Methods for reducing nonspecific amplification in nucleic acid amplification reactions

Abstract

A method for reducing nonspecific amplification in a nucleic acid amplification reaction according to one aspect of the present invention comprises a step for obtaining a nucleic acid extraction solution by mixing a sample containing a nucleic acid with a dodecyl alcohol sulfate ester salt and a nonionic surfactant. The nonionic surfactant is polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (10) octylphenyl ether, or n-octanoyl-N-methyl-D-glucamine. According to said method, it is possible to reduce nonspecific amplification in a nucleic acid amplification reaction when said amplification reaction is performed without performing purification of a nucleic acid extraction solution.

Description

Technical Field

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[0001] The present invention relates to a method for reducing non-specific amplification in nucleic acid amplification reactions.

Background Art

[0002] Generally, as a method for amplifying nucleic acids contained in a biological sample, a method is known in which nucleic acids are extracted (eluted) from a biological sample using a surfactant, a protein denaturant such as guanidine, or an organic solvent such as phenol or chloroform, the nucleic acids are purified from the obtained nucleic acid extract, and then amplified (for example, Patent Document 1). Since protein denaturants and organic solvents inhibit nucleic acid amplification reactions, this method requires a purification step for removing protein denaturants and organic solvents after the extraction step.

[0003] On the other hand, nucleic acid amplification methods that do not necessarily require a purification step after the nucleic acid extraction step are also known. Patent Document 2 describes a nucleic acid amplification method including a procedure of dissolving a solid-phase reagent containing at least a DNA polymerase, cyclodextrin, and a binder in a liquid containing nucleic acids, and a procedure of amplifying nucleic acids. According to this method, even when an ionic surfactant is used for nucleic acid extraction, inhibition of the nucleic acid amplification reaction by the ionic surfactant can be reduced by cyclodextrin contained in the solid-phase reagent.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] According to the method described in Patent Document 2, although the time required for nucleic acid amplification can be shortened by omitting the purification step, it has the drawback of causing nonspecific amplification during the nucleic acid amplification reaction.

[0006] Therefore, the present invention aims to reduce nonspecific amplification in nucleic acid amplification reactions when the amplification reaction is carried out without purifying the nucleic acid extract. [Means for solving the problem]

[0007] As a result of diligent research, the inventors discovered that by combining a dodecyl alcohol sulfate salt, which has the effect of extracting nucleic acids, with a specific nonionic surfactant, nonspecific amplification in the nucleic acid amplification reaction can be reduced even when the amplification reaction is carried out without purifying the nucleic acid extract, thus completing the present invention.

[0008] A method for reducing nonspecific amplification in a nucleic acid amplification reaction, according to one aspect of the present invention, comprises the step of mixing a sample containing nucleic acid with a dodecyl alcohol sulfate salt and a nonionic surfactant to obtain a nucleic acid extract, wherein the nonionic surfactant is polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (10) octylphenyl ether, or n-octanoyl-N-methyl-D-glucamine. The nonionic surfactant is preferably n-octanoyl-N-methyl-D-glucamine. The mixed solution of n-octanoyl-N-methyl-D-glucamine and a dodecyl alcohol sulfate salt has excellent storage stability in that it can maintain its effect of reducing nonspecific amplification even when stored for a long period of time (e.g., 35 days or more at 60°C). n-octanoyl-N-methyl-D-glucamine is also preferred from the viewpoint of environmental toxicity.

[0009] In the step of mixing a nucleic acid-containing sample with dodecyl alcohol sulfate and a nonionic surfactant, an amount of nonionic surfactant may be mixed so that the concentration of the nonionic surfactant in the nucleic acid extract is 0.3% (w / v) to 5.0% (w / v). In the step of mixing a nucleic acid-containing sample with dodecyl alcohol sulfate and a nonionic surfactant, from the viewpoint of reducing nonspecific amplification in the nucleic acid amplification reaction and efficiently amplifying nucleic acids, it is preferable to mix an amount of nonionic surfactant so that the concentration of polyoxyethylene (20) sorbitan monolaurate in the nucleic acid extract is 1.25% (w / v) to 5.0% (w / v), the concentration of polyoxyethylene (10) octylphenyl ether in the nucleic acid extract is 0.63% (w / v) to 2.5% (w / v), or the concentration of n-octanoyl-N-methyl-D-glucamine in the nucleic acid extract is 0.5% (w / v) to 1.5% (w / v). The sample containing nucleic acids may be saliva or nasal swab. Nonspecific amplification may be caused by substances other than nucleic acids in the nucleic acid-containing sample.

[0010] A nucleic acid extraction reagent according to one aspect of the present invention comprises a dodecyl alcohol sulfate salt and a nonionic surfactant. The nonionic surfactant is polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (10) octylphenyl ether, or n-octanoyl-N-methyl-D-glucamine, preferably n-octanoyl-N-methyl-D-glucamine. A nucleic acid extraction reagent containing n-octanoyl-N-methyl-D-glucamine as the nonionic surfactant exhibits excellent storage stability, in that it can maintain its effect of reducing nonspecific amplification even when stored for a long period of time (e.g., 35 days or more at 60°C). n-octanoyl-N-methyl-D-glucamine is also preferred from the viewpoint of environmental toxicity.

[0011] From the viewpoint of reducing nonspecific amplification in nucleic acid amplification reactions and efficiently amplifying nucleic acids, the concentration of n-octanoyl-N-methyl-D-glucamine is preferably 0.5% (w / v) to 1.5% (w / v).

[0012] A nucleic acid extraction kit according to one aspect of the present invention comprises a dodecyl alcohol sulfate salt and a nonionic surfactant. Furthermore, a method for extracting nucleic acids according to one aspect of the present invention includes the step of mixing a sample containing nucleic acids with a dodecyl alcohol sulfate salt and a nonionic surfactant to obtain a nucleic acid extract. The nonionic surfactant is polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (10) octylphenyl ether, or n-octanoyl-N-methyl-D-glucamine, preferably n-octanoyl-N-methyl-D-glucamine. The mixed solution of n-octanoyl-N-methyl-D-glucamine and dodecyl alcohol sulfate salt exhibits excellent storage stability, maintaining its effect of reducing nonspecific amplification even when stored for a long period (e.g., 35 days or more at 60°C). n-octanoyl-N-methyl-D-glucamine is also preferred from the viewpoint of environmental toxicity. [Effects of the Invention]

[0013] According to the present invention, even when the amplification reaction is carried out without purifying the nucleic acid extract, nonspecific amplification in the nucleic acid amplification reaction can be reduced. [Brief explanation of the drawing]

[0014] [Figure 1] This graph shows nonspecific amplification in RT-LAMP for saliva, saliva fractions with 3kDa ≥, and saliva fractions with 3kDa ≤. [Figure 2] This graph shows the relationship between protein concentration in saliva and nonspecific amplification in RT-LAMP. [Figure 3] This graph shows the effect of substances such as surfactants on nonspecific amplification in RT-LAMP. [Figure 4] This graph shows the effect of substances such as surfactants on nonspecific amplification in RT-LAMP. [Figure 5] This graph shows the effect of substances such as surfactants on nonspecific amplification in RT-LAMP. [Figure 6] It is a graph showing the results of an accelerated stability test of a nucleic acid extraction reagent containing SDS and Tween (registered trademark) 20 or Triton (registered trademark) X-100. [Figure 7] It is a graph showing the results of an accelerated stability test of a nucleic acid extraction reagent containing SDS and MEGA-8. [Figure 8] It is a graph showing the relationship between the concentration of MEGA-8 in a nucleic acid extraction reagent and non-specific amplification in RT-LAMP. [Figure 9] (A) of FIG. 9 is a graph showing the relationship between the concentration of Tween (registered trademark) 20 in a nucleic acid extraction reagent and non-specific amplification in RT-LAMP. (B) of FIG. 9 is a graph showing the relationship between the concentration of Triton (registered trademark) X-100 in a nucleic acid extraction reagent and non-specific amplification in RT-LAMP. [Figure 10] (A) of FIG. 10 is a graph showing the effect of the combination of SDS and a specific nonionic surfactant on non-specific amplification in RT-LAMP, and (B) of FIG. 10 is a graph showing that the combination of SDS and a specific nonionic surfactant did not affect the amplification of an RNA template. [Figure 11] It is a graph showing the effect of the combination of LDS and a specific nonionic surfactant on non-specific amplification in RT-LAMP. [Figure 12] It is a graph showing the results of an accelerated stability test of a nucleic acid extraction reagent containing LDS and a specific nonionic surfactant.

Mode for Carrying Out the Invention

[0015] A method for reducing non-specific amplification in a nucleic acid amplification reaction according to one aspect of the present invention includes a step of mixing a sample containing nucleic acid with a dodecyl alcohol sulfate salt and a specific nonionic surfactant to obtain a nucleic acid extract (hereinafter, also referred to as an extraction step). More specifically, non-specific amplification may be non-specific amplification caused by substances other than nucleic acid in a sample containing nucleic acid. Examples of substances other than nucleic acid (more specifically, biological substances other than nucleic acid) include proteins.

[0016] The dodecyl alcohol sulfate salt is not particularly limited as long as it is a salt of dodecyl alcohol sulfate, and for example, it may be sodium dodecyl sulfate (SDS) or lithium dodecyl sulfate (LDS).

[0017] The above specific nonionic surfactant is polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (10) octylphenyl ether, or n-octanoyl-N-methyl-D-glucamine. Polyoxyethylene (20) sorbitan monolaurate is commercially available as "Tween (registered trademark) 20", polyoxyethylene (10) octylphenyl ether is commercially available as "Triton (registered trademark) X-100", and n-octanoyl-N-methyl-D-glucamine is commercially available as "MEGA-8" (manufactured by Dojindo Laboratories). From the viewpoint of the storage stability of the mixed solution with the dodecyl alcohol sulfate salt, the nonionic surfactant is preferably n-octanoyl-N-methyl-D-glucamine or polyoxyethylene (10) octylphenyl ether, and more preferably n-octanoyl-N-methyl-D-glucamine. The mixed solution of n-octanoyl-N-methyl-D-glucamine and the dodecyl alcohol sulfate salt is excellent in storage stability in that it can maintain the effect of reducing non-specific amplification even when stored for a long period (for example, 35 days or more at 60°C). From the viewpoint of environmental harmfulness, the nonionic surfactant is preferably n-octanoyl-N-methyl-D-glucamine or polyoxyethylene (20) sorbitan monolaurate. As the nonionic surfactant, a plurality of nonionic surfactants may be used.

[0018] The combination of dodecyl alcohol sulfate and the above-mentioned specific nonionic surfactant is not particularly limited, and may include, for example, a combination of SDS and polyoxyethylene (20) sorbitan monolaurate, a combination of SDS and polyoxyethylene (10) octylphenyl ether, a combination of SDS and n-octanoyl-N-methyl-D-glucamine, a combination of LDS and polyoxyethylene (20) sorbitan monolaurate, a combination of LDS and polyoxyethylene (10) octylphenyl ether, or a combination of LDS and n-octanoyl-N-methyl-D-glucamine. The combination of dodecyl alcohol sulfate and the above-mentioned specific nonionic surfactant is preferably a combination of SDS and n-octanoyl-N-methyl-D-glucamine or a combination of LDS and n-octanoyl-N-methyl-D-glucamine.

[0019] The nucleic acid-containing sample is not particularly limited and may be a biological sample such as sputum, body fluids, feces, tissues, or suspensions thereof. Body fluids may be, for example, nasal secretions, saliva, blood, serum, plasma, cerebrospinal fluid, urine, semen, or amniotic fluid. The sample may also be a bronchial lavage fluid, bronchoalveolar lavage fluid, nasal aspirate, nasal lavage fluid, nasal swab, pharyngeal swab, or gargle solution. According to the inventors' studies, nonspecific amplification tends to increase when the amount of protein in the sample is high, but with this method, nonspecific amplification can be reduced even when using protein-containing samples such as saliva or nasal swab.

[0020] The type of nucleic acid contained in the sample is not particularly limited. The nucleic acid may be, for example, DNA or RNA, and may be single-stranded or double-stranded. The origin of the nucleic acid is also not limited; the nucleic acid may be derived from, for example, animals, plants, fungi, bacteria, or viruses.

[0021] The concentration of dodecyl alcohol sulfate in the nucleic acid extract is not particularly limited as long as it is a concentration that can extract nucleic acids. For example, it may be 0.01 to 1% (w / v), 0.1 to 1% (w / v), or 0.2 to 1% (w / v). Therefore, the amount of dodecyl alcohol sulfate mixed in the extraction process may be such that the concentration of dodecyl alcohol sulfate in the nucleic acid extract falls within the above concentration range.

[0022] The concentration of the nonionic surfactant in the nucleic acid extract may be, for example, 0.3 to 5.0% (w / v) or 1.25 to 1.5% (w / v). When the nonionic surfactant includes n-octanoyl-N-methyl-D-glucamine, the concentration of n-octanoyl-N-methyl-D-glucamine in the nucleic acid extract is preferably 0.5% (w / v) or more, more preferably 1.0% (w / v) or more, from the viewpoint of reducing nonspecific amplification in the nucleic acid amplification reaction, and preferably 1.5% (w / v) or less, more preferably 1.25% (w / v) or less, from the viewpoint of efficiently amplifying nucleic acids. The concentration of n-octanoyl-N-methyl-D-glucamine in the nucleic acid extract may be, for example, 0.5 to 1.5% (w / v) or 0.5 to 1.25% (w / v). When polyoxyethylene (20) sorbitan monolaurate is included as a nonionic surfactant, the concentration of polyoxyethylene (20) sorbitan monolaurate in the nucleic acid extract is preferably 0.3% (w / v) or more, 0.6% (w / v) or more, more preferably 1.25% (w / v) or more, even more preferably 2.5% (w / v) or more, and particularly preferably 5.0% (w / v) or more, from the viewpoint of reducing nonspecific amplification in the nucleic acid amplification reaction, and preferably 5.0% (w / v) or less, and more preferably 2.5% (w / v) or less, from the viewpoint of efficiently amplifying nucleic acids. The concentration of polyoxyethylene (20) sorbitan monolaurate in the nucleic acid extract may be, for example, 0.3 to 5.0% (w / v), 1.25 to 5.0% (w / v), or 1.25 to 2.5% (w / v). When polyoxyethylene(10) octylphenyl ether is included as a nonionic surfactant, the concentration of polyoxyethylene(10) octylphenyl ether in the nucleic acid extract is preferably 0.3% (w / v) or higher, more preferably 0.6% (w / v) or higher, and even more preferably 1.25% (w / v) or higher, from the viewpoint of reducing nonspecific amplification in the nucleic acid amplification reaction, and preferably 2.5% (w / v) or lower, from the viewpoint of reducing nonspecific amplification in the nucleic acid amplification reaction and efficiently amplifying nucleic acids. The concentration of polyoxyethylene(10) octylphenyl ether in the nucleic acid extract may be, for example, 0.3 to 2.5% (w / v) or 0.63 to 2.5% (w / v).Therefore, the amount of nonionic surfactant mixed in the extraction process may be such that the concentration of the nonionic surfactant in the nucleic acid extract falls within the above concentration range.

[0023] The order in which the sample, dodecyl alcohol sulfate, and nonionic surfactant are mixed is not particularly limited, and each component can be mixed in any order. Alternatively, each component may be mixed simultaneously. In one embodiment, the dodecyl alcohol sulfate and nonionic surfactant may be mixed in advance to prepare the nucleic acid extraction reagent described later, and this nucleic acid extraction reagent may be mixed with the sample.

[0024] The temperature of the extraction process is not particularly limited and may be, for example, 0 to 100°C or 15 to 30°C.

[0025] In one embodiment, a method for reducing nonspecific amplification in a nucleic acid amplification reaction may further include a step of amplifying nucleic acids in a nucleic acid extract (hereinafter also referred to as the amplification step). Nucleic acid amplification can be carried out using conventionally known nucleic acid amplification methods such as PCR (polymerase chain reaction), RT-PCR, LAMP (loop-mediated isothermal amplification), RT-LAMP, and TRC (transcription-reverse transcription concerted reaction).

[0026] The amplification step may include mixing known reagents for carrying out nucleic acid amplification reactions with the nucleic acid extract. Examples of known reagents for carrying out nucleic acid amplification reactions include nucleic acid synthases such as reverse transcriptase and DNA polymerase, deoxynucleoside triphosphates (dNTPs: dATP, dTTP, dCTP, and dGTP), and primers. Other known reagents for carrying out nucleic acid amplification reactions include, for example, buffers that provide suitable conditions for the enzymatic reaction, protective agents such as dithiothreitol (DTT) that stabilize the enzyme or template, and cyclodextrins that are useful for efficiently amplifying nucleic acids. The amplification step is preferably carried out in the presence of cyclodextrins.

[0027] Examples of cyclodextrins include α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, and their derivatives. A derivative of cyclodextrin is a molecule in which some of the hydroxyl groups are replaced with OR groups, where R is, for example, a hydrocarbon group or a hydroxyalkyl group. As an example, the cyclodextrin may be hydroxypropyl-β-cyclodextrin. The amount of cyclodextrin to be mixed with the nucleic acid extract may be, for example, an amount such that the concentration of cyclodextrin in the nucleic acid extract is 8 times or more the concentration of dodecyl alcohol sulfate. In other words, the amount of cyclodextrin may be 8 parts by mass or more per 1 part by mass of dodecyl alcohol sulfate in the nucleic acid extract. Since cyclodextrin has the effect of encapsulating dodecyl alcohol sulfate, it can reduce the influence of dodecyl alcohol sulfate on the nucleic acid amplification reaction. This makes it possible to efficiently amplify nucleic acids.

[0028] The method for reducing nonspecific amplification in nucleic acid amplification reactions described herein uses dodecyl alcohol sulfate and the above-mentioned specific nonionic surfactant for nucleic acid extraction, thus eliminating the need to separate and purify nucleic acids from the nucleic acid extract before amplification. Therefore, in one embodiment, the method for reducing nonspecific amplification in nucleic acid amplification reactions does not require a step of separating or purifying nucleic acids from the nucleic acid extract between the extraction step and the amplification step. According to this method, the step of separating or purifying nucleic acids can be omitted, allowing for simple, rapid, and highly efficient amplification of nucleic acids.

[0029] Another aspect of the present invention provides a method for extracting nucleic acids, comprising the step of mixing a sample containing nucleic acids with a dodecyl alcohol sulfate salt and the above-mentioned specific nonionic surfactant to obtain a nucleic acid extract (extraction step). Details of the extraction step are as described above.

[0030] Another aspect of the present invention provides a method for amplifying nucleic acids, comprising the steps of obtaining a nucleic acid extract by the above-described method for extracting nucleic acids (extraction step) and amplifying the nucleic acids in the nucleic acid extract (amplification step). Details of the amplification step are as described above. The method for amplifying nucleic acids according to this aspect does not require a step of separating or purifying nucleic acids from the nucleic acid extract between the extraction step and the amplification step.

[0031] According to the above method for extracting nucleic acids and the above method for amplifying nucleic acids, nonspecific amplification in the nucleic acid amplification reaction can be reduced even when the amplification reaction is carried out without purifying the nucleic acid extract.

[0032] A nucleic acid extraction reagent according to one aspect of the present invention comprises a dodecyl alcohol sulfate salt and the above-mentioned specific nonionic surfactant. The specific nonionic surfactant is polyoxyethylene(20) sorbitan monolaurate, polyoxyethylene(10) octylphenyl ether, or n-octanoyl-N-methyl-D-glucamine. From the viewpoint of storage stability of the nucleic acid extraction reagent, the nonionic surfactant is preferably n-octanoyl-N-methyl-D-glucamine or polyoxyethylene(10) octylphenyl ether, and more preferably n-octanoyl-N-methyl-D-glucamine. A nucleic acid extraction reagent containing n-octanoyl-N-methyl-D-glucamine as the nonionic surfactant has excellent storage stability in that it can maintain the effect of reducing nonspecific amplification even when stored for a long period of time (for example, 35 days or more at 60°C). From the viewpoint of environmental toxicity, n-octanoyl-N-methyl-D-glucamine or polyoxyethylene(20) sorbitan monolaurate are preferred as nonionic surfactants. The nonionic surfactant may also contain multiple nonionic surfactants.

[0033] The combination of dodecyl alcohol sulfate and the above-mentioned specific nonionic surfactant is not particularly limited, and may include, for example, a combination of SDS and polyoxyethylene (20) sorbitan monolaurate, a combination of SDS and polyoxyethylene (10) octylphenyl ether, a combination of SDS and n-octanoyl-N-methyl-D-glucamine, a combination of LDS and polyoxyethylene (20) sorbitan monolaurate, a combination of LDS and polyoxyethylene (10) octylphenyl ether, or a combination of LDS and n-octanoyl-N-methyl-D-glucamine. The combination of dodecyl alcohol sulfate and the above-mentioned specific nonionic surfactant is preferably a combination of SDS and n-octanoyl-N-methyl-D-glucamine or a combination of LDS and n-octanoyl-N-methyl-D-glucamine.

[0034] The concentration of dodecyl alcohol sulfate in the nucleic acid extraction reagent is not particularly limited as long as it is a concentration that can extract nucleic acids, and may be, for example, 0.01 to 1% (w / v), 0.1 to 1% (w / v), or 0.2 to 1% (w / v).

[0035] The concentration of the nonionic surfactant in the nucleic acid extraction reagent may be, for example, 0.3 to 5.0% (w / v) or 1.25 to 1.5% (w / v). When the nonionic surfactant includes n-octanoyl-N-methyl-D-glucamine, the concentration of n-octanoyl-N-methyl-D-glucamine in the nucleic acid extraction reagent is preferably 0.5% (w / v) or more, more preferably 1.0% (w / v) or more, from the viewpoint of reducing nonspecific amplification in the nucleic acid amplification reaction, and preferably 1.5% (w / v) or less, more preferably 1.25% (w / v) or less, from the viewpoint of efficiently amplifying nucleic acids. The concentration of n-octanoyl-N-methyl-D-glucamine in the nucleic acid extraction reagent may be, for example, 0.5 to 1.5% (w / v) or 0.5 to 1.25% (w / v). When polyoxyethylene (20) sorbitan monolaurate is included as a nonionic surfactant, the concentration of polyoxyethylene (20) sorbitan monolaurate in the nucleic acid extraction reagent is preferably 0.3% (w / v) or more, 0.6% (w / v) or more, more preferably 1.25% (w / v) or more, even more preferably 2.5% (w / v) or more, and particularly preferably 5.0% (w / v) or more, from the viewpoint of reducing nonspecific amplification in the nucleic acid amplification reaction, and preferably 5.0% (w / v) or less, and more preferably 2.5% (w / v) or less, from the viewpoint of efficiently amplifying nucleic acids. The concentration of polyoxyethylene (20) sorbitan monolaurate in the nucleic acid extraction reagent may be, for example, 0.3 to 5.0% (w / v), 1.25 to 5.0% (w / v), or 1.25 to 2.5% (w / v). When polyoxyethylene(10) octylphenyl ether is included as a nonionic surfactant, the concentration of polyoxyethylene(10) octylphenyl ether in the nucleic acid extraction reagent is preferably 0.3% (w / v) or higher, more preferably 0.6% (w / v) or higher, and even more preferably 1.25% (w / v) or higher, from the viewpoint of reducing nonspecific amplification in the nucleic acid amplification reaction, and preferably 2.5% (w / v) or lower, from the viewpoint of reducing nonspecific amplification in the nucleic acid amplification reaction and efficiently amplifying nucleic acids. The concentration of polyoxyethylene(10) octylphenyl ether in the nucleic acid extraction reagent may be, for example, 0.3 to 2.5% (w / v) or 0.63% to 2.5% (w / v).

[0036] The nucleic acid extraction reagent can be used on any sample containing nucleic acids, and the above-mentioned samples can be used. Furthermore, as mentioned above, the type of nucleic acid is not particularly limited. The above-mentioned nucleic acid extraction reagent can reduce nonspecific amplification even when using samples containing proteins, such as saliva or nasal swabs. In other words, in one embodiment, the nucleic acid extraction reagent is suitable as a reagent for extracting nucleic acids from a sample selected from the group consisting of saliva and nasal swabs.

[0037] The nucleic acid extraction reagent described above is a composition comprising a dodecyl alcohol sulfate salt and a specific nonionic surfactant. However, in another aspect of the present invention, the dodecyl alcohol sulfate salt and the specific nonionic surfactant may be provided as separate reagents. That is, one aspect of the present invention provides a nucleic acid extraction kit comprising a dodecyl alcohol sulfate salt and the above-mentioned specific nonionic surfactant. [Examples]

[0038] In the following test examples, % refers to mass-volume %, i.e., %(w / v), unless otherwise specified. Unless otherwise specified, each operation was performed at room temperature (20-25°C).

[0039] <rt-lamp> In the following test example, RT-LAMP was performed using the Loopamp® SARS-CoV-2 Detection Reagent Kit (manufactured by Eiken Chemical Co., Ltd.) as follows: First, 15 μL of primer mix and then 10 μL of sample were added to a tube containing the RNA amplification drying reagent (buffer, MgSO4, dNTP, AMV Reverse Transcriptase (manufactured by Roche), and BstDNA Polymerase (manufactured by New England Biolabs)) included in the kit. After restoring the drying reagent for 5 minutes, the mixture was stirred and centrifuged. The reaction tube was placed in a real-time turbidimeter Loopamp LA-320C or LoopampEXIA® (both manufactured by Teramex Co., Ltd.), and the RT-LAMP reaction was performed at 62.5°C for 90 minutes. The time until the turbidity reached 0.05 (hereinafter referred to as the Tt value) was measured. The positive control reagent included in the kit was used as the positive control (PC). The positive control reagent contains either the SARS-CoV-2 N gene or the RNA-dependent RNA polymerase (RdRp) gene. A 0.2% SDS solution was used as the negative control (NC) (except for Test Example 7, where a 0.2% LDS solution was used as the negative control). Table 1 shows the components of the primer mix. Table 2 shows the sequences of the primer sets included in the primer mix.

[0040] [Table 1] [Table 2]

[0041] <BCAアッセイ> In the following test examples, the BCA (bicinchoninic acid) assay was performed using the TaKaRa BCA Protein Assay Kit (Takara Bio Inc.) as follows: First, the Working Solution prepared according to the kit protocol was dispensed into a 384-well plate, and 5 μL of sample was added to each well. The well plate was incubated in a 37°C incubator for 30 minutes, and the absorbance at 562 nm was measured using a plate reader.

[0042] <Test Example 1> Identifying the cause of nonspecific amplification Four saliva samples collected from healthy individuals were ultrafiltered as follows to separate them into fractions of less than 3 kDa and fractions of 3 kDa or more. First, to prevent filter clogging, the saliva was centrifuged at 12000 × g for 5 minutes, and the supernatant was collected. 100 μL of the supernatant was added to an Amicon® Ultra 0.5 mL-3 kDa (Merck) filter pre-rinsed with physiological saline, and centrifuged at 14000 × g for 30 minutes. The flow-through (fraction of less than 3 kDa) was collected. 300 μL of physiological saline was added to the above filter, and centrifuged at 14000 × g for 30 minutes. This was repeated three times. The sample on the filter was collected, and the volume was adjusted to 100 μL with physiological saline (fraction of 3 kDa or more).

[0043] 5 μL of saliva, a fraction with a molecular weight of 3 kDa or less, or a fraction with a molecular weight of 3 kDa or more was mixed with 200 μL of 0.2% SDS solution. RT-LAMP was performed on each sample, and the Tt value was measured. The results are shown in Figure 1. In the graph below, white circles represent measured values, and bars or black circles represent average values. In three of the four saliva samples, the average Tt value of the fraction with a molecular weight of 3 kDa or more was faster than the average Tt value of the fraction with a molecular weight of 3 kDa or less. This indicates that the nonspecific amplification reaction was accelerated in the fraction with a molecular weight of 3 kDa or more compared to the fraction with a molecular weight of 3 kDa or less. This result suggests that the cause of nonspecific amplification may be high molecular weight substances, rather than low molecular weight substances such as metal ions.

[0044] Since saliva contains proteins, it was hypothesized that proteins were the cause of nonspecific amplification. Therefore, the correlation between the amount of protein in saliva and nonspecific amplification was analyzed. First, two saliva samples collected from healthy individuals were centrifuged at 12000 × g for 5 minutes to separate them into supernatant and sediment. The amount of protein was confirmed by BCA assay for each of the following: supernatant, sediment, mixture of supernatant and sediment, and a sample without saliva. In addition, 5 μL of each sample was mixed with 200 μL of 0.2% SDS solution, and the Tt value was measured by RT-LAMP reaction. The results are shown in Figure 2. A tendency was observed for earlier detection of nonspecific amplification as the protein concentration increased. From these results, it was suggested that proteins are the cause of nonspecific amplification.

[0045] <Test Example 2> Search for substances that reduce nonspecific amplification We searched for substances that can reduce nonspecific amplification among 21 surfactants, betaine, and dimethyl sulfode (DMSO) known to function in protein solubilization and dispersion. Betaine and DMSO reduce primer nonspecific annealing by lowering the Tm value. The concentrations of each substance that do not inhibit RNA amplification are shown in Tables 3 and 4.

[0046] [Table 3] [Table 4]

[0047] 5 μL of saliva samples collected from healthy individuals were mixed with 200 μL of 0.2% SDS solution containing surfactant, betaine, or DMSO at the concentrations shown in Table 3 or Table 4. RT-LAMP was performed on each sample, and the Tt value was measured. The results are shown in Figures 3 to 5. When using SDS solutions containing Tween® 20, Triton® X-100, or MEGA-8, the average Tt value was significantly delayed compared to when using SDS solutions without surfactant, betaine, or DMSO. This result indicates that nonspecific amplification could be reproducibly reduced by using SDS solutions containing Tween® 20, Triton® X-100, or MEGA-8.

[0048] <Test Example 3> Accelerated Stability Test Accelerated stability tests were conducted using Tween® 20, Triton® X-100, and MEGA-8. Specifically, nucleic acid extraction reagents were prepared by adding Tween® 20, Triton® X-100, or MEGA-8 at the concentrations shown in Table 3 to a 0.2% SDS solution, and the nucleic acid extraction reagents were stored at 60°C. Before storage at 60°C (before the accelerated stability test), and on days 7, 8, and 35 of the accelerated stability test, 5 μL of saliva samples collected from healthy individuals were mixed with 200 μL of nucleic acid extraction reagent, and RT-LAMP was performed. The results are shown in Figures 6 and 7. In the case of Tween® 20, the effect of reducing nonspecific amplification was lost on day 7, whereas in the case of Triton® X-100, the effect of reducing nonspecific amplification was maintained even on day 7, and in the case of MEGA-8, it was maintained even on day 35.

[0049] <Test Example 4> Examination of Effective Concentration Nucleic acid extraction reagents were prepared by adding MEGA-8 to a 0.2% SDS solution at concentrations of 0%, 0.13%, 0.25%, 0.5%, 1.0%, or 1.5%. 5 μL of saliva samples collected from healthy individuals were mixed with 200 μL of the nucleic acid extraction reagent, and RT-LAMP was performed on the resulting samples. The results are shown in Figure 8. Nonspecific amplification was further reduced when the MEGA-8 concentration in the nucleic acid extraction reagent was 0.5% or higher.

[0050] The same tests as above were performed using nucleic acid extraction reagents containing Tween® 20 or Triton® X-100 at concentrations of 0%, 0.31%, 0.63%, 1.25%, 2.50%, or 5.00% instead of MEGA-8. The results are shown in Figures 9(A) and 9(B).

[0051] Nucleic acid extraction reagents were prepared by adding MEGA-8 to a 0.2% SDS solution at the concentrations shown in Table 5. 1 μL of a solution containing 2500 copies of the SARS-CoV-2 N gene or RdRp gene was mixed with 100 μL of the nucleic acid extraction reagent, and 10 μL of this mixture was used for RT-LAMP for 35 minutes. The results are shown in Table 5. When the concentration of MEGA-8 in the nucleic acid extraction reagent was 1.75% or higher, amplification of the template RNA (SARS-CoV-2 gene) was inhibited.

[0052] [Table 5]

[0053] <Test Example 5> Examination of the specimen A nucleic acid extraction reagent was prepared containing 0.2% SDS, 1% MEGA-8, 2.5% Tween® 20, or 2.5% Triton® X-100. Saliva or nasal swab samples collected from healthy individuals were mixed with the nucleic acid extraction reagent, and RT-LAMP was performed on the resulting samples. Samples containing saliva were prepared by mixing the nucleic acid extraction reagent and the sample in a volume ratio of 40:1. Samples containing nasal swabs were prepared by immersing a nasal swab in 4 mL of nucleic acid extraction reagent and stirring.

[0054] The results of RT-LAMP are shown in Figure 10(A). It was confirmed that nonspecific amplification can be reduced not only when saliva is used as a sample, but also when nasal swab fluid is used.

[0055] The above samples, obtained by mixing the specimens with nucleic acid extraction reagents, were then treated with a solution containing the SARS-CoV-2 N gene or RdRp gene, and RT-LAMP was performed in the presence of 250 copies of the N gene or RdRp gene. The results of RT-LAMP are shown in Figure 10(B). In all samples, there was little inhibition of the template RNA amplification reaction.

[0056] <Test Example 6> Investigation of dodecyl alcohol sulfate salts A nucleic acid extraction reagent was prepared containing 0.2% LDS and MEGA-8, Tween® 20, or Triton® X-100 at the concentrations shown in Table 3. Saliva or nasal swab samples collected from healthy individuals were mixed with the nucleic acid extraction reagent, and RT-LAMP was performed on the resulting samples. Each sample was prepared in the same manner as in Test Example 5.

[0057] The results of RT-LAMP are shown in Figure 11. It was confirmed that nonspecific amplification could be reduced in samples containing saliva or nasal swabs even when LDS was used instead of SDS as the dodecyl alcohol sulfate ester.

[0058] <Test Example 7> Accelerated Stability Test 2 Nucleic acid extraction reagents were prepared containing 0.2% LDS and MEGA-8, Tween® 20, or Triton® X-100 at the concentrations shown in Table 3, and the reagents were stored at 60°C. Before storage at 60°C (before the accelerated stability test), and on days 7 and 36 of the accelerated stability test, 5 μL of saliva samples collected from healthy individuals were mixed with 200 μL of nucleic acid extraction reagent, and RT-LAMP was performed. The results are shown in Figure 12. In the case of Triton® X-100 and MEGA-8, the reduction effect on nonspecific amplification was maintained even on day 36. On the other hand, in the case of Tween® 20, the reduction effect on nonspecific amplification was lost on day 7, but a reduction in nonspecific amplification was observed again on day 36.

[0059] A solution containing the SARS-CoV-2 N gene or RdRp gene was added to the nucleic acid extraction reagent on days 7 and 36 of the accelerated stability test, and RT-LAMP was performed on the resulting samples (copy number of N gene or RdRp gene in the reaction solution: 250 copies). The regeneration time for the RNA amplification drying reagent was shortened from 5 minutes to 2 minutes, and the RT-LAMP reaction time was also shortened from 90 minutes to 35 minutes. The results are shown in Table 6.

[0060] [Table 6]

[0061] In the tables herein, "ND" means not detected. When the nucleic acid extraction reagent contained Tween® 20 along with LDS, the amplification of template RNA was significantly inhibited. This result suggests that when the sample contains the combination of Tween® 20 and LDS, accelerated stability testing may generate some substance that inhibits the RT-LAMP reaction. The reduction in nonspecific amplification observed on day 36 in the sample containing the combination of Tween® 20 and LDS in the above accelerated stability testing is also presumed to be due to this inhibitor.

[0062] <Test Example 8> Confirmation of RNA template amplification Nucleic acid extraction reagents were prepared containing 0.2% SDS or LDS and MEGA-8, Tween® 20, or Triton® X-100 at concentrations shown in Tables 7-9. A solution containing the SARS-CoV-2 N gene or RdRp gene was added to the nucleic acid extraction reagent, and RT-LAMP was performed on the resulting sample (copy number of N gene or RdRp gene in the reaction solution: 250 copies). The regeneration time for the RNA amplification drying reagent was shortened from 5 minutes to 2 minutes, and the RT-LAMP reaction time was also shortened from 90 minutes to 35 minutes. The results are shown in Tables 7-9.

[0063] [Table 7]

[0064] [Table 8]

[0065] [Table 9]

[0066] Amplification of template RNA was inhibited when the concentration of MEGA-8 in the nucleic acid extraction reagent was 2.0% or higher, and when the concentration of Tween® 20 in the nucleic acid extraction reagent was 5.0%. At other concentrations, amplification of template RNA proceeded well. When using Triton® X-100, amplification of template RNA proceeded well at all concentrations.

Claims

1. A method for reducing nonspecific amplification in nucleic acid amplification reactions, The process includes the step of mixing a sample containing nucleic acids with a dodecyl alcohol sulfate salt and a nonionic surfactant to obtain a nucleic acid extract. Samples containing nucleic acids are saliva or nasal swab samples. The nonionic surfactant is polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (10) octylphenyl ether, or n-octanoyl-N-methyl-D-glucamine. In the process of mixing a sample containing nucleic acids with a dodecyl alcohol sulfate salt and a nonionic surfactant, The concentration of polyoxyethylene (20) sorbitan monolaurate in the nucleic acid extract is between 1.25% (w / v) and 5.0% (w / v), The concentration of polyoxyethylene (10) octylphenyl ether in the nucleic acid extract is between 0.63% (w / v) and 2.5% (w / v), A method comprising mixing a nonionic surfactant in an amount such that the concentration of n-octanoyl-N-methyl-D-glucamine in the nucleic acid extract is 0.5% (w / v) to 1.5% (w / v).

2. The method according to claim 1, wherein the nonionic surfactant is n-octanoyl-N-methyl-D-glucamine.

3. The method according to claim 1 or 2, wherein the nonspecific amplification is due to a substance other than nucleic acid in the sample containing nucleic acid.

4. A nucleic acid extraction reagent comprising dodecyl alcohol sulfate and 0.5% (w / v) to 1.5% (w / v) of n-octanoyl-N-methyl-D-glucamine.

5. A nucleic acid extraction kit containing dodecyl alcohol sulfate and 0.5% (w / v) to 1.5% (w / v) of n-octanoyl-N-methyl-D-glucamine.

6. The process includes a step of mixing a sample containing nucleic acids with dodecyl alcohol sulfate and n-octanoyl-N-methyl-D-glucamine to obtain a nucleic acid extract. In the step of mixing a sample containing nucleic acids with dodecyl alcohol sulfate and n-octanoyl-N-methyl-D-glucamine, A method for extracting nucleic acids, comprising mixing an amount of n-octanoyl-N-methyl-D-glucamine in the nucleic acid extract solution such that the concentration of n-octanoyl-N-methyl-D-glucamine in the nucleic acid extract solution is 0.5% (w / v) to 1.5% (w / v).

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