A preserving solution for ammonium bisulfite, a preserving method and a kit

Abstract

The present application relates to a high concentration of ammonium bisulfite preservation solution, the preservation solution components include ammonium bisulfite and ethanol, the concentration of ammonium bisulfite is about 13 mol / L to about 15 mol / L, the concentration of ethanol is about 0.3 mol / L to about 1.5 mol / L. The present application also provides a method and kit for preserving high concentration of ammonium bisulfite solution.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to a method for preserving ammonium bisulfite solution. Background Technology

[0002] Ammonium bisulfite has many advantages in promoting DNA transformation. It is an effective conversion reagent for cytosine base transformation. Using ammonium bisulfite conversion reagent can shorten the processing time of other conversion reagents in existing methods from several hours to several minutes to tens of minutes while ensuring a low loss rate of DNA samples. In addition, the composition of ammonium bisulfite conversion reagent is simple and easy to prepare, and it does not need to be prepared on the spot.

[0003] However, high-concentration ammonium bisulfite solutions are easily oxidized when exposed to air, leading to decreased solution stability, loss of components, and consequently affecting the base conversion function of ammonium bisulfite. In practical applications, ammonium bisulfite exists at a high concentration of 68%-70%, and during production and storage, it is easily exposed to air. Prolonged contact with oxygen can cause oxidation, resulting in reduced conversion efficiency. Summary of the Invention

[0004] This application provides a method for producing a base conversion kit. The high-concentration ammonium bisulfite component in the conversion reagent remains stable during the production process. Even after long-term storage, the conversion reagent still exhibits high conversion efficiency during DNA conversion, indicating that the high-concentration ammonium bisulfite component remains stable. The production method is simple to operate, the solution is easy to prepare, and the resulting kit can be stably stored at room temperature.

[0005] This invention provides a method for preserving high-concentration ammonium bisulfite. By adding ethanol to the ammonium bisulfite solution, a preservation solution is prepared with an ammonium bisulfite concentration of approximately 13 mol / L to approximately 15 mol / L and an ethanol concentration of approximately 0.3 mol / L to approximately 1.5 mol / L, thereby greatly extending the shelf life of the ammonium bisulfite conversion reagent.

[0006] The existing approach is to use stronger reducing agents such as quinones and phenols to replace ammonium bisulfite in the solution for reaction with air, thus preventing the ammonium bisulfite from being oxidized. However, the addition of strong reducing agents such as quinones and phenols to the conversion reagent raises concerns about whether it will affect the reagent's function, namely its ability to convert cytosine bases.

[0007] This application reveals that by adding ethanol of a specific component to an ammonium bisulfite solution, the ammonium bisulfite is prevented from being oxidized, extending the shelf life of the solution. The addition of ethanol does not affect the function of the conversion reagent; the conversion reagent can still convert cytosine bases even with prolonged storage. Furthermore, the described preservation method is simple to operate, the solution is easy to prepare, and the resulting kit can be stably stored at room temperature.

[0008] In a first aspect, this application provides a preservation solution for ammonium bisulfite, the components of which include ammonium bisulfite and ethanol, wherein the concentration of ammonium bisulfite is from 13 mol / L to 15 mol / L and the concentration of ethanol is from 0.3 mol / L to 1.5 mol / L.

[0009] In some embodiments, the concentration of ammonium bisulfite is from 13.7 mol / L to 14.1 mol / L. In some embodiments, the concentration of ammonium bisulfite is from 66% (w / w) to 70% (w / w). In some embodiments, the concentration of ammonium bisulfite is from 68% (w / w) to 70% (w / w).

[0010] In some embodiments, the concentration of ethanol is from 0.66 mol / L to 0.70 mol / L. In some embodiments, the concentration of ethanol is 68%. In some embodiments, the concentration of ethanol is from 3.8% (w / w) to 4.2% (w / w). In some embodiments, the concentration of ethanol is 4% (w / w).

[0011] In some embodiments, the preservation solution does not contain other stabilizers, such as hydroquinone or vitamin E.

[0012] Secondly, this application provides a method for preserving an ammonium bisulfite solution, the method comprising: (1) adding ethanol to the ammonium bisulfite solution to be preserved, and (2) mixing, wherein the concentration of ammonium bisulfite in the mixed solution is 13 mol / L to 15 mol / L, and the concentration of ethanol in the mixed solution is 0.3 mol / L to 1.5 mol / L. In some embodiments, the concentration of the ammonium bisulfite solution to be preserved is 68% (w / w) to 70% (w / w).

[0013] In some embodiments, (1) the added ethanol is anhydrous ethanol or any ethanol solution with a concentration of less than 99.9%.

[0014] In some implementations, the storage period is 3 to 12 months.

[0015] In some embodiments, the storage temperature is 0°C to 40°C. In some embodiments, the storage temperature is 20-37°C. In some embodiments, the storage temperature is room temperature.

[0016] Thirdly, this application provides a cytosine conversion kit, which includes a conversion agent, a washing buffer, a desulfurization buffer, an elution buffer, a purification column, and a collection tube, wherein the conversion agent is the ammonium bisulfite preservation solution described in this application.

[0017] Thirdly, this application provides a method for converting cytosine bases in a nucleic acid sample, the method comprising deamination of a reaction mixture by heating the reaction mixture at a temperature of 95°C to 98°C for 1 to 20 minutes to obtain a deamination reaction product, the reaction mixture comprising the nucleic acid sample and a converting agent, the converting agent comprising 50% (w / v) to 70% (w / v) of the total volume of the reaction mixture, wherein the converting agent is the ammonium bisulfite preservation solution described in this application.

[0018] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0019] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and accompanying drawings described in detail below. A brief description of the drawings is as follows:

[0020] Figure 1 The image shows the state of the ammonium bisulfite solution after 3, 6, and 12 months of storage, with and without the addition of ethanol stabilizer as described in this application.

[0021] Figures 2-4 The figures show the unconverted cytosine content of nucleic acid samples after being treated with ammonium bisulfite solution stored for 3, 6, and 12 months, with and without the addition of ethanol stabilizer, as described in this application. Figure 2 The results show the non-conversion rate of cytosine after nucleic acid samples were treated with ammonium bisulfite solution after being stored for 3 months. Figure 3 The results show the non-conversion rate of cytosine after nucleic acid samples were treated with ammonium bisulfite solution after being stored for 6 months. Figure 4The results show the non-conversion rate of cytosine after nucleic acid samples were treated with ammonium bisulfite solution for 12 months.

[0022] Figure 5 The image shows the state of the ammonium bisulfite solution after 3, 6, and 12 months of storage when ethanol, water-soluble vitamin E (Ve), and hydroquinone are added as stabilizers, as described in this application. Detailed Implementation

[0023] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0024] Terminology Definition

[0025] In this application, the terms “comprising” and “including” are used interchangeably, and generally refer to the subject that is defined by “comprising” and “including” having specific, specified characteristics, but without excluding other components or elements.

[0026] In this application, the term "preservation" generally refers to the operation of preventing the components and / or functions of a chemical reagent from changing due to the increase of the reagent's storage time. When a chemical reagent is "preserved," its components and / or functions do not change, or its shelf life is extended compared to when it is not "preserved." The reagent may be a chemical reagent, and may be an organic or inorganic chemical reagent.

[0027] In this application, the term "nucleic acid" is used interchangeably with "nucleic acid molecule" or "polynucleotide," and generally refers to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), a combination of DNA or RNA, or a polymer of the above substances in single-stranded or double-stranded form. The term "nucleic acid" also includes genes, sense or antisense strands of genomic DNA and synthetic forms and mixed polymers of both, cDNA, and / or mRNA. For example, nucleic acid molecules can be synthesized in vitro (e.g., chemically synthesized) or intracellularly (e.g., recombinant synthesis).

[0028] In this application, the term "ammonium bisulfite" generally refers to a reagent capable of distinguishing between modified and unmodified DNA regions. For example, ammonium bisulfite can deamination of the amino group of unmodified cytosine to distinguish it from modified cytosine. The ammonium bisulfite also includes analogs of ammonium bisulfite. In this application, the term "analyte" generally refers to a substance having a similar structure and / or function. For example, an analog of ammonium bisulfite may have a similar structure to ammonium bisulfite, or it may refer to a reagent that can also distinguish between modified and unmodified DNA regions.

[0029] In this application, the term "sample" generally refers to a specimen containing a substance to be analyzed, such as microorganisms, viruses, nucleic acids (e.g., genes), or components thereof, which may contain nucleic acids isolated from the analyte. Samples can be from any source, such as biological specimens or environmental sources. Biological specimens include any tissue or material obtained from a living or dead organism, which may contain the analyte or nucleic acids in or obtained from the analyte. Examples of biological samples include respiratory tissue, exudates (e.g., bronchoalveolar lavage fluid), biopsies, sputum, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, feces, urine, cerebrospinal fluid, tissue fluid (e.g., pleural and peritoneal fluid), lavage fluid (e.g., alveoli), or other fluids, tissues, or materials. The sample may also include tissues, such as tissues obtained from biopsies (e.g., tissues obtained from liquid biopsies), formalin-fixed and paraffin-embedded (FFPE) tissues, etc.

[0030] In this application, the terms "recovery" and "separation" or "purification" are used interchangeably and generally refer to the operation of removing one or more components from a mixture (e.g., a sample) to separate them from one or more other components in the mixture. Sample components include nucleic acids, and may include cell fragments, proteins, carbohydrates, lipids, and other compounds. For example, "recovery" can be the separation of nucleic acids from other components in a sample.

[0031] In this application, the term "elution" generally refers to the action of removing biological material from a substrate or solution. In some embodiments, the removal operation can be performed using a liquid or fluid, such as a suitable buffer system.

[0032] In this application, the term "about" is used to mean approximately, around, roughly, or about. When the term "about" is used in conjunction with a numerical value or range, it can modify and expand the range by extending the upper and lower limits of the listed numerical value or range. Typically, the term "about" is used herein to modify a numerical value by a variation of 10%, 8%, 5%, 2%, 1%, 0.5%, or 0.1% above or below the specified value.

[0033] In this application, the term "conversion" generally refers to a change in the bases of a nucleic acid molecule, that is, the change from one type of base to another under specific reaction conditions. For example, cytosine bases are converted to uracil bases under the catalysis of bisulfite. In some embodiments, the modified bases cannot be changed, such as methylated cytosine, which cannot be converted to uracil under the catalysis of bisulfite. Invention Details

[0035] Ammonium bisulfite preservation solution and cytosine conversion kit

[0036] This application provides a preservation solution for high-concentration ammonium bisulfite, the preservation solution comprising ammonium bisulfite and ethanol, wherein the concentration of ammonium bisulfite may be about 13 mol / L to about 15 mol / L, and the concentration of ethanol may be about 0.3 mol / L to about 1.5 mol / L.

[0037] This application also provides a kit comprising a conversion reagent capable of converting cytosine bases in a nucleic acid sample and instructions for use. The conversion reagent comprises ammonium bisulfite and ethanol, wherein the concentration of the ammonium bisulfite solution in the conversion reagent is approximately 13 mol / L to approximately 15 mol / L, and the concentration of the ethanol in the conversion reagent is approximately 0.3 mol / L to approximately 1.5 mol / L.

[0038] Meanwhile, this application provides a method for preserving a high-concentration ammonium bisulfite solution, comprising adding ethanol to the ammonium bisulfite solution, wherein the concentration of the ammonium bisulfite solution in the mixed solution is about 13 mol / L to about 15 mol / L, and the concentration of the ethanol in the mixed solution is about 0.3 mol / L to about 1.5 mol / L.

[0039] Meanwhile, this application provides a method for producing the above-mentioned cytosine conversion kit, the method comprising the preparation of a conversion reagent, wherein the preparation of the conversion reagent comprises adding ethanol to a high-concentration ammonium bisulfite solution, wherein the concentration of the ammonium bisulfite solution in the conversion reagent is about 13 mol / L to about 15 mol / L, and the concentration of the ethanol in the conversion reagent is about 0.3 mol / L to about 1.5 mol / L.

[0040] Based on the molar mass of ammonium bisulfite (99.1%), a 68%-70% ammonium bisulfite solution corresponds to a molar mass concentration of 13.7-14.1 mol / L. Similarly, based on the molar mass and density of ethanol, when the volume ratio of ethanol to ammonium bisulfite in the mixture is 2%-8% (volume ratio of anhydrous ethanol to ammonium bisulfite solution), the ethanol concentration in the mixture corresponds to 0.344-1.376 mol / L after conversion. When the volume ratio of ethanol to ammonium bisulfite in the mixture is 2%, 4%, 6%, and 8% (volume ratio of anhydrous ethanol to ammonium bisulfite solution), the ethanol concentration in the mixture corresponds to 0.344, 0.688, 1.032, and 1.376 mol / L, respectively.

[0041] Specifically, the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent can be approximately 13.1 mol / L to approximately 15 mol / L, approximately 13.3 mol / L to approximately 15 mol / L, approximately 13.5 mol / L to approximately 15 mol / L, approximately 13.7 mol / L to approximately 15 mol / L, approximately 13.7 mol / L to approximately 15 mol / L, approximately 13.9 mol / L to approximately 15 mol / L, approximately 14.1 mol / L to approximately 15 mol / L, approximately 14.3 mol / L to approximately 15 mol / L, approximately 14.5 mol / L to approximately 15 mol / L, approximately 14.7 mol / L to approximately 15 mol / L, or approximately 14.9 mol / L to approximately 15 mol / L.

[0042] Specifically, the concentration of ethanol in the preservation solution and / or conversion reagent may be approximately 0.3 mol / L to approximately 1.5 mol / L, approximately 0.4 mol / L to approximately 1.5 mol / L, approximately 0.5 mol / L to approximately 1.5 mol / L, approximately 0.6 mol / L to approximately 1.5 mol / L, approximately 0.7 mol / L to approximately 1.5 mol / L, approximately 0.8 mol / L to approximately 1.5 mol / L, approximately 0.9 mol / L to approximately 1.5 mol / L, approximately 1 mol / L to approximately 1.5 mol / L, approximately 1.1 mol / L to approximately 1.5 mol / L, approximately 1.2 mol / L to approximately 1.5 mol / L, approximately 1.3 mol / L to approximately 1.5 mol / L, or approximately 1.4 mol / L to approximately 1.5 mol / L.

[0043] In some embodiments, the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent described in this application may be from about 13.7 mol / L to about 14.1 mol / L.

[0044] In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent described in this application may be from about 0.3 mol / L to about 1.3 mol / L. Specifically, the concentration of ethanol in the preservation solution and / or conversion reagent can be approximately 0.34 mol / L to approximately 0.68 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent can be approximately 13.1 mol / L to approximately 15 mol / L, approximately 13.2 mol / L to approximately 15 mol / L, approximately 13.3 mol / L to approximately 15 mol / L, approximately 13.4 mol / L to approximately 15 mol / L, approximately 13.5 mol / L to approximately 15 mol / L, approximately 13.7 mol / L to approximately 15 mol / L, approximately 13.8 mol / L to approximately 15 mol / L, approximately 13.9 mol / L to approximately 15 mol / L, approximately 14 mol / L to approximately 15 mol / L, approximately 14.1 mol / L to approximately 15 mol / L, approximately 14.2 mol / L to approximately 15 mol / L, approximately 14.3 mol / L to approximately 15 mol / L. mol / L, approximately 14.4 mol / L to approximately 15 mol / L, approximately 14.5 mol / L to approximately 15 mol / L, approximately 14.6 mol / L to approximately 15 mol / L, approximately 14.7 mol / L to approximately 15 mol / L, approximately 14.8 mol / L to approximately 15 mol / L, or approximately 14.9 mol / L to approximately 15 mol / L.

[0045] Specifically, the concentration of ethanol in the preservation solution and / or conversion reagent can be approximately 0.68 mol / L to approximately 1.02 mol / L, approximately 0.34 mol / L to approximately 0.344 mol / L, approximately 0.344 mol / L to approximately 0.68 mol / L, approximately 0.68 mol / L to approximately 0.688 mol / L, approximately 1.02 mol / L to approximately 1.032 mol / L, 1.032 mol / L to approximately 1.37 mol / L, or approximately 1.37 mol / L to approximately 1.376 mol / L.

[0046] In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.344 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.7 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.344 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.8 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.344 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.9 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.344 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 14 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.344 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 14.1 mol / L.

[0047] In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.688 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.7 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.688 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.8 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.688 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.9 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.688 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 14 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 0.688 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 14.1 mol / L.

[0048] In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.032 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.7 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.032 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.8 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.032 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.9 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.032 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 14 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.032 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 14.1 mol / L.

[0049] In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.376 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.7 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.376 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.8 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.376 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 13.9 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.376 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 14 mol / L. In some embodiments, the concentration of ethanol in the preservation solution and / or conversion reagent may be about 1.376 mol / L, and the concentration of ammonium bisulfite in the preservation solution and / or conversion reagent may be about 14.1 mol / L.

[0050] Specifically, the preservation solution and / or conversion reagent described in this application can be stored for approximately 1 to approximately 30 months, or approximately 2 to approximately 30 months. Specifically, the preservation solution and / or conversion reagent described in this application can be stored for approximately 3 to approximately 24 months. Specifically, the preservation solution and / or conversion reagent described in this application can be stored for approximately 3 to approximately 18 months. Specifically, the preservation solution and / or conversion reagent described in this application can be stored for approximately 3 to approximately 12 months. Specifically, the preservation solution and / or conversion reagent described in this application can be stored for approximately 3 to approximately 6 months. Specifically, the preservation solution and / or conversion reagent described in this application maintain a clear state and / or do not crystallize within approximately 3 to approximately 24 months.

[0051] The preservation solution and / or conversion reagent can maintain the clarity of the solution or prevent crystallization within the aforementioned preservation time. The solution state can be observed by placing the preservation solution and / or conversion reagent at room temperature in the dark, and by comparing it with an ammonium bisulfite solution without ethanol, a white precipitate or crystallization can be observed. Comparative observation demonstrates that the preservation solution and / or conversion reagent provided in this application can maintain the stability and function of the solution within the preservation time, thereby significantly extending the preservation time of the ammonium bisulfite solution.

[0052] The storage temperature of the preservation solution and / or conversion reagent can be approximately 0°C to approximately 40°C. Specifically, the storage temperature of the preservation solution and / or conversion reagent can be approximately 15°C to approximately 25°C. Specifically, the storage temperature of the preservation solution and / or conversion reagent can be approximately 15°C to approximately 16°C, 15°C to approximately 17°C, 15°C to approximately 18°C, 15°C to approximately 19°C, 15°C to approximately 20°C, 15°C to approximately 21°C, 15°C to approximately 22°C, 15°C to approximately 23°C, or 15°C to approximately 24°C. Specifically, the storage temperature of the preservation solution and / or conversion reagent can be approximately 4°C to approximately 15°C.

[0053] Specifically, the storage temperature for the preservation solution and / or conversion reagent can be room temperature.

[0054] The preservation solution and / or kit can be used to transform cytosine bases in nucleic acid samples, wherein the conversion efficiency of cytosine bases to uracil bases can be about 90% or more, for example, about 92% or more, about 94% or more, about 96% or more, about 98% or more, or about 99% or more. For example, about 99%.

[0055] The kit provided in this application also includes instructions for use. The instructions for use include deamination of the reaction mixture of the conversion reagent and the nucleic acid sample in the kit, wherein the conversion reagent comprises approximately 50% (w / v) to approximately 70% (w / v) of the total volume of the reaction mixture, and the deamination is performed by heating the reaction mixture at a temperature of at least 90°C for approximately 1 minute to approximately 20 minutes.

[0056] To remove the amino group from the cytosine bases in the nucleic acid sample to generate the deamination reaction product, specifically, the deamination treatment described in the instructions may be performed by heating the reaction mixture at a temperature of at least 90°C for about 1-20 minutes, about 2-20 minutes, about 3-20 minutes, about 4-20 minutes, about 5-20 minutes, about 6-20 minutes, about 7-20 minutes, about 8-20 minutes, about 9-20 minutes, about 10-20 minutes, about 11-20 minutes, about 12-20 minutes, about 13-20 minutes, about 14-20 minutes, about 15-20 minutes, about 16-20 minutes, about 17-20 minutes, about 18-20 minutes, or about 19-20 minutes. In some embodiments, the deamination treatment may be performed by heating the reaction mixture at a temperature of at least 90°C for about 5 minutes to about 15 minutes to obtain the deamination product, i.e., heating for about 5-15 minutes, about 6-15 minutes, about 7-15 minutes, about 8-15 minutes, about 9-15 minutes, about 10-15 minutes, about 11-15 minutes, about 12-15 minutes, about 13-15 minutes, or about 14-15 minutes. For example, the deamination treatment may be performed by heating the reaction mixture at the stated temperature for about 10 minutes to obtain the deamination product. In some embodiments, the deamination treatment may be performed by heating the reaction mixture at a temperature of about 90-100°C, about 91-100°C, about 92-100°C, about 93-100°C, about 94-100°C, about 95-100°C, about 96-100°C, about 97-100°C, about 98-100°C, about 99-100°C, or higher. In some embodiments, the deamination treatment involves heating the reaction mixture at a temperature of about 95°C to about 98°C to obtain the deamination product. Specifically, the deamination treatment may be heating the reaction mixture at a temperature of about 95-98°C, about 96-98°C, or about 97-98°C to obtain the deamination product. More specifically, for example, the deamination treatment involves heating the reaction mixture at a temperature of about 98°C to obtain the deamination product. During the heating period of about 1 minute to about 20 minutes at a temperature of at least 90°C described in this application, the same temperature may be maintained, or the temperature may be varied, but the method does not include the step of heating the reaction mixture at a temperature of 85°C or below.

[0057] The method described in the instructions for use, wherein the reaction mixture comprises the nucleic acid sample and the conversion reagent, specifically, the conversion reagent may constitute approximately 50-70% (w / v), 51-70% (w / v), 52-70% (w / v), 53-70% (w / v), 54-70% (w / v), 55-70% (w / v), 56-70% (w / v), or 57-70% (w / v) of the total volume of the reaction mixture. The concentrations are approximately 58-70% (w / v), 59-70% (w / v), 60-70% (w / v), 61-70% (w / v), 62-70% (w / v), 63-70% (w / v), 64-70% (w / v), 65-70% (w / v), 66-70% (w / v), 67-70% (w / v), 68-70% (w / v), or 69-70% (w / v). In some embodiments, the conversion agent constitutes approximately 60% (w / v) to approximately 70% (w / v) of the total volume of the reaction mixture. Specifically, the conversion reagent may account for approximately 60-70% (w / v), 61-70% (w / v), 62-70% (w / v), 63-70% (w / v), 64-70% (w / v), 65-70% (w / v), 66-70% (w / v), 67-70% (w / v), 68-70% (w / v), or 69-70% (w / v) of the total volume of the reaction mixture.

[0058] In some embodiments, the conversion agent comprises about 61% (w / v) to about 63% (w / v) of the total volume of the reaction mixture. For example, the conversion agent may comprise about 60% (w / v), about 60.1-63% (w / v), about 60.2-63% (w / v), about 60.3-63% (w / v), about 60.4-63% (w / v), about 60.5-63% (w / v), about 60.6-63% (w / v), about 60.7-63% (w / v), about 60.8-63% (w / v), about 60.9-63% (w / v), about 61-63% (w / v), about 61.1-63% (w / v), about 61.2-63% (w / v), about 61.3-63% (w / v), about 61.4% (w / v), etc., of the total volume of the reaction mixture. -63% (w / v), approximately 61.5-63% (w / v), approximately 61.6-63% (w / v), approximately 61.7-63% (w / v), approximately 61.8-63% (w / v), approximately 61.9-63% (w / v), approximately 62-63% (w / v), approximately 62.1-63% (w / v), approximately 62.2-63% (w / v), approximately 62.3-63% (w / v), approximately 62.4-63% (w / v), approximately 62.5-63% (w / v), approximately 62.6-63% (w / v), approximately 62.7-63% (w / v), approximately 62.8-63% (w / v), or approximately 62.9-63% (w / v).

[0059] In some embodiments, the volume molar concentration of bisulfite ions in the reaction mixture is from about 7 mol / L to about 12 mol / L. For example, the volume molar concentration of bisulfite ions in the reaction mixture may be about 7.0-12 mol / L, about 8.0-12 mol / L, about 9.0-12 mol / L, about 10.0-12 mol / L, or about 11.0-12 mol / L.

[0060] The kit described in this application may also include components that assist in the deamination process. For example, the assistance may refer to promoting the target reaction (e.g., the cytosine base deamination reaction in this application) or increasing its reaction rate, introducing protecting groups to prevent side reactions of non-target reactants or to prevent the degradation of target reactants (e.g., nucleic acid molecules), providing a suitable pH environment for the target reaction, and promoting the separation and purification of the target reaction products. In some embodiments, the deamination process may further include adding one or more protecting agents optionally selected from the following: hydroquinone, trehalose, spermine, quinoline dimethacrylate, and their analogues and derivatives. In some embodiments, the deamination process may further include adding a desulfurization accelerator and / or a buffer solution suitable for carrying out the deamination process.

[0061] In some embodiments, the kit may further comprise a purification component. For example, the purification component may comprise one or more of the following: a component capable of removing sulfite groups from the deamination-treated product; a washing component; and a component for recovering the nucleic acid. For example, the recovery component may comprise a solid support and an elution component. In some embodiments,

[0062] The recovered components may also contain a coupling agent. In some embodiments, the kit further comprises one or more other components selected from the following: hydroquinone, trehalose, spermine, quinoline dimethacrylate, and their analogues and derivatives; a desulfurization accelerator; and a buffer suitable for carrying out the deamination treatment.

[0063] In some embodiments, the conversion component, the desulfite group component, the washing component, the recovery component, and the other components are not mixed with each other. For example, the conversion component, the desulfite group component, the washing component, the recovery component, and the other components are each independently contained in separate packages or devices.

[0064] Methods for preserving ammonium bisulfite solution and manufacturing methods for cytosine conversion kits.

[0065] The method for preserving the high-concentration ammonium bisulfite solution includes adding anhydrous ethanol at a volume ratio of about 2% to about 8% to an ammonium bisulfite solution with a mass fraction of about 68% to about 70% (volume ratio of anhydrous ethanol to ammonium bisulfite solution).

[0066] Specifically, the method for preserving the high-concentration ammonium bisulfite solution includes adding anhydrous ethanol (volume ratio of anhydrous ethanol to ammonium bisulfite solution) to an ammonium bisulfite solution with a mass fraction of about 68% to about 70%, at a volume ratio of about 2% to about 8%, about 3% to about 8%, about 4% to about 8%, about 5% to about 8%, about 6% to about 8%, or about 7% to about 8%.

[0067] Specifically, the method for preserving the high-concentration ammonium bisulfite solution includes adding anhydrous ethanol (volume ratio of anhydrous ethanol to ammonium bisulfite solution) to an ammonium bisulfite solution with a mass fraction of about 68% to about 70%, at a volume ratio of about 2% to about 8%, about 3% to about 8%, about 4% to about 8%, about 5% to about 8%, about 6% to about 8%, or about 7% to about 8%.

[0068] Specifically, the method for preserving the high-concentration ammonium bisulfite solution includes adding anhydrous ethanol (volume ratio of anhydrous ethanol to ammonium bisulfite solution) to an ammonium bisulfite solution with a mass fraction of about 68% to about 70%.

[0069] use

[0070] This application provides the use of the above-described preservation solution and kit in the conversion of cytosine bases, wherein the converted cytosine bases are uracil bases.

[0071] On the other hand, this application provides the following implementation methods:

[0072] 1. A preservation solution for ammonium bisulfite, the preservation solution comprising ammonium bisulfite and ethanol, wherein the concentration of ammonium bisulfite is 13 mol / L to 15 mol / L and the concentration of ethanol is 0.3 mol / L to 1.5 mol / L.

[0073] 2. The preservation solution according to Embodiment 1, wherein the concentration of ammonium bisulfite is from 13.7 mol / L to 14.1 mol / L, and the concentration of ethanol is from 0.3 mol / L to 1.3 mol / L.

[0074] 3. The preservation solution according to any one of Embodiments 1-2, wherein the concentration of ethanol in the preservation solution is about 0.68 mol / L.

[0075] 4. The preservation solution according to any one of embodiments 1-3 has a preservation time of about 3 months to about 12 months.

[0076] 5. The preservation solution according to any one of embodiments 1-5, wherein the preservation temperature of the preservation solution is about 0°C to about 40°C.

[0077] 6. The preservation solution according to any one of embodiments 1-6, wherein the preservation temperature of the preservation solution is about 15°C to about 25°C.

[0078] 7. The preservation solution according to any one of embodiments 1-7, wherein the preservation temperature of the preservation solution is about 4°C to about 15°C.

[0079] 8. The preservation solution according to any one of embodiments 1-8, wherein the preservation solution is used to convert cytosine bases in a nucleic acid sample, and the conversion efficiency of the cytosine bases to uracil bases is about 99%.

[0080] 9. A method for preserving a high-concentration ammonium bisulfite solution, comprising adding ethanol to the ammonium bisulfite solution, wherein the concentration of the ammonium bisulfite solution in the mixed solution is about 13 mol / L to about 15 mol / L, and the concentration of the ethanol in the mixed solution is about 0.3 mol / L to about 1.5 mol / L.

[0081] 10. According to the method of embodiment 10, the added ethanol is anhydrous ethanol or any ethanol solution with a concentration of less than 99.9%.

[0082] 11. Use of ethanol in the preservation of high-concentration ammonium bisulfite solutions, including adding ethanol to an ammonium bisulfite solution, wherein the concentration of the ammonium bisulfite solution in the mixed solution is about 13 mol / L to about 15 mol / L, and the concentration of ethanol in the mixed solution is about 0.3 mol / L to about 1.5 mol / L.

[0083] 12. A kit comprising a conversion reagent capable of converting cytosine bases in a nucleic acid sample and instructions for use, the conversion reagent comprising ammonium bisulfite and ethanol, wherein the concentration of the ammonium bisulfite solution in the conversion reagent is about 13 mol / L to about 15 mol / L, and the concentration of the ethanol in the conversion reagent is about 0.3 mol / L to about 1.5 mol / L.

[0084] 13. The kit according to embodiment 13, wherein the instruction manual includes deamination of the reaction mixture of the conversion reagent and the nucleic acid sample in the kit, wherein the conversion reagent comprises about 50% (w / v) to about 70% (w / v) of the total volume of the reaction mixture, and the deamination is performed by heating the reaction mixture at a temperature of at least 90°C for about 1 minute to about 20 minutes.

[0085] 14. The kit according to any one of embodiments 13-14, wherein the kit further comprises a purified component, a desulfurization accelerator, and any buffer suitable for performing the deamination treatment.

[0086] 15. The kit according to any one of embodiments 13-15, wherein the kit further comprises one or more other components as described below: hydroquinone, trehalose, spermine, quinoline dimethacrylate, and analogues and derivatives thereof.

[0087] 16. A method for producing a kit according to any one of embodiments 13-16, the method comprising the preparation of a conversion reagent, the preparation of the conversion reagent comprising adding ethanol to an ammonium bisulfite solution, wherein the concentration of the ammonium bisulfite solution in the conversion reagent is about 13 mol / L to about 15 mol / L, and the concentration of the ethanol in the conversion reagent is about 0.3 mol / L to about 1.5 mol / L.

[0088] 17. Use of the preservation solution of any one of Embodiments 1-9 and the kit of any one of Embodiments 13-16 in the transformation of cytosine bases in nucleic acid samples.

[0089] 18. According to the use described in Embodiment 18, the preservation solution and kit have a shelf life of approximately 3 to 24 months.

[0090] 19. According to any one of embodiments 18-19, the preservation solution and kit have a shelf life of about 3 months to 18 months.

[0091] 20. According to any one of embodiments 18-20, the preservation solution and kit are stored for about 3 to 12 months.

[0092] 21. According to any one of embodiments 18-21, the preservation solution and kit are stored for about 3 to 6 months.

[0093] 22. The preservation solution according to any one of embodiments 18-22, wherein the preservation solution remains clear and / or does not crystallize during the preservation period.

[0094] 23. In the use according to any one of embodiments 18-23, the preservation solution and kit are stored at a temperature of about 0°C to about 40°C.

[0095] 24. In accordance with any one of embodiments 18-24, the preservation solution and kit are stored at a temperature of about 15°C to about 25°C.

[0096] 25. In the use according to any one of embodiments 18-25, the preservation solution and kit are stored at a temperature of about 4°C to about 15°C.

[0097] 26. According to any one of embodiments 18-26, the preservation solution and kit have a conversion efficiency of about 99% for converting cytosine bases to uracil bases.

[0098] 27. The use according to any one of embodiments 18-27 includes deamination of the reaction mixture, said deamination being performed by heating the reaction mixture at a temperature of at least 90°C for about 1 minute to about 20 minutes to obtain a deamination reaction product, said reaction mixture comprising the nucleic acid sample and a conversion reagent, said conversion reagent comprising about 50% (w / v) to about 70% (w / v) of the total volume of the reaction mixture.

[0099] 28. According to the use described in Embodiment 28, the deamination treatment is performed by heating the reaction mixture at a temperature of about 95°C to about 98°C to obtain the deamination reaction product.

[0100] 29. In the use according to any one of embodiments 28-29, the deamination treatment is performed by heating the reaction mixture at a temperature of about 98°C to obtain the deamination reaction product.

[0101] 30. In the use according to any one of embodiments 28-30, the deamination treatment is to heat the reaction mixture at the stated temperature for about 5 minutes to about 15 minutes to obtain the deamination reaction product.

[0102] 31. According to any one of embodiments 28-31, the deamination treatment is to heat the reaction mixture at the stated temperature for about 10 minutes to obtain the deamination reaction product.

[0103] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0104] Example

[0105] Example 1: Preparation of Ethanol-Ammonium Bisulfite Preservative Solution

[0106] The ammonium bisulfite solution used in this application was purchased from Shanghai Maclean Biochemical Technology Co., Ltd., with a concentration of 68-70%. Anhydrous ethanol was also purchased from Shanghai Maclean Biochemical Technology Co., Ltd. 400 μL, 800 μL, 1200 μL, and 1600 μL of anhydrous ethanol were added as stabilizers (approximately 2%, 4%, 6%, and 8% by volume) to 20 ml of the 68%-70% ammonium bisulfite solution, and mixed thoroughly to obtain the ammonium bisulfite preservation solution.

[0107] The results of the following examples are presented using 800 μL / 4% as an example. The results are similar when the volume of anhydrous ethanol added is 2%, 6%, and 8%.

[0108] Example 2: Evaluation of the effect of ethanol-ammonium bisulfite preservation solution on DNA samples

[0109] Dispense the ammonium bisulfite solution with or without anhydrous ethanol into 4ml brown glass bottles and store at room temperature away from light for 3 months, 6 months, and 12 months, respectively.

[0110] Observe the state of the ammonium bisulfite solution. After 3 months of storage, the ammonium bisulfite solution without ethanol had developed a white precipitate, while the ammonium bisulfite solution with ethanol remained clear. Figure 1 Above); after 6 months of storage, the ammonium bisulfite solution without ethanol showed more white precipitate, while the ammonium bisulfite solution with ethanol remained clear. Figure 1 (In Chinese); after 12 months of storage, the ammonium bisulfite solution without ethanol showed a large amount of white flocculent precipitate, while the ammonium bisulfite solution with added ethanol remained clear. Figure 1 Down).

[0111] At the same time, the base conversion rate was detected after 3 months, 6 months and 12 months of storage, respectively, according to the following method.

[0112] 1) Take 10 ng of DNA from RKO (human colon adenocarcinoma cell line) cells and divide it into two portions. Add water to both DNA samples to bring the volume to 20 uL.

[0113] 2) Sample processing: Add 180 μL of the ethanol-ammonium bisulfite solution and 68%-70% ammonium bisulfite solution from Example 1 to each of the two samples, and mix well. Then heat at 98°C for 10 minutes to perform base conversion.

[0114] 3) Purification: All processed DNA samples were purified using the purification column, binding buffer, washing buffer, desulfurization buffer, and elution buffer in the EZ-DNA-Methylation-Gold kit. Add the transformed sample to the purification column, centrifuge at 13000g for 30 seconds, and discard the filtrate. Add 100µL of washing buffer to the purification column, centrifuge at 13000g for 30 seconds, and discard the filtrate. Add 200µL of desulfurization solution (formulation: 30ml 1.3% NaOH solution + 70ml isopropanol) to the purification column, let it stand at room temperature for 20 minutes, then centrifuge at 13000g for 30 seconds and discard the filtrate. Add 200µL of washing buffer to the purification column, centrifuge at 13000g for 30 seconds, and discard the filtrate. Add 200µL of washing buffer to the purification column, centrifuge at 13000g for 1 minute, and discard the filtrate. Add 25µL of elution buffer to the purification column, centrifuge at 13000g for 1 minute, and collect the filtrate, which is the nucleic acid after cytosine transformation.

[0115] 4) Use qPCR to detect the cytosine transformation efficiency of the sample; based on the sequence of the ACTB gene in the human genome, find a fragment containing only C, and design and synthesize a set of primers and fluorescently labeled probes for transformation according to the rules of cytosine to thymine (as shown in the table below:).

[0116] Table 1 Primer and probe sequences

[0117]

[0118] Prepare the reaction system according to the formula in the table below:

[0119] Table 2 Reaction System

[0120]

[0121] Perform the qPCR reaction according to the following procedure using a Roche Light Cycler 480 II qPCR instrument:

[0122] Table 3 qPCR program

[0123]

[0124] The test results are shown in the table below. Figure 2-4 As shown:

[0125] Table 4. Cutoff values ​​for qPCR

[0126]

[0127] The results showed that 68%-70% ammonium bisulfite solutions stored for 3 months could complete cytosine conversion regardless of whether a stabilizer (anhydrous ethanol) was added. For 68%-70% ammonium bisulfite solutions stored for 6 months, solutions with ethanol as a stabilizer could complete cytosine conversion, while solutions without ethanol as a stabilizer did not produce detectable conversion products, indicating that ammonium bisulfite is unstable. These results demonstrate that adding ethanol can stabilize 68-70% ammonium bisulfite solutions and extend their storage time.

[0128] Example 3: Preparation of different ammonium bisulfite preservation solutions

[0129] Hydroquinone and vitamin E are commonly used solution stabilizers in this field.

[0130] Weigh 0.280g of hydroquinone and prepare 5ml of 500mM hydroquinone aqueous solution; add 40uL of 500mM hydroquinone aqueous solution as a stabilizer to 20ml of 68%-70% ammonium bisulfite solution (final hydroquinone concentration is 1mM), mix well to form hydroquinone-ammonium bisulfite preservation solution, which is used as a base conversion reagent.

[0131] Weigh 0.251g of water-soluble vitamin E (Ve) and dissolve it in 1ml of DMSO to prepare a 1mol / L water-soluble Ve stock solution; then add 40uL of 200mM Ve aqueous solution as a stabilizer to 20ml of 68%-70% ammonium bisulfite solution (final concentration of water-soluble Ve is 2mM), mix well to obtain a water-soluble Ve-ammonium bisulfite preservation solution, which is used as a base conversion reagent.

[0132] Example 4: Effect of different protective agents on the stability of conversion reagents

[0133] Ammonium bisulfite solution with or without stabilizer was dispensed into 4ml brown glass bottles and stored at room temperature away from light for 3 months, 6 months, and 12 months, respectively.

[0134] Observe the state of the conversion solution; after 3 months of storage, all three reagents are in a clear state. Figure 5Above); after 6 months of storage, the conversion reagent containing hydroquinone and water-soluble Ve showed a small amount of crystalline precipitate, while the conversion solution containing ethanol remained clear. Figure 5 (In Chinese); after 12 months of storage, the conversion reagent containing hydroquinone and water-soluble Ve showed more crystal precipitation, while the conversion solution containing ethanol remained clear. Figure 5 Down).

[0135] Conversion rates were tested at 3 months, 6 months and 12 months of storage using the methods described below.

[0136] Take 10 ng of DNA from RKO (human colon adenocarcinoma cell line) cells and divide it into three portions. Add water to each of the three DNA samples to bring the volume to 20 uL.

[0137] Sample preparation: Add 180 μL of the ethanol-ammonium bisulfite preservation solution, hydroquinone-ammonium bisulfite preservation solution, and Ve-ammonium bisulfite preservation solution from Examples 1 and 3 to each of the three samples, and mix well. Then heat at 98°C for 10 minutes.

[0138] Purification: All processed DNA samples were purified using the purification column, binding buffer, washing buffer, desulfurization buffer, and elution buffer from the EZ-DNA-Methylation-Gold kit. Then, the transformed sample was added to a purification column, centrifuged at 13000g for 30 seconds, and the filtrate was discarded. 100µL of washing buffer (10mM Tris-HCl, pH 8.0 buffer) was added to the purification column, and centrifuged at 13000g for 30 seconds, and the filtrate was discarded. 200µL of desulfurization solution (formulation: 30ml 1.3% NaOH solution + 70ml isopropanol) was added to the purification column, and the column was allowed to stand at room temperature for 20 minutes. Then, it was centrifuged at 13000g for 30 seconds, and the filtrate was discarded. 200µL of washing buffer was added to the purification column, and centrifuged at 13000g for 30 seconds, and the filtrate was discarded. 200µL of washing buffer was added to the purification column, and the column was centrifuged at 13000g for 1 minute, and the filtrate was discarded. 25µL of elution buffer was added to the purification column, and the column was centrifuged at 13000g for 1 minute. The filtrate was collected, which is the nucleic acid after cytosine transformation.

[0139] The cytosine transformation efficiency of the samples was detected using qPCR; the transformation rate was detected using the probe primers from Example 2.

[0140] Prepare the reaction system according to the formula in the table below:

[0141] Table 5 Reaction System

[0142]

[0143] Perform the qPCR reaction according to the following procedure using a Roche Light Cycler 480 II qPCR instrument:

[0144] Table 6 qPCR Procedure

[0145]

[0146] The test results are shown in the table below:

[0147] Table 7 Cutoff values ​​for qPCR

[0148]

[0149] The results showed that after 6 months of storage, cytosine conversion was completed in 68%-70% ammonium bisulfite solutions using hydroquinone, water-soluble vitamin E, and ethanol as stabilizers. For 68%-70% ammonium bisulfite solutions stored for 12 months, cytosine conversion was completed in solutions with ethanol as a stabilizer, while the conversion products were undetectable in solutions with hydroquinone and water-soluble vitamin E as stabilizers. These results indicate that the ethanol stabilizer provided in this application has the best preservation effect on 68%-70% ammonium bisulfite solutions compared to hydroquinone and vitamin E.

Claims

1. A method of converting cytosine bases in a nucleic acid sample, the method comprising subjecting a reaction mixture to a deamination treatment by heating the reaction mixture at a temperature of 95 °C to 98 °C for 1 minute to 20 minutes to obtain a deamination reaction product, the reaction mixture comprising the nucleic acid sample and a conversion agent, the conversion agent having a mass of 50% w / v to 70% w / v of the total volume of the reaction mixture, wherein, The conversion agent is an ammonium bisulfite solution including ammonium bisulfite and ethanol, wherein, in the ammonium bisulfite solution, the concentration of the ammonium bisulfite is 13 mol / L to 15 mol / L, and the concentration of the ethanol is 0.3 mol / L to 1.5 mol / L.

2. The method according to claim 1, wherein, in the ammonium bisulfite solution, the concentration of the ammonium bisulfite is 13.7 mol / L to 14.1 mol / L.

3. The method according to claim 1, wherein, in the ammonium bisulfite solution, the concentration of the ethanol is 0.66 mol / L to 0.70 mol / L.

4. The method according to claim 1, wherein the deamination treatment is heating the reaction mixture at a temperature of 98°C to obtain the deamination reaction product.

5. The method according to claim 1, wherein the deamination treatment is heating the reaction mixture at the temperature for 5 minutes to 15 minutes to obtain the deamination reaction product.

6. The method according to claim 1, wherein the deamination treatment is heating the reaction mixture at the temperature for 10 minutes to obtain the deamination reaction product.

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