Preserving solution for preserving RNA (Ribonucleic Acid) nucleic acid in biological sample at normal temperature

Abstract

The invention discloses a preserving fluid for preserving RNA (Ribonucleic Acid) nucleic acid in a biological sample at normal temperature, and belongs to the technical field of biological preserving reagents. The preservation solution comprises the following components in percentage by mass volume: 10-30% of dimethylacetamide, 90-200mM of inorganic salt, 1-10M of cracking agent, 20-100mM of chelating agent, 0.5-5% of reducing agent, 1-5M of stabilizing agent and 30-200mM of buffer salt, wherein the pH (Potential of Hydrogen) of the preservation solution is 6.0-9.0. According to the invention, dimethylacetamide is used as a preservative, so that a biological sample can be preserved at normal temperature for 15 days without degrading RNA nucleic acid. The preserving fluid provided by the invention is very suitable for preserving RNA nucleic acids, especially miRNA, can provide important guarantee for medical detection based on miRNA, and has very important clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preservation reagents, and specifically relates to a preservation solution for preserving RNA nucleic acids in biological samples at room temperature. Background Art

[0002] In recent years, with the development of the molecular detection industry in China, the popularization of early cancer screening and home detection, and the breakthrough of sample preservation technology, the market scale of nucleic acid sample preservation reagents in China has been growing steadily.

[0003] At present, there are many institutions in China that can collect samples, but the number of institutions that can perform nucleic acid detection is limited. Therefore, the samples need to be transported for a certain period before entering the detection process; or when the number of samples is small, the sampling institution will collect samples for a period of time and then transport the samples to the nucleic acid detection institution for testing. Therefore, the samples need to be preserved and transported for a certain period of time before entering the detection process. For a long time, the preservation of RNA nucleic acid samples has relied on cryogenic transportation such as ultra-low temperature liquid nitrogen or dry ice. The cryogenic transportation method has limitations such as high transportation cost, high equipment cost, and low convenience.

[0004] At present, for example, the preservation solution for RNA nucleic acid samples of viruses on the market has a short preservation time at room temperature, and can generally preserve samples at room temperature for about 3 to 7 days. Long-term preservation will affect subsequent nucleic acid detection. For example: when the virus load of the sample is low (samples near the detection limit of nucleic acid detection reagents), after long-term preservation at room temperature, it may not be detected, resulting in false negative results, affecting subsequent treatment and timely implementation of isolation and other measures.

[0005] Therefore, there is an urgent need in the art for a preservation reagent that can preserve RNA nucleic acids for a long time at room temperature. Summary of the Invention

[0006] In order to solve the above technical problems, through long-term exploration and research, the inventors unexpectedly found that when dimethylacetamide is used as a preservative, biological samples can be preserved at room temperature for 15 days without degradation of RNA nucleic acids, and subsequent detection is not affected, thus completing the present invention. The technical solution adopted by the present invention is as follows:

[0007] The first aspect of the present invention provides a preservation solution for preserving RNA nucleic acids in biological samples at room temperature, comprising dimethylacetamide with a mass-volume percentage of 10-30%, inorganic salts with a molar concentration of 90-200 mM, a lysing agent with a molar concentration of 1-10 M, a chelating agent with a molar concentration of 20-100 mM, a reducing agent with a mass-volume percentage of 0.5-5%, a stabilizer with a molar concentration of 1-5 M, and a buffer salt with a molar concentration of 30-200 mM, and the pH is 6.0-9.0.

[0008] In the present invention, the inventors unexpectedly found that dimethylacetamide alone as a protective agent, through reasonable configuration with inorganic salts, lysing agents, chelating agents, reducing agents, buffer salts, etc., can be used to preserve RNA nucleic acids in biological samples at room temperature.

[0009] In some embodiments of the present invention, the inorganic salt is selected from at least one of the group consisting of sodium chloride, sodium sulfate, potassium chloride, potassium carbonate, and lithium chloride.

[0010] In some embodiments of the present invention, the lysing agent is selected from at least one of the group consisting of urea, guanidine hydrochloride, and guanidine isothiocyanate.

[0011] In some embodiments of the present invention, the chelating agent is selected from at least one of the group consisting of ethylenediaminetetraacetic acid (EDTA), citric acid, and ethylene glycol bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA).

[0012] In some embodiments of the present invention, the reducing agent is selected from at least one of the group consisting of vitamin E, coenzyme Q10, thioglycerol, dibutylhydroxytoluene, and propyl gallate.

[0013] In some embodiments of the present invention, the stabilizer is selected from at least one of the group consisting of bovine serum albumin, casein, and gelatin.

[0014] In some embodiments of the present invention, the buffer salt is selected from at least one of the group consisting of Tris, sodium citrate, MOPS, HEPES, and PBS.

[0015] The second aspect of the present invention provides the use of dimethylacetamide in the preparation of a preservation solution for preserving RNA nucleic acids in biological samples at room temperature, wherein the mass-volume percentage of dimethylacetamide in the preservation solution is 10-30%.

[0016] In some embodiments of the present invention, dimethylacetamide is the sole preservative in the preservation solution.

[0017] In the present invention, the biological sample is selected from one of the group consisting of serum samples, plasma samples, urine samples, and saliva samples.

[0018] In the present invention, the "RNA nucleic acids" are selected from at least one of the group consisting of mRNA, tRNA, rRNA, lncRNA, miRNA, siRNA, piRNA, saRNA, TERC, snRNA, scRNA, snoRNA, eRNA, circRNA, and ribozymes.

[0019] In some preferred embodiments of the present invention, the RNA-based nucleic acid is miRNA. miRNA has a short nucleic acid chain, is easily degraded, and its molecular weight is often relatively small. Therefore, it is of great significance to preserve miRNA at room temperature.

[0020] In the present invention, the "room temperature" also referred to as "ambient temperature" means 10 - 35°C.

[0021] Advantages of the present invention

[0022] Compared with the prior art, the present invention has the following advantages:

[0023] The present invention uses dimethylacetamide alone as a preservative, has no requirements for other components of the preservation solution, and can preserve biological samples at room temperature for 15 days without degradation of RNA-based nucleic acids.

[0024] The preservation solution of the present invention is very suitable for the preservation of RNA-based nucleic acids, especially miRNA, and can provide an important guarantee for miRNA-based medical detection, having very important clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shows the CT values of qPCR detection of serum samples preserved by preservation solutions 1.1 - 1.4 in Example 1 of the present invention at 0 day and 15 days.

[0026] Figure 2 Shows the CT values of qPCR detection of serum samples preserved by preservation solutions 2.1 - 2.8 in Example 2 of the present invention at 0 day and 15 days.

[0027] Figure 3 Shows the CT values of qPCR detection of plasma samples preserved by preservation solutions 2.1 - 2.8 in Example 3 of the present invention at 0 day and 15 days.

[0028] Figure 4 Shows the CT values of qPCR detection of urine samples preserved by preservation solutions 2.1 - 2.8 in Example 3 of the present invention at 0 day and 15 days.

[0029] Figure 5 Shows the CT values of qPCR detection of saliva samples preserved by preservation solutions 2.1 - 2.8 in Example 3 of the present invention at 0 day and 15 days.

[0030] Figure 6 Shows the changing trend of CT values of qPCR detection of miRNA in serum samples preserved by preservation solutions 2.1 - 2.8 in Example 4 of the present invention from 0 to 15 days.

[0031] Figure 7It shows the change trend of CT values detected by qPCR of RNA in serum samples stored with preservation solution 2.1 - 2.8 for 0 - 15 days in Example 4 of the present invention. Detailed implementation manners

[0032] Unless otherwise specified, implied from the context or in accordance with the convention of the prior art, all parts and percentages in this application are based on weight, and the test and characterization methods used are synchronized with the filing date of this application. Where applicable, any patents, patent applications or published contents referred to in this application are incorporated herein by reference in their entirety, and their equivalent family patents are also incorporated by reference, especially the definitions of relevant terms in the art disclosed in these documents. If the definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition of the term provided in this application shall prevail.

[0033] The numerical ranges in this application are approximate values, so unless otherwise specified, they may include values outside the ranges. The numerical ranges include all values from the lower limit value to the upper limit value increased by 1 unit, provided that there is an interval of at least 2 units between any lower value and any higher value. For ranges containing values less than 1 or fractions greater than 1 (such as 1.1, 1.5, etc.), 1 unit is appropriately regarded as 0.0001, 0.001, 0.01 or 0.1. For ranges containing single - digit numbers less than 10 (such as 1 to 5), 1 unit is usually regarded as 0.1. These are only specific examples of what is intended to be expressed, and all possible combinations of the values between the lowest and highest values listed are considered to be clearly recorded in this application.

[0034] The terms "comprising", "including", "having" and their derivatives do not exclude the existence of any other components, steps or processes, and are independent of whether these other components, steps or processes are disclosed in this application. To eliminate any doubt, unless explicitly stated, all compositions using the terms "comprising", "including", or "having" in this application may contain any additional additives, excipients or compounds. In contrast, the term "consisting essentially of" excludes any other components, steps or processes from the scope described below any such term, except those necessary for the operating performance. The term "consisting of" does not include any components, steps or processes not specifically described or listed. Unless explicitly stated, the term "or" refers to the individual members listed or any combination thereof.

[0035] In order to make the technical problems, technical solutions and beneficial effects solved by the present invention more clearly understood, the present invention will be further described in detail below with reference to the embodiments.

[0036] Embodiment

[0037] The following examples are used herein to demonstrate the preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to implement the present invention, and thus can be regarded as preferred embodiments for implementing the present invention. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein and still obtain the same or similar results without departing from the spirit or scope of the present invention.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications, patent applications, patents, and other references mentioned herein and their incorporated materials will be incorporated by reference.

[0039] Those skilled in the art will recognize or be able to ascertain many equivalent techniques to the specific embodiments of the invention described herein through routine experimentation. Such equivalents will be included in the claims.

[0040] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the test materials used in the following examples are obtained from conventional biochemical reagent stores unless otherwise specified.

[0041] Example 1 Four Kinds of Sample Preservation Liquids 1.1 - 1.4

[0042] Sample Preservation Liquid 1.1: This example provides a sample preservation liquid, which includes 20% dimethylacetamide by mass - volume percentage, potassium chloride with a final concentration of 180 mM, guanidine isothiocyanate with a final concentration of 3 M, EDTA with a final concentration of 40 mM, thioglycerol with a mass - volume percentage of 3%, bovine serum albumin with a final concentration of 4 M, Tris - HCl with a final concentration of 70 mM, prepared with DEPC water, and the pH is 8.40.

[0043] Sample Preservation Liquid 1.2: The difference from Sample Preservation Liquid 1.1 is that 3 M guanidine isothiocyanate is replaced with 3 M guanidine hydrochloride, and the other components and concentrations are the same.

[0044] Sample Preservation Liquid 1.3: The difference from Sample Preservation Liquid 1.1 is that Tris - HCl with a final concentration of 70 mM is replaced with sodium citrate with a final concentration of 70 mM, and the other components and concentrations are the same.

[0045] Sample Preservation Liquid 1.4: The difference from Sample Preservation Liquid 1.1 is that EDTA with a final concentration of 40 mM is replaced with nitrilotriacetic acid with a final concentration of 40 mM, and the other components and concentrations are the same.

[0046] Serum samples were added to 4 kinds of sample preservation solutions respectively. The ratio of the sample preservation solution to the serum sample was 3:1. RNA was extracted before room temperature preservation and 15 days after preservation for fluorescence quantitative qPCR detection. Two serum samples were preserved with each kind of sample preservation solution, and the detection was repeated 2 times. The CT values of the detection results are shown in Table 1, and the average CT values are as Figure 1 shown.

[0047] Table 1 Comparison table of CT values detected for serum samples preserved by preservation solutions 1.1 - 1.4

[0048]

[0049]

[0050] Among them, G1 - G12 represent different genes, and the same applies hereinafter.

[0051] From Table 1 and Figure 1 it can be seen that when using 4 kinds of sample preservation solutions to preserve serum samples at room temperature, the CT values of qPCR detection of RNA did not increase significantly on the 15th day compared with the 0th day. It shows that the protection effects of the 4 kinds of sample preservation solutions on the RNA nucleic acid of serum samples are all relatively ideal, indicating that when using dimethylacetamide as a preservative, there are no special requirements for other tissues in the preservation solution such as buffer salts.

[0052] Example 2 Cross - use of different protective agents

[0053] This example provides 8 kinds of sample preservation solutions 2.1 - 2.8, specifically as follows:

[0054] Sample preservation solution 2.1: The same as sample preservation solution 1.1.

[0055] Sample preservation solution 2.2: The difference from sample preservation solution 1.1 is that dimethylacetamide is not added.

[0056] Sample preservation solution 2.3: The difference from sample preservation solution 1.1 is that 20% dimethylacetamide is replaced by 20% dimethyl sulfoxide, and the other components and concentrations are the same.

[0057] Sample preservation solution 2.4: The difference from sample preservation solution 1.1 is that 20% dimethylacetamide is replaced by acetonitrile with a final concentration of 20%, and the other components and concentrations are the same.

[0058] Sample preservation solution 2.5: The difference from sample preservation solution 1.1 is that 20% dimethylacetamide is replaced by 10% dimethyl sulfoxide and 10% acetonitrile, and the other components and concentrations are the same.

[0059] Sample preservation solution 2.6: The difference between it and sample preservation solution 1.1 is that on the basis of sample preservation solution 1.1, it also includes 10% dimethyl sulfoxide, and the remaining components and concentrations are the same.

[0060] Sample preservation solution 2.7: The difference between it and sample preservation solution 1.1 is that on the basis of sample preservation solution 1.1, it also includes acetonitrile with a final concentration of 10%, and the remaining components and concentrations are the same.

[0061] Sample preservation solution 2.8: The difference between it and sample preservation solution 1.1 is that on the basis of sample preservation solution 1.1, it also includes 5% dimethyl sulfoxide and 5% aqueous acetonitrile solution, and the remaining components and concentrations are the same.

[0062] Serum samples were added to 8 kinds of sample preservation solutions respectively, and the ratio of sample preservation solution to serum sample was 3:1. RNA was extracted and detected by fluorescence quantitative qPCR at 0 day and 15 days of storage at room temperature respectively. Each sample preservation solution was used to preserve 2 serum samples, and the detection was repeated 2 times. The CT values of the detection results are shown in Table 2, and the average CT values are as Figure 2 shown.

[0063] Table 2 Comparison table of CT values for detecting serum samples preserved by preservation solutions 2.1 - 2.8

[0064]

[0065]

[0066] It can be seen from Table 2 and Figure 2 that:

[0067] (1) Dimethylacetamide is not added (sample preservation solution 2.2);

[0068] (2) Dimethyl sulfoxide and acetonitrile are used alone (sample preservation solutions 2.3 and 2.4);

[0069] (3) Three preservatives, dimethylacetamide, dimethyl sulfoxide and acetonitrile, are used crosswise in pairs (sample preservation solutions 2.5 - 2.7);

[0070] (4) Three preservatives, dimethylacetamide, dimethyl sulfoxide and acetonitrile, are used simultaneously (sample preservation solution 2.8),

[0071] The protection effects of the above 4 situations on RNA in serum are not as good as those of the sample preservation solution using dimethylacetamide alone (sample preservation solution 2.1), which is different from the general perception of those skilled in the art (the more preservatives, the better), and is unexpected to those skilled in the art.

[0072] Example 3 Sample preservation solution - Different sample types

[0073] In this embodiment, the sample preservation solution 2.1 (i.e., the sample preservation solution 1.1)-2.8 is applied to the preservation of other different types of samples. Specifically, plasma samples, urine samples, and saliva samples are respectively added to the sample preservation solution, and RNA nucleic acids are extracted and fluorescence quantitative qPCR detection is carried out at 0 days and 15 days of room temperature storage. Two serum samples are preserved in each sample preservation solution, and the detection is repeated 2 times. The detection results are shown in Table 3 to Table 5 and Figures 3 to 5 as shown.

[0074] Table 3 Comparison table of CT values for detecting plasma samples preserved by preservation solutions 2.1-2.8

[0075]

[0076]

[0077] Table 4 Comparison table of CT values for detecting urine samples preserved by preservation solutions 2.1-2.8

[0078]

[0079]

[0080] Table 5 Comparison table of CT values for detecting saliva samples preserved by preservation solutions 2.1-2.8

[0081]

[0082] From Table 2 to Table 5 and Figures 2 to 5 it can be seen that for the preservation of serum samples, plasma samples, urine samples, and saliva samples by the sample preservation solutions 2.1-2.8, after 0 days and 15 days of room temperature storage, the preservation solution 2.1 has the best preservation effect. It shows that the sample preservation solution 2.1 can better preserve the RNA nucleic acids in serum, plasma, urine, and saliva samples, prevent degradation, and the preservation effect is ideal.

[0083] Example 4 Room temperature stability of the sample preservation solution - RNA and miRNA experiment comparison

[0084] In this embodiment, the preservation effects of the sample preservation solutions 2.1 (i.e., the sample preservation solution 1.1) to 2.8 on RNA and miRNA in serum samples are explored. Specifically, serum samples are respectively added to the sample preservation solutions 2.1-2.8, and RNA nucleic acids are extracted and fluorescence quantitative qPCR detection is carried out at 0 days, 5 days, 10 days, and 15 days of room temperature storage. The qPCR detection includes RNA detection and miRNA detection. Four serum samples are preserved in each sample preservation solution, and the detection is repeated 2 times. The detection results are shown in Table 6 to Table 7 and Figures 6 to 7 as shown.

[0085] Table 6 Comparison table of CT values for the detection of miRNAs stored in preservation solution 2.1 - 2.8

[0086]

[0087]

[0088] Table 7 Comparison table of CT values for the detection of RNAs stored in preservation solution 2.1 - 2.8

[0089]

[0090]

[0091] From Tables 6 to 72 and Figures 6 to 7 it can be seen that when using preservation solution 2.1 to preserve serum samples, after storing at room temperature for 15 days, the CT values of qPCR detection of RNA stored in preservation solution 2.1 show no significant change, and the CT values of qPCR detection of miRNA remain more stable. After storing at room temperature for 15 days, for both RNA and miRNA stored in preservation solutions 2.2 - 2.8, the CT values are larger than those of preservation solution 2.1, and the CT values increase from day 0 to day 15. The above results indicate that preservation solution 2.1 can preserve RNA, especially miRNA, in serum samples.

[0092] All documents mentioned in the present invention are cited herein as references, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. A preservation solution for preserving RNA nucleic acids in biological samples at room temperature, characterized in that, It includes dimethylacetamide with a mass-volume percentage of 10 to 30%, inorganic salts with a molar concentration of 90 to 200 mM, a lysing agent with a molar concentration of 1 to 10 M, a chelating agent with a molar concentration of 20 to 100 mM, a reducing agent with a mass-volume percentage of 0.5 to 5%, a stabilizer with a molar concentration of 1 to 5 M, and a buffer salt with a molar concentration of 30 to 200 mM, and the pH is 6.0 to 9.

0.

2. The preservation solution for RNA nucleic acid in biological samples for room temperature preservation according to claim 1, wherein The inorganic salt is selected from at least one of the group consisting of sodium chloride, sodium sulfate, potassium chloride, potassium carbonate, and lithium chloride.

3. A preservation solution for preserving RNA nucleic acids in biological samples at room temperature according to claim 1, characterized in that, The lysing agent is selected from at least one of the group consisting of urea, guanidine hydrochloride, and guanidine isothiocyanate.

4. A preservation solution for preserving RNA nucleic acids in biological samples at room temperature according to claim 1, wherein The chelating agent is selected from at least one of the group consisting of ethylenediaminetetraacetic acid (EDTA), citric acid, and ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA).

5. The preservation solution for RNA nucleic acids in biological samples for room temperature preservation according to claim 1, wherein, The reducing agent is selected from at least one of the group consisting of vitamin E, coenzyme Q10, thioglycerol, butylated hydroxytoluene, and propyl gallate.

6. The preservation solution for RNA nucleic acids in biological samples for room temperature preservation according to claim 1, wherein, The stabilizer is selected from at least one of the group consisting of bovine serum albumin, casein, and gelatin.

7. A preservation solution for preserving RNA nucleic acids in biological samples at room temperature according to claim 1, characterized in that, The buffer salt is selected from at least one of the group consisting of Tris, sodium citrate, MOPS, HEPES, and PBS.

8. Use of dimethylacetamide in the preparation of a preservation solution for preserving RNA nucleic acids in biological samples at room temperature, characterized in that, The mass-volume percentage of the dimethylacetamide in the preservation solution is 10 to 30%.

9. The application according to claim 8, wherein The biological sample is selected from one of the group consisting of serum samples, plasma samples, urine samples, and saliva samples.

10. The application according to claim 8 or 9, characterized in that, The RNA-based nucleic acid is miRNA.

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