 10: preservation solution V5

The invention provides a preservation solution, which comprises the following components: brij 58 with the mass percent by volume of 0.75%, lithium dodecyl sulfate with the mass percent by volume of 0.05%, trihydroxyaminomethane with the mass percent by volume of 20mM, potassium chloride with the mass percent of 150mM, TCEP with the mass percent of 1mM, ethanol with the mass percent by volume of 0.5%, anthocyanin with the mass percent by volume of 0.05%, DOPC-R with the mass percent by volume of 0.05% and pH 7.4.

Comparative example 4 preserving fluid B1

20mM of trishydroxyaminomethane, 150mM of potassium chloride, 0.1% BSA, pH 7.4.

Comparative example 5 preserving fluid B2

20mM of trihydroxyaminomethane, 150mM of potassium chloride, 0.75% of Brij 58, 0.05% of lithium dodecyl sulfate and 20mM of trihydroxyaminomethane, and the pH value is 7.4.

Example 11: storage and direct amplification assays were performed using the storage solutions and deionized water prepared in examples 7-10, and comparative examples 4 and 5

Referring to the second aspect of the present invention, there is provided a method of using a virus specimen directly amplified preservation solution, comprising adding 10 to 1mL of different preservation solutions^*5Pseudovirus (COVID-19-pseudovirus), upside down for 10 times until mixing, after standing for 10min at room temperature, using the Luna univariate probe step RT-qPCRkit (Cat # E3006S) of NEB company, taking 2 muL of a preservation solution sample containing the pseudovirus and adding the sample into a 20 muL system by using a method referring to a kit instruction, and analyzing the Ct values of the pseudovirus sample at the room temperature (25 ℃) for preservation on days 0, 3 and 5 in different preservation solutions by detecting a pseudovirus OFR1ab gene fragment. From the data in Table 3, we have found that the initial Ct value is lower when different gene releasing agents are added, indicating that the addition of the gene releasing agent is more favorable for releasing nucleic acid from the virus sample. Surprisingly, BSA, as a component of formulations for preservation of viral and cellular integrity by UTM and VTM preservation solutions, served to protect cells, but in our formulation testing it was found that preservation solution B2 alone also served as a means of releasing nucleic acids from viruses. In addition, DOPC-R and DOPC have more excellent gene release effects as gene releasing agents than BSA.

Table 3: preservation of viral RNA in different sample preserving solutions at room temperature and direct amplification detection

Example 12: direct amplification of different concentrations of virus using preservative fluid V5

To 1mL of the storage solution V5 prepared in example 10 was added 10^*5、10^*4、10^*3And pseudovirus (COVID-19-pseu)dovirus), reversing the upside down for 10 times to mix evenly, standing for 10min at room temperature, adding 2 mu L of a pseudovirus-containing preservation solution sample into a 20 mu L system by using a Luna elementary probe one step RT-qPCR kit (Cat # E3006S) of NEB company, and analyzing the detection results of the pseudovirus samples with different titers in the preservation solution by detecting the pseudovirus OFR1ab gene segment, wherein the result is shown in figure 1, and the sample preservation solution V5 has a good release effect on the virus RNA in the pseudoviruses with different titers.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention. The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended 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.

It should be noted that the above embodiments can be freely combined as necessary. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A virus sample direct amplification type preservation solution comprises a surfactant, a buffer, salt ions, a reducing agent, alcohol and a nuclease inhibitor; wherein, the mass volume percentage of the surfactant is 0.01-5%; the final concentration of the buffer is 2-250 mM; the concentration of salt ions is 10-500 mM; the concentration of the reducing agent is 0.5-50 mM; the mass volume percentage of the alcohol is 0.1-15%; the mass volume percentage of the nuclease inhibitor is 0.01-1%; the pH value of the preservation solution is 5.5-9.5.

2. The preservation solution for directly amplifying virus samples according to claim 1, wherein the preservation solution further comprises a gene releasing agent, wherein the gene releasing agent is Bovine Serum Albumin (BSA), 1, 2-dioleoyl lecithin derivatives and/or combinations thereof.

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

the 1, 2-dioleoyl lecithin derivative has the following structure:

3. the preservation solution for directly amplifying virus samples according to claim 2, wherein the gene releasing agent is bovine serum albumin with a mass volume percentage of 0.05-0.5%, 1, 2-dioleoyl lecithin with a mass volume percentage of 0.01-0.5%, 1, 2-dioleoyl lecithin derivatives with a mass volume percentage of 0.01-0.35%, and/or combinations thereof.

4. The virus sample direct amplification type preservation solution according to claim 1, wherein the surfactant is triethanolamine dodecyl sulfate, triethanolamine dodecyl benzene, Brij 35, Brij 56, Brij 58, sodium dodecyl sulfate, NP40, polyethylene glycol octylphenyl ether, Span-80, Span-20, lithium dodecyl sulfate, sodium dodecyl benzene sulfonate, sodium dodecyl sulfonate, sarcosyl, octadecyl amine polyoxyethylene ether biquaternary ammonium salt, cetyl alcohol polyoxyethylene ether dimethyloctane ammonium chloride, octyl polyoxyethylene tetradecyl ammonium chloride, octyl polyoxyethylene dodecyl ammonium chloride, and/or a combination thereof.

5. The virus sample direct amplification type preservation solution according to claim 1, wherein the buffer is tris, 4-hydroxyethyl piperazine 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, bicine, 3- (cyclohexylamine) -1-propanesulfonic acid, 3-morpholinopropanesulfonic acid, and/or a combination thereof.

6. The preservation solution for direct viral sample amplification according to claim 1, wherein the salt ion is sodium chloride, ammonium chloride, sodium sulfate, ammonium sulfate, potassium chloride, lithium chloride, sodium acetate, and/or a combination thereof.

7. The preservation solution for directly amplifying viral samples according to claim 1, wherein 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, thioglycolic acid and/or combinations thereof.

8. The preservation solution for directly amplifying virus samples according to claim 1, wherein 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 8000 and/or a combination thereof.

9. The preservation solution for directly amplifying viral samples according to claim 1, wherein the nuclease inhibitor is diethyl pyrophosphate (DEPC), guanidinium isothiocyanate, vanadyl riboside complex, RNase protein inhibitor (RNase), urea, theaflavin, anthocyanidin, flavonoid, lignan, N-acetamido hexose, hexuronic acid and/or a combination thereof.

10. A method for using a preservation solution for directly amplifying a virus sample, wherein the preservation solution is the preservation solution according to any one of claims 1 to 9; the method is characterized by comprising the following steps:

obtaining a sample that may contain a virus;