Composition for pretreatment of nucleic acid amplification

Abstract

This invention provides a novel technology that can suppress the time-dependent degradation of RNA in a mixture containing a sample used for nucleic acid amplification and an alkaline solution. [Solution] A pretreatment composition for nucleic acid amplification comprising an alkaline solution and a disaccharide or water-soluble polysaccharide contained in the alkaline solution.

Description

Technical Field

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[0001] The present invention relates to the treatment of a sample to be subjected to nucleic acid amplification, which contains RNA (Ribonucleic acid).

Background Art

[0002] When amplifying nucleic acids in a biological sample by a method such as PCR and detecting the amplified nucleic acids, pretreatment is performed to suppress the influence of biological substances other than nucleic acids contained in the biological sample. As one of such pretreatments, it is known to prepare a mixed solution of a biological sample and an alkaline solution (for example, Patent Document 1). By amplifying nucleic acids in the alkaline mixed solution, it is possible to suppress the influence of biological substances other than nucleic acids and amplify nucleic acids.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] One object of the present invention is to provide a novel technique capable of suppressing the time-dependent degradation of RNA in a mixed solution containing a sample to be subjected to nucleic acid amplification and an alkaline solution.

Means for Solving the Problems

[0005] The present inventor noticed that when the pretreatment of preparing an alkaline mixed solution is performed on a virus-positive specimen as a sample, the virus RNA to be measured may also be hydrolyzed over time and it may become impossible to measure even if nucleic acid amplification by PCR is performed. As a result of diligent research, the inventors have discovered that RNA degradation can be suppressed by including at least one of disaccharides and water-soluble polysaccharides in an alkaline mixture.

[0006] The gist of this invention is as follows: [1] Alkaline solution and A pretreatment composition for nucleic acid amplification, comprising a disaccharide or water-soluble polysaccharide contained in the aforementioned alkaline solution. [2] The pretreatment composition according to [1], further comprising a sample to be subjected to nucleic acid amplification. [3] A pretreatment composition according to [1] or [2], wherein the concentration of the disaccharide or water-soluble polysaccharide is 10 mg / mL or more and 500 mg / mL or less. [4] A pretreatment composition according to [1] or [2], wherein the concentration of the disaccharide or water-soluble polysaccharide is 50 mg / mL or more and 300 mg / mL or less. [5] A pretreatment composition according to any one of [1] to [4], comprising at least one disaccharide selected from the group consisting of sucrose, trehalose, and maltose. [6] A pretreatment composition containing trehalose, as described in any one of [1] to [4]. [7] A pretreatment composition containing dextran, as described in any one of [1] to [4]. [8] A pretreatment composition according to any one of [1] to [7], wherein the alkaline solution concentration is 0.1% by weight / volume or more and 0.4% by weight / volume or less. [9] A method for suppressing the time-dependent degradation of RNA in a mixture containing a sample to be used for nucleic acid amplification and an alkaline solution, The method comprising including a disaccharide or a water-soluble polysaccharide in the aforementioned mixture.

[10] The method according to [9], wherein the concentration of the disaccharide or water-soluble polysaccharide in the mixture is 10 mg / mL or more and 500 mg / mL or less.

[11] The method according to [9], wherein the concentration of the disaccharide or water-soluble polysaccharide in the mixture is 50 mg / mL or more and 300 mg / mL or less.

[12] The method according to any one of [9] to

[11] , wherein the mixture contains at least one disaccharide selected from the group consisting of sucrose, trehalose, and maltose.

[13] The method according to any one of [9] to

[11] , wherein trehalose is contained in the mixture.

[14] The method according to any one of [9] to

[11] , wherein dextran is contained in the mixture.

[15] The method according to any one of [9] to

[14] , wherein the alkaline solution concentration in the mixture is 0.1% by weight / volume or more and 0.4% by weight / volume or less.

[16] A method for detecting viruses contained in a sample, The aforementioned sample is mixed with an alkaline solution and a disaccharide or water-soluble polysaccharide to form a mixture. The method comprising nucleic acid amplification from RNA derived from a virus in the aforementioned mixture.

[17] The method according to

[16] , wherein the concentration of trehalose or water-soluble polysaccharide in the mixture is 10 mg / mL or more and 500 mg / mL or less.

[18] The method according to

[16] , wherein the concentration of trehalose or water-soluble polysaccharide in the mixture is 50 mg / mL or more and 300 mg / mL or less.

[19] The method according to any one of

[16] to

[18] , wherein the mixture contains at least one disaccharide selected from the group consisting of sucrose, trehalose, and maltose.

[20] The method according to any one of

[16] to

[18] , wherein trehalose is contained in the mixture.

[21] The method according to any one of

[16] to

[18] , wherein dextran is contained in the mixture.

[22] The method according to any one of

[16] to

[21] , wherein the concentration of the alkaline solution in the mixture is 0.1% by weight / volume% or more and 0.4% by weight / volume% or less. [Effect of the Invention]

[0007] According to the present invention, it is possible to provide a novel technique capable of suppressing the degradation of RNA over time in a mixture containing a sample to be subjected to nucleic acid amplification and an alkaline solution. [Brief Description of the Drawings]

[0008] [Figure 1] It is a graph showing the amplification curve of Test Example 1 (GeneSoC SARS-CoV-2 N2 detection kit, SARS-CoV-2, swab non-mixed). The vertical axis represents the fluorescence intensity, and the horizontal axis represents the cycle number. [Figure 2] It is a graph showing the amplification curve of Test Example 2 (GeneSoC SARS-CoV-2 N2 detection kit, SARS-CoV-2, and swab are mixed and used). The vertical axis represents the fluorescence intensity, and the horizontal axis represents the cycle number. [Figure 3] It is a graph showing the amplification curve of Test Example 3 (forward primer, reverse primer, and probe for SARS-CoV-2 detection and influenza detection, SARS-CoV-2, and swab are mixed and used). The vertical axis represents the fluorescence intensity, and the horizontal axis represents the cycle number. [Figure 4] It is a graph showing the amplification curve of Test Example 4 (forward primer, reverse primer, and probe for SARS-CoV-2 detection and influenza detection, Flu A, and swab are mixed and used). The vertical axis represents the fluorescence intensity, and the horizontal axis represents the cycle number. [Figure 5]This graph shows the amplification curve for Test Example 5 (using a mixture of forward primers, reverse primers, and probes for SARS-CoV-2 and influenza detection, Flu B, and a swab). The vertical axis represents fluorescence intensity, and the horizontal axis represents the number of cycles. [Figure 6] This graph shows the amplification curve for Test Example 6 (using a mixture of primers and probes for influenza A and influenza B detection, Flu A, and a swab). The vertical axis represents fluorescence intensity, and the horizontal axis represents the number of cycles. [Figure 7] This graph shows the amplification curve for Test Example 7 (using a mixture of primers and probes for influenza A and influenza B detection, Flu B, and a swab). The vertical axis represents fluorescence intensity, and the horizontal axis represents the number of cycles. [Figure 8] This graph shows the amplification curve for Test Example 8. The vertical axis represents fluorescence intensity, and the horizontal axis represents the number of cycles. [Figure 9] This graph shows the amplification curve for Test Example 9 (using a mixture of primers and probes for SARS-CoV-2 and RSV detection, RSV, and swabs). The vertical axis represents fluorescence intensity, and the horizontal axis represents the number of cycles. [Figure 10] This graph shows the amplification curve for Test Example 10 (using a mixture of primers and probes for influenza A and influenza B detection, Flu A, and a swab). The vertical axis represents fluorescence intensity, and the horizontal axis represents the number of cycles. [Modes for carrying out the invention]

[0009] One embodiment of the present invention will be described in detail below. This embodiment relates to a pretreatment composition to be mixed with a sample to be subjected to nucleic acid amplification, wherein the pretreatment composition comprises an alkaline solution and a disaccharide or water-soluble polysaccharide contained in the alkaline solution.

[0010] In this specification, an alkaline solution is a solution in which an alkaline substance is dissolved, and an alkaline substance is a compound that, when dissolved in water, has a pH greater than 7. Examples of alkaline substances include sodium hydroxide, lithium hydroxide, potassium hydroxide, rubidium hydroxide, cesium hydroxide, magnesium hydroxide, calcium hydroxide, lithium carbonate, sodium carbonate, potassium carbonate, rubidium carbonate, cesium carbonate, magnesium carbonate, calcium carbonate, lithium bicarbonate, sodium bicarbonate, potassium bicarbonate, rubidium bicarbonate, and cesium bicarbonate. For example, the alkaline solution according to this embodiment may be prepared using one or more of these substances.

[0011] The alkali concentration of the alkaline solution constituting the pretreatment composition of this embodiment is not particularly limited and can be set as appropriate by those skilled in the art. However, from the viewpoint of reducing the influence of sample-derived substances other than nucleic acids on the PCR reaction, it is preferable that the alkali concentration when the pretreatment composition is mixed with the sample is 0.1 w / v% (w / v%) or more and 0.4 w / v% or less, more preferably 0.25 w / v% (w / v%) or more and 0.35 w / v% or less, and even more preferably 0.3 w / v%.

[0012] In this specification, alkalinity is expressed as weight / volume %, and can be defined as the weight (total amount if multiple types of alkaline substances are present) of the alkaline substance contained in 100 ml of alkaline solution, expressed in grams (g).

[0013] Disaccharides are a type of carbohydrate that has a structure in which two monosaccharides are linked by a glycosidic bond. Examples of disaccharides include sucrose, trehalose, maltose, isomaltose, and cellobiose. Of these, from the viewpoint of suppressing the degradation of RNA over time, it is preferable to include at least one selected from the group consisting of sucrose, trehalose, and maltose, and it is more preferable to include trehalose. Water-soluble polysaccharides are high-molecular-weight compounds formed by the polymerization of multiple (specifically more than 10) monosaccharides via glycosidic bonds, and which dissolve in water. Examples of water-soluble polysaccharides include dextran, pullulan, gum arabic, guar gum, carrageenan, and alginic acid. Of these, it is preferable to include dextran from the viewpoint of suppressing the degradation of RNA over time.

[0014] In the pretreatment composition of this embodiment, the disaccharide or water-soluble polysaccharide can be dissolved, for example, in the alkaline solution described above. The concentration of the disaccharide or water-soluble polysaccharide is not particularly limited and can be set as appropriate by those skilled in the art.

[0015] Furthermore, from the viewpoint of suppressing the degradation of RNA over time, the concentration of disaccharides or water-soluble polysaccharides when the pretreatment composition of this embodiment is mixed with the sample is preferably 10 mg / mL or more, and more preferably 50 mg / mL or more. Also, from the viewpoint of improving amplification efficiency, the concentration of disaccharides or water-soluble polysaccharides when the pretreatment composition is mixed with the sample is preferably 500 mg / mL or less, and more preferably 300 mg / mL or less.

[0016] The pretreatment composition of this embodiment may contain other components in addition to the alkaline solution and disaccharides or water-soluble polysaccharides, as long as the objectives of the present invention can be achieved, and is not particularly limited. Other components may include, for example, protein denaturants such as urea or sodium dodecyl sulfate. Furthermore, the pretreatment composition of this embodiment may further include a sample to be subjected to nucleic acid amplification. A sample to be subjected to nucleic acid amplification, such as the PCR method described later, means a sample that is to be subjected to nucleic acid amplification treatment, such as the PCR method described later, and which may contain template RNA. Examples of samples include, as described later, animal and plant tissues, bodily fluids, excrement, cells, bacteria, viruses, and liquids or solids that may contain these.

[0017] In one embodiment, the pretreatment composition of this embodiment can be prepared by mixing an alkaline solution with a disaccharide or water-soluble polysaccharide and other components as needed. Furthermore, the pretreatment composition of this embodiment is not limited to this embodiment, and may be prepared on a made-to-order basis. For example, the alkaline solution, the disaccharide or water-soluble polysaccharide, and one or more of the other components as needed may be stored separately and mixed together when mixed with the sample.

[0018] In one aspect of the present invention, an RNA degradation inhibitor in a sample, which is mixed with an alkaline solution in a sample subjected to nucleic acid amplification, is provided, and which includes a disaccharide or a water-soluble polysaccharide. Furthermore, in one aspect of the present invention, a method for suppressing the time-dependent degradation of RNA in a mixture containing a sample to be used for nucleic acid amplification and an alkaline solution can be provided, which includes including a disaccharide or a water-soluble polysaccharide in the mixture.

[0019] Next, we will describe the detection of viruses that may be present in a biological sample using the composition of this embodiment.

[0020] The composition of this embodiment can be used, for example, to detect a virus while suppressing the degradation of virus-derived RNA in a sample. Therefore, in one aspect of the present invention, a method for detecting viruses can be provided, which involves mixing a sample taken from a living organism, food, or the environment with an alkaline solution and a disaccharide or water-soluble polysaccharide to form a mixture, and then amplifying nucleic acid from RNA derived from a virus in the mixture.

[0021] The viruses that can be detected are not particularly limited, and examples include SARS coronavirus 2 (SARS-CoV-2), influenza virus, respiratory syncytial virus (RSV), adenovirus, norovirus, rotavirus, sapovirus, and diarrheal adenovirus. Furthermore, the viruses to be detected may be a single virus or two or more viruses.

[0022] As mentioned above, the sample can be a sample taken from living organisms, food, or the environment, and examples include animal and plant tissues, bodily fluids, excrement, cells, bacteria, viruses, and liquids or solids that may contain these. Examples of living organisms from which samples can be collected include animal and plant tissues, body fluids, excretions, and cells. More specifically, these include pharyngeal swabs, nasal swabs, nasopharyngeal swabs, pleural fluid, rectal swabs, sputum, blood, plasma, serum, blood culture medium, urine, saliva, amniotic fluid, pus, cerebrospinal fluid, tissue sections, skin, vomit, feces, tympanotomy fluid, alveolar lavage fluid, gastric lavage fluid, bowel lavage fluid, cervical swabs, urethral swabs, organ extracts, tissue extract isolation culture colonies, bronchial lavage fluid, and catheter lavage fluid.

[0023] Biological samples can be, for example, samples derived from humans or animals other than humans. Examples of animals other than humans include non-human mammals such as dogs, cats, mice, rats, guinea pigs, hamsters, rabbits, pigs, cows, sheep, and goats, as well as fish, amphibians, reptiles, and birds. The method for collecting biological samples is not particularly limited and can be appropriately determined by those skilled in the art depending on the type, size, and purpose of the sample. For example, samples can be collected using collection tools such as cotton swabs, cotton swabs, platinum loops, droppers, spatulas, or spoons.

[0024] Other examples of food samples to be collected include water, soft drinks, alcoholic beverages, vegetables, livestock products such as raw meat, seafood such as raw fish, processed egg products, dairy products, and prepared foods. In addition, samples collected from the environment include those taken from water, ice, and soil, as well as samples taken from areas that have come into contact with animals, plants, or food, including humans. The method of collecting samples from food or the environment is not particularly limited and can be set as appropriate by those skilled in the art.

[0025] Furthermore, the sample may undergo one or more treatments for purposes such as sample preservation or improving the sensitivity of virus detection, and is not particularly limited. Examples of such processing include dissolution or suspension in a liquid, purification and concentration of components contained in the sample, addition of salts or the like to the sample, and storage at room temperature or below.

[0026] In this embodiment, a sample such as a biological sample is mixed with an alkaline solution and a disaccharide or water-soluble polysaccharide to prepare a mixed solution. The mixture can be, for example, a solution or turbidity of the sample. The method for preparing the mixture as a solution or turbidity when the sample is a solid or concentrated substance is not particularly limited and can be appropriately determined by those skilled in the art. In the mixture, as described above, the alkaline solution concentration is preferably 0.1% w / v% or more and 0.4% w / v% or less, and the concentration of disaccharides or water-soluble polysaccharides is preferably 10 mg / mL or more and 500 mg / mL or less. Furthermore, the pH of the mixture is not particularly limited as long as it is alkaline, but a pH of 8.0 or more and 14.0 or less is preferred.

[0027] The mixture may be subjected to treatments such as filtration before the nucleic acid amplification treatment described later, for example, to improve the sensitivity of virus detection. When filtration is performed, the filter media, filtration method, etc., are not particularly limited and can be appropriately determined by those skilled in the art.

[0028] Next, nucleic acid amplification is performed based on RNA in the aforementioned mixture, and the virus in the sample is detected by detecting the amplified nucleic acid of the target virus. When performing nucleic acid amplification processing, various components can be added to the mixture as appropriate to advance the amplification or detection of nucleic acids before processing. Examples of such components include nucleic acid amplification reagent sets containing DNA polymerase, reverse transcriptase, nucleic acid primer pairs, dNTPs, nucleic acid probes, etc.

[0029] The nucleic acid amplification method used is not particularly limited as long as it allows for nucleic acid amplification from RNA, and can be appropriately determined by those skilled in the art. Specific examples of nucleic acid amplification methods include PCR (Polymerase Chain Reaction), SDA (Strand Displacement Amplification), ICAN (Isothermal and Chimeric Primer-Initiated Amplification of Nucleic Acids), and LAMP (Loop-mediated Isothermal Amplification). Nucleic acid amplification conditions, such as temperature, should be appropriately set according to the nucleic acid amplification method and the type of virus to be detected.

[0030] The method for detecting amplified nucleic acids is not particularly limited. Examples of detection methods include agarose gel electrophoresis, nucleic acid probe methods, intercalator methods, SSCP methods, and RFLP methods.

[0031] According to this embodiment, it is possible to detect viruses in a sample while suppressing the degradation of RNA over time due to the effects of alkaline solutions, and therefore it is expected to contribute to suppressing false negatives, for example. [Examples]

[0032] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0033] [Preparation of pretreatment composition] 110.5 mg of trehalose dihydrate (Wako Pure Chemical Industries, 2022-18452) was mixed with 600 μL of 0.5 wt / vol% sodium hydroxide (NaOH) solution (Hayashi Pure Chemical Industries, EP44025095). After dissolving the trehalose dihydrate, the solution was diluted to 1000 μL with nuclease-free water (Ambion Nuclease-Free Water (Thermo, AM9937)) to obtain the pretreatment composition for the example.

[0034] [Test Example 1] The RT-PCR master mix was prepared by mixing the 100 μM SCoV-2 N2 forward primer, 100 μM SCoV-2 N2 reverse primer, 100 μM SCoV-2 N2 probe, PCR buffer, enzyme mix (containing DNA polymerase and reverse transcriptase), and RNase-free water included with the GeneSoC SARS-CoV-2 N2 detection kit (manufactured by Kyorin Pharmaceutical Co., Ltd.) according to the instructions provided. Thermoinactivated SARS-CoV-2 (manufactured by ATCC) was diluted with physiological saline to prepare a 400 copies / μL sample. The sample was mixed with the pretreatment composition used in the example to obtain pretreatment solutions with a sodium hydroxide concentration of 0.3% and trehalose concentrations of 10 mg / mL, 50 mg / mL, or 100 mg / mL. The pretreatment solution was left to stand at room temperature for 0 hours (0h, no standing), 1 hour (1h), 2 hours (2h), or 4 hours (4h). Then, 15 μL of RT-PCR master mix was mixed in, and real-time PCR was performed using GeneSoC mini (manufactured by Kyorin Pharmaceutical Co., Ltd.) and its dedicated chip. Furthermore, a pretreatment solution prepared in the same manner as above, except for the absence of trehalose, was used as a control. All experimental conditions were performed with N=3. The PCR conditions were as follows: activation reaction: 96°C, 10 seconds; denaturation reaction (DN): 96°C, 4 seconds; annealing and extension reaction (AE): 58°C, 8 seconds. The DN and AE were performed for 50 cycles, and the presence or absence of amplification was determined by visually observing the amplification curve displayed on the GeneSoC mini (Kyorin Pharmaceutical Co., Ltd.) screen. The results are shown in Figure 1.

[0035] As can be seen from Figure 1, the pretreatment solution obtained by mixing the pretreatment composition and sample in the examples showed a clear amplification curve even after standing, indicating that RNA degradation over time was suppressed.

[0036] [Test Example 2] The RT-PCR master mix was prepared using the same method as in Test Example 1. A nasopharyngeal swab suspension was prepared by suspending one nasopharyngeal swab (BIOMEDICA) collected from a healthy individual in 1 mL of physiological saline. Thermoinactivated SARS-CoV-2 (ATCC) was diluted with this swab suspension, and a 100 copies / μL nasopharyngeal swab suspension was used as the sample. The sample and the pretreatment composition were mixed to obtain a pretreatment solution with a sodium hydroxide concentration of 0.3% and a trehalose concentration of 50 mg / mL or 100 mg / mL. After allowing the pretreatment solution to stand at room temperature for 0 hours (0h, no standing), 1 hour (1h), or 4 hours (4h), 15 μL of RT-PCR master mix was mixed in, and real-time PCR was performed using GeneSoC mini (manufactured by Kyorin Pharmaceutical Co., Ltd.) and its dedicated chip. Furthermore, a pretreatment solution prepared in the same manner as above, except for the absence of trehalose, was used as a control. All experimental conditions were performed with N=3. The PCR conditions were the same as in Test Example 1. The results are shown in Figure 2.

[0037] As can be seen from Figure 2, the pretreatment solution obtained by mixing the pretreatment composition and sample in the example showed a clear amplification curve even after standing, indicating that RNA degradation over time was suppressed.

[0038] [Test Example 3] A solution was prepared by mixing PCR buffer, enzyme mix (containing DNA polymerase and reverse transcriptase), forward primers, reverse primers, and probes for SARS-CoV-2 detection and influenza detection, and this mixture was used as the RT-PCR master mix. Real-time PCR was performed in the same manner as in Test Example 2, except that the trehalose concentration in the pretreatment solution was set to 100 mg / mL, and the standing time of the pretreatment solution was set to 0 hours (0h, no standing), 1 hour (1h), or 3 hours (4h). The results are shown in Figure 3.

[0039] As can be seen from Figure 3, a clear amplification curve was observed even after standing for the pretreatment solution obtained by mixing the pretreatment composition and sample in the example, indicating that RNA degradation over time was suppressed.

[0040] [Test Example 4] Real-time PCR was performed in the same manner as in Test Example 3, except that influenza virus A (cultured Flu A) was diluted in a swab suspension and a 200 copies / μL nasopharyngeal swab suspension was used as the sample. The results are shown in Figure 4.

[0041] As can be seen from Figure 4, the pretreatment solution obtained by mixing the pretreatment composition and sample in the example showed a clear amplification curve even after standing, indicating that RNA degradation over time was suppressed.

[0042] [Test Example 5] Real-time PCR was performed in the same manner as in Test Example 3, except that influenza virus B (cultured Flu B) was diluted in a swab suspension and a 200 copies / μL nasopharyngeal swab suspension was used as the sample. The results are shown in Figure 5.

[0043] As can be seen from Figure 5, the pretreatment solution obtained by mixing the pretreatment composition and sample in the example showed a clear amplification curve even after standing, indicating that RNA degradation over time was suppressed. [Test Example 6] Real-time PCR was performed in the same manner as in Test Example 2, except that the primers and probes of the RT-PCR master mix in Test Example 3 were replaced with primers and probes for detecting influenza A and influenza B, and influenza A virus (cultured Flu A) was diluted in a swab suspension to obtain a 200 copies / μL nasopharyngeal swab suspension as the sample. The results are shown in Figure 6.

[0044] As can be seen from Figure 6, the pretreatment solution obtained by mixing the pretreatment composition and sample in the example showed a clear amplification curve even after standing, indicating that RNA degradation over time was suppressed.

[0045] [Test Example 7] Real-time PCR was performed in the same manner as in Test Example 6, except that influenza virus B (cultured Flu B) was diluted with a swab suspension to obtain a 200 copies / μL nasopharyngeal swab suspension as the sample. The results are shown in Figure 7.

[0046] As can be seen from Figure 7, a clear amplification curve was observed even after standing for the pretreatment solution obtained by mixing the pretreatment composition and sample in the example, indicating that RNA degradation over time was suppressed.

[0047] [Test Example 8] Real-time PCR was performed in the same manner as in Test Example 2, except that the trehalose concentration in the pretreatment solution was set to 100 mg / mL, 200 mg / mL, 300 mg / mL, 400 mg / mL, or 500 mg / mL, and 15 μL of RT-PCR master mix was mixed into the pretreatment solution without allowing it to stand. The results are shown in Figure 8.

[0048] As can be seen from Figure 8, a clear amplification curve was observed in all pretreatment solutions obtained by mixing pretreatment compositions with different trehalose concentrations.

[0049] [Test Example 9] The primers and probes of the RT-PCR master mix from Test Example 2 were replaced with primers and probes for SARS-CoV-2 and RSV detection to create the RT-PCR master mix. Real-time PCR was performed in the same manner as in Test Example 2, except that RSV (cultured RSV) was diluted in a swab suspension to obtain a 100 copies / μL nasopharyngeal swab suspension as the sample. The results are shown in Figure 9.

[0050] As can be seen from Figure 9, the pretreatment solution obtained by mixing the pretreatment composition and sample in the example showed a clear amplification curve even after standing, indicating that RNA degradation over time was suppressed.

[0051] [Test Example 10] Real-time PCR was performed in the same manner as in Test Example 6, except that trehalose was replaced with dextran (80 mg / mL) and the standing time of the pretreatment solution was set to 20 minutes, 40 minutes, or 60 minutes. The results are shown in Figure 10.

[0052] As can be seen from Figure 10, a clear amplification curve was observed even after standing for the pretreatment solution obtained by mixing the pretreatment composition and sample in the example, indicating that RNA degradation over time was suppressed.

Claims

1. Alkaline solution and A pretreatment composition for nucleic acid amplification, comprising a disaccharide or water-soluble polysaccharide contained in the aforementioned alkaline solution.

2. The pretreatment composition according to claim 1, further comprising a sample to be subjected to nucleic acid amplification.

3. The pretreatment composition according to claim 1 or 2, wherein the concentration of the disaccharide or water-soluble polysaccharide is 10 mg / mL or more and 500 mg / mL or less.

4. The pretreatment composition according to claim 1 or 2, wherein the concentration of the disaccharide or water-soluble polysaccharide is 50 mg / mL or more and 300 mg / mL or less.

5. The pretreatment composition according to claim 1 or 2, comprising at least one disaccharide selected from the group consisting of sucrose, trehalose, and maltose.

6. A pretreatment composition according to claim 1 or 2, comprising trehalose.

7. A pretreatment composition according to claim 1 or 2, containing dextran.

8. The pretreatment composition according to claim 1 or 2, wherein the alkaline solution concentration is 0.1% by weight / vol.% or more and 0.4% by weight / vol.% or less.

9. A method for suppressing the time-dependent degradation of RNA in a mixture containing a sample to be used for nucleic acid amplification and an alkaline solution, The method comprising including a disaccharide or a water-soluble polysaccharide in the aforementioned mixture.

10. The method according to claim 9, wherein the concentration of the disaccharide or water-soluble polysaccharide in the mixture is 10 mg / mL or more and 500 mg / mL or less.

11. The method according to claim 9, wherein the concentration of the disaccharide or water-soluble polysaccharide in the mixture is 50 mg / mL or more and 300 mg / mL or less.

12. The method according to any one of claims 9 to 11, wherein the mixture contains at least one disaccharide selected from the group consisting of sucrose, trehalose, and maltose.

13. The method according to any one of claims 9 to 11, wherein trehalose is included in the mixed liquid.

14. The method according to any one of claims 9 to 11, wherein dextran is contained in the mixed solution.

15. The method according to any one of claims 9 to 11, wherein the alkaline solution concentration in the mixed liquid is 0.1% by weight / volume or more and 0.4% by weight / volume or less.

16. A method for detecting viruses contained in a sample, The aforementioned sample is mixed with an alkaline solution and a disaccharide or water-soluble polysaccharide to form a mixture. The method comprising nucleic acid amplification from RNA derived from a virus in the aforementioned mixture.

17. The method according to claim 16, wherein the concentration of trehalose or water-soluble polysaccharide in the mixture is 10 mg / mL or more and 500 mg / mL or less.

18. The method according to claim 16, wherein the concentration of trehalose or water-soluble polysaccharide in the mixture is 50 mg / mL or more and 300 mg / mL or less.

19. The method according to any one of claims 16 to 18, wherein the mixture contains at least one disaccharide selected from the group consisting of sucrose, trehalose, and maltose.

20. The method according to any one of claims 16 to 18, wherein trehalose is included in the mixed liquid.

21. The method according to any one of claims 16 to 18, wherein dextran is included in the mixed solution.

22. The method according to any one of claims 16 to 18, wherein the alkaline solution concentration in the mixed liquid is 0.1% by weight / volume or more and 0.4% by weight / volume or less.

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