WO2020059792A1

WO2020059792A1 - Composition for nucleic acid amplification reaction

Info

Publication number: WO2020059792A1
Application number: PCT/JP2019/036731
Authority: WO
Inventors: 貴圭肥山
Original assignee: 東洋紡株式会社
Priority date: 2018-09-21
Filing date: 2019-09-19
Publication date: 2020-03-26
Also published as: JPWO2020059792A1

Abstract

A purpose of the present invention is to provide methods for effectively performing a nucleic acid amplification reaction. Additionally, another purpose of the present invention is to provide nucleic acid amplification methods that are capable of maintaining highly efficient amplification even when a long-chain nucleic acid having a 200 bp or longer amplification chain is targeted. In a specific example, as a method for amplifying a target nucleic acid having a 200 bp or longer amplification chain length, the present invention provides a nucleic acid amplification method characterized by performing a nucleic acid amplification reaction in a reaction solution that includes at least one diol compound selected from the group consisting of 1,2-propandiol and 1,2-ethandiol. In an additional example, the reaction solution further includes a tetramethylammonium salt.

Description

Composition for nucleic acid amplification reaction

The present invention relates to a composition and a kit for a nucleic acid amplification reaction, which can improve nucleic acid amplification efficiency and detection sensitivity and can perform efficient nucleic acid amplification, a nucleic acid amplification method, and the like. In particular, the present invention relates to a nucleic acid amplification method and a kit capable of efficiently amplifying a long-chain target nucleic acid.

Nucleic acid amplification is a technology that amplifies several copies of a target nucleic acid to a level at which it can be visualized, that is, to hundreds of millions or more. It is also widely used in microbial tests and the like. A typical nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR includes (1) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (2) annealing of primer to single-stranded DNA template, and (3) the above-mentioned primer using DNA polymerase. This is a method of amplifying a target nucleic acid in a sample by repeating the three steps of elongation as one cycle and repeating this cycle.

In recent years, among PCRs, the real-time PCR method has been widely practiced. The real-time PCR method can analyze an amplified nucleic acid over time, secure high quantitativeness, and perform highly sensitive detection. When performing such real-time PCR, it is important to improve the nucleic acid amplification efficiency to increase the quantitativeness and the detection sensitivity.

There have been reported methods for adding various reagents to a nucleic acid amplification reaction solution in order to improve the efficiency and sensitivity of the nucleic acid amplification method.

For example, in Patent Document 1, the nucleic acid amplification efficiency and detection sensitivity are improved by using a nucleic acid amplification composition characterized by containing a tetramethylammonium-resistant DNA polymerase together with a tetramethylammonium salt having a final concentration of 50 mM or more. For effective nucleic acid amplification. Non-Patent Document 1 also describes that a tetramethylammonium salt improves the amplification efficiency and specificity of a nucleic acid amplification reaction. According to the report of Non-Patent Document 1, the concentration at which the amplification efficiency is maximum is 5 mM for tetramethylammonium chloride and 10 mM or less for tetramethylammonium acetate, and the concentration at which the specificity is maximum is tetramethylammonium chloride and tetramethylammonium acetate. Ammonium is set to 20 mM. Furthermore, tetramethylammonium salts are also said to inhibit enzymes at high concentrations. According to Non-Patent Document 1, when Taq DNA polymerase is used, tetramethylammonium chloride exceeds 35 mM and tetramethylammonium acetate exceeds 40 mM. And that nucleic acid amplification is inhibited by 90%.

Further, for example, melting temperature regulators such as dimethyl sulfoxide (DMSO), betaine derivatives, and ionic liquids have been reported as methods for improving nucleic acid amplification efficiency. All of these are known to improve nucleic acid amplification efficiency by lowering the melting temperature of nucleic acids (Patent Documents 2, 3, and 4).

Further, Patent Documents 5 and 6 describe a method in which a glycol is added during a nucleic acid amplification reaction to lower the melting temperature of nucleic acid and suppress nonspecific amplification. However, Patent Documents 5 and 6 do not specifically describe that the use of glycols improves the Ct value and the PCR efficiency.

By the way, the design of the primer is an important factor that determines the success or failure of the experiment. For example, in real-time PCR, in order to increase amplification efficiency during a nucleic acid amplification reaction, primers are designed so that the length of the amplified chain length (amplified chain length) is smaller than 200 bp (for example, 50 bp or more and less than 200 bp). That is common.

However, due to non-specific amplification and the occurrence of primer dimers, the design site of the primer is limited, so that a situation where the amplified chain length must be 200 bp or more often occurs. In such a case, there has been a problem that the nucleic acid amplification efficiency and the detection sensitivity are reduced with a normal real-time PCR reagent.

Furthermore, in recent years, analysis using the multiplex PCR method has been performed in gene diagnosis applications and gene testing applications. The multiplex PCR method is a method in which several primer pairs are used simultaneously in one PCR assay. This method of simultaneously amplifying multiple regions of DNA in a single reaction mixture saves labor, time, and money because it requires minimal sample handling and reduces the risk of cross-contamination. However, on the other hand, the addition of a plurality of primers causes a problem that a primer dimer is easily formed, which makes it very difficult to design a primer. In such a case, it is assumed that the design site of the primer is limited in order to avoid the primer dimer, and the chain length to be amplified becomes 200 bp or more.

In addition, in the multiplex PCR method, when the lengths of the respective amplified chains of the DNA regions that are simultaneously amplified in the reaction mixture are different, there is a concern that the difference in the length of the amplification product may cause variation in amplification efficiency. Accordingly, there is a possibility that an accurate quantification result cannot be obtained, or a problem such as a decrease in target DNA detection sensitivity may occur.

In the multiplex PCR method using the intercalator method, a method is employed in which the presence or absence of a target DNA is determined by changing the Tm value of an amplification product in a melting curve analysis for each target DNA region. In such a case, the chain length of the amplification product is generally changed for the purpose of distinguishing the Tm value of the amplification product. At this time, it is assumed that the chain length to be amplified is 200 bp or more. For example, in Non-Patent Document 2, for the purpose of analyzing by fusion curve analysis the velotoxin gene from mixed feces, the Salmonella enterotoxin gene, Shigella ipaH gene, and the product derived from the internal control, the respective amplified products are 171 bp, 264 bp, Primers are designed to be 242 bp and 540 bp.

JP 2017-108735 A Japanese Patent No. 4761265 Japanese Patent No. 4300321 JP 2014-27934 A JP 2010-246528 A JP 2010-246529 A

In the nucleic acid amplification method, a nucleic acid amplification method for improving the nucleic acid amplification efficiency and the detection sensitivity has been studied. However, depending on the nucleic acid sequence to be amplified, a decrease in amplification efficiency or sensitivity may be observed, and further improvement is required. In particular, when the amplification chain length is long, there has been a problem that the amplification efficiency and the detection sensitivity are reduced.

Accordingly, one object of the present invention is to provide a composition for a nucleic acid amplification reaction (eg, a PCR reaction composition) that can be amplified with high efficiency in a nucleic acid amplification method. Still another object of the present invention is to provide a nucleic acid amplification reaction composition that can maintain highly efficient amplification even when the length of the amplified chain is long.

MEANS TO SOLVE THE PROBLEM As a result of earnest research, the present inventors have solved the above-mentioned problem and found that a nucleic acid amplification reaction was performed in a reaction solution containing at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol. By carrying out, the present inventors have found that it is possible to carry out a PCR reaction with high efficiency even when, for example, amplifying a long target nucleic acid of 200 bp or more, and have accomplished the present invention. This effect is more effective when the reaction is performed using a reaction solution containing a combination of the diol compound and a tetramethylammonium salt. Furthermore, it has been found that this highly efficient nucleic acid amplification method is remarkable when a Thermus thermophilus-derived DNA polymerase is used as the DNA polymerase.

That is, the outline of the present invention is as follows.

[Item 1] A method for amplifying a target nucleic acid having an amplification chain length of 200 bp or more, wherein the reaction comprises at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol. A nucleic acid amplification method comprising performing a nucleic acid amplification reaction in a liquid.

[Item 2] The method according to Item 1, wherein the reaction solution further contains a tetramethylammonium salt.

[Item 3] The method according to Item 1 or 2, wherein the tetramethylammonium salt is at least one selected from the group consisting of tetramethylammonium acetate, tetramethylammonium chloride, and tetramethylammonium hydroxide.

[Item 4] The method according to any one of Items 1 to 3, wherein the amplified chain length is from 200 bp to 1000 bp.

[Item 5] The method according to any one of Items 1 to 4, wherein the amplified chain length is 400 bp or more and 500 bp or less.

[Item 6] The method according to any one of Items 1 to 5, wherein the nucleic acid amplification reaction is real-time PCR.

[Item 7] The method according to any one of Items 1 to 6, wherein the elongation time in the nucleic acid amplification reaction is 60 seconds or less.

[Item 8] The method according to any one of Items 1 to 7, wherein the extension time in the nucleic acid amplification reaction is 30 seconds or less.

[Item 9] The method according to any one of Items 1 to 8, wherein the reaction solution further contains a DNA polymerase derived from Thermus thermophilus.

[Item 10] A composition for a nucleic acid amplification reaction comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol, and a tetramethylammonium salt.

[Item 11] The composition for a nucleic acid amplification reaction according to Item 10, wherein the concentration of the diol compound in the reaction solution at the time of the nucleic acid amplification reaction is adjusted to 1 to 20% by volume based on the whole reaction solution. .

[Item 12] The nucleic acid amplification reaction according to

Item

10 or 11, wherein the concentration of the diol compound in the reaction solution at the time of the nucleic acid amplification reaction is adjusted to 3 to 10% by volume based on the entire reaction solution. Composition.

[Item 13] The nucleic acid amplification according to any one of Items 10 to 12, wherein the tetramethylammonium salt is at least one selected from the group consisting of tetramethylammonium acetate, tetramethylammonium chloride, and tetramethylammonium hydroxide. Reaction composition.

[Item 14] The nucleic acid according to any one of Items 10 to 13, wherein the concentration of the tetramethylammonium salt in the reaction solution at the time of the nucleic acid amplification reaction is adjusted to 50 to 200 mM based on the whole reaction solution. Composition for amplification reaction.

[Item 15] The composition for a nucleic acid amplification reaction according to any one of Items 10 to 14, further comprising a DNA polymerase derived from Thermus thermophilus.

[Item 16] The composition for a nucleic acid amplification reaction according to any one of Items 10 to 15, wherein the nucleic acid amplification reaction is a real-time PCR reaction.

[Item 17] The composition for a nucleic acid amplification reaction according to any one of Items 10 to 16, which is used for amplifying a target nucleic acid having an amplification chain length of 200 bp or more.

[Item 18] A reagent for amplifying a target nucleic acid having an amplification chain length of 200 bp or more, comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol.

[Item 19] A kit for amplifying a target nucleic acid having an amplification chain length of 200 bp or more, comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol.

[Item 20] A kit for nucleic acid amplification reaction, comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol, and a tetramethylammonium salt.

[Item 21] The kit for a nucleic acid amplification reaction according to Item 20, which is used for amplifying a target nucleic acid having an amplification chain length of 200 bp or more.

According to the present invention, nucleic acid amplification can be performed with high efficiency. INDUSTRIAL APPLICABILITY The present invention is particularly effective in real-time PCR, and is particularly effective when amplifying a target having a long amplification chain length (for example, a target nucleic acid having an amplification chain length of 200 bp or more), which was conventionally difficult to perform efficiently. Is remarkable.

It is a figure which shows the result of having investigated the amplification efficiency of the target which amplification chain length becomes 200bp, 300bp, 400bp, and 500bp using a commercially available real-time PCR reagent. FIG. 9 is a diagram showing the results of examining the amplification efficiency of a target having an amplification chain length of 200 bp, 300 bp, and 400 bp by using real-time PCR without adding 1,2-propanediol or by adding 7% by volume. It is a figure which shows the result of having investigated the amplification efficiency of the target which 500 bp of amplification chain lengths added by adding 7 volume% of various diol compounds using real-time PCR method. It is a figure which shows the result of having investigated the amplification efficiency of the target which has added 1,2-propanediol in various concentrations and the amplification chain length is 500 bp using the real-time PCR method. It is a figure which shows the result of having investigated the amplification efficiency of the target of 500 bp of amplification chain length by the real-time PCR method, without adding the density | concentration of tetramethylammonium acetate, or adding 50 mM or 100 mM.

Hereinafter, the present invention will be described in detail while showing embodiments of the present invention, but the present invention is not limited to these. In addition, all of the non-patent documents and patent documents described in this specification are incorporated herein by reference. In addition, “to” in the present specification means “more than or equal to or less”. For example, when “X to Y” is described in the specification, it means “more than X and less than or equal to Y”. Further, “and / or” in the present specification means either one or both.

In one aspect, the present invention relates to a method for amplifying a target nucleic acid having an amplified chain length of 200 bp or more, wherein at least one diol selected from the group consisting of 1,2-propanediol and 1,2-ethanediol Provided is a nucleic acid amplification method, wherein a nucleic acid amplification reaction is performed in a reaction solution containing a compound. The reaction solution used in the present invention may be, for example, a nucleic acid amplification reaction solution provided as a nucleic acid amplification reaction reagent. The nucleic acid amplification reaction solution may be a reagent which is prepared in advance and can be used as it is during the nucleic acid amplification reaction, or may be a reagent which is prepared before use before the nucleic acid amplification reaction. In the present specification, the nucleic acid amplification reaction solution as described above is also referred to as a nucleic acid amplification reaction composition, and these can be used interchangeably. For example, the nucleic acid amplification reaction solution used in the present invention may be provided in the form of a PCR reagent, a real-time PCR reagent, an RT-PCR reagent, a real-time RT-PCR reagent, or the like.

As used herein, the nucleic acid amplification reaction refers to a reaction for synthesizing a nucleic acid having a sequence complementary to a template nucleic acid in a sequence-dependent manner, and the manner thereof is not particularly limited. Chain reaction (PCR) method, Loop-Mediated Isolation Amplification (LAMP) method, Transcription Reversal Transcription Reconciliation Reaction (TRC) method, Amplification of Nucleic Acid Sequence method of Nucleic Acid Sequence method, etc. Method. The present invention can be used for any of the nucleic acid amplification reactions, but is preferably used for a method using a polymerase chain reaction (PCR) method.

Examples of the PCR method include not only a normal PCR method but also real-time PCR, RT-PCR (Reverse Transcription PCR), LA-PCR (Long and Accurate PCR), competitive PCR, in situ PCR, RNA-primed PCR, and multiplex PCR. Various PCR methods are known in the art, such as, for example, shuttle PCR, PCR / GC-calmp method, stretch PCR, Alu PCR, megaprimer PCR, Immuno PCR. The present invention can be used for any PCR method known in the art, but is preferably used for a real-time PCR method.

The PCR method as described above usually proceeds by repeating each reaction step of heat denaturation, annealing and extension. Thermal denaturation is a step for separating double-stranded DNA, annealing is a step of annealing a primer to the separated DNA, and elongation is a step of synthesizing a complementary strand with a DNA polymerase. In the PCR method performed in the present invention, the annealing temperature and the extension temperature may be different temperatures or may be the same temperature. In the present invention, when the annealing temperature and the elongation temperature are performed at the same temperature, the elongation reaction holding time is the holding time obtained by adding the annealing reaction time and the elongation reaction time. The extension reaction holding time is not particularly limited, but may be, for example, 60 seconds or less, preferably 50 seconds or less, more preferably 40 seconds or less, and still more preferably 30 seconds or less. The lower limit of the elongation reaction holding time is not particularly limited as long as the effects of the present invention are exhibited, but may be, for example, 1 second or longer, preferably 5 seconds or longer, more preferably 10 seconds or longer. Here, the holding time may be the same time in all cycles, or may be different.

The diol compound is a kind of alcohol (polyol) and is a compound in which one carbon group is present in each of two carbon atoms among carbon atoms forming a chain or cyclic aliphatic hydrocarbon. , And glycols. In addition, ethylene glycol having the simplest structure may be simply referred to as glycol, but here, it refers to the above-mentioned glycols in a broad sense.

In the present invention, 1,2-ethanediol and 1,2-propanediol are used as the diol compound. The present inventors surprisingly amplify long-chain target nucleic acids of 200 bp or more (eg, 400-500 bp target nucleic acids) by using 1,2-propanediol and / or 1,2-ethanediol. Even in this case, it has been found for the first time that a decrease in nucleic acid amplification efficiency and detection sensitivity is suppressed, and nucleic acid amplification can be performed efficiently. Although these diol compounds have different production methods depending on their structures, industrial production methods have been established for all of them, and they are inexpensive and easily available. One of these diol compounds may be used alone, or the effects of the present invention are exhibited even when two or more diol compounds are used in combination. Furthermore, it has also been found that the effect observed when amplifying a long-chain target nucleic acid by 1,2-propanediol and / or 1,2-ethanediol is remarkable when combined with a tetramethylammonium salt. .

Since 1,2-propanediol and / or 1,2-ethanediol that can be used in the present invention has a low viscosity, operability due to the addition of a nucleic acid amplification reaction solution is extremely low. In addition, it is also excellent in that it can be stored for a long time and is stable. In addition, these diol compounds are also advantageous in that they have the effect of lowering the melting temperature of nucleic acids.

The concentration of at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol in the reaction solution at the time of the nucleic acid amplification reaction is 1 to 20 vol. %, More preferably 3 to 10% by volume. When the concentration of these diol compounds in the reaction solution is less than 1% by volume, the effect of improving amplification efficiency and detection sensitivity tends to be small even when combined with, for example, a tetramethylammonium salt. When the concentration is higher than 20% by volume, the disadvantage that handling becomes worse is greater than the effect. Therefore, the nucleic acid amplification reaction solution used in the present invention is preferably prepared such that the concentration of the diol compound in the reaction solution at the time of the nucleic acid amplification reaction is within the above range.

The nucleic acid amplification reaction solution in the present invention may be, for example, one prepared as a concentrated nucleic acid amplification reaction reagent for storage. Such a nucleic acid amplification reaction solution may be, for example, a 2 to 10-fold concentrated solution. In such a case, 1,2-propanediol and / or 1,2-ethanediol are contained in a concentration 2 to 10 times the final concentration in the reaction solution so that the final concentration in the reaction solution of the nucleic acid amplification reaction becomes a preferable concentration. You should let it.

In recent years, a double concentration (or higher concentration) of a premic reagent has been suitably used for the purpose of improving workability. The premix reagent is a reagent in which a reaction buffer, a reaction substrate (dNTPs), magnesium ions, a polymerase and the like are mixed in advance. The nucleic acid amplification reaction solution prepared in a concentrated state as described above is suitable when provided as such a premix reagent. In addition, the premix reagent may be inferior in stability because various components are preliminarily mixed and stored for a long period of time, but the nucleic acid amplification reaction solution used in the present invention may be in a mixed state for a long time (for example, 1 to 1). It has been found to be stable when stored for more than a month.

For detection and concentration measurement of a diol compound in a solution or a solid, a method using mass spectrometry, liquid chromatography or gas chromatography, a method using a polyol dehydrogenase, and the like are generally used. Preferably, the detection and concentration measurement of the diol compound can be performed by a method combining mass spectrometry, liquid chromatography analysis, and gas chromatography analysis.

In a specific preferred embodiment, the nucleic acid amplification method of the present invention performs a nucleic acid amplification reaction in the presence of a tetramethylammonium salt in a reaction solution. By performing the nucleic acid amplification reaction in a reaction solution further containing a tetramethylammonium salt, the effects of the present invention can be more effectively obtained.

As the tetramethylammonium salt that can be used in the present invention, any tetramethylammonium salt may be used as long as the effects of the present invention are exhibited. From the viewpoint that a higher effect of the present invention can be easily obtained when used in combination with 1,2-propanediol and / or 1,2-ethanediol, preferably, tetramethylammonium salt is tetramethylhydroxide. Ammonium, tetramethylammonium chloride, and tetramethylammonium acetate are used, and more preferably, tetramethylammonium acetate is used. Although these tetramethylammonium salts have different production methods depending on their structures, industrial production methods have been established for all of them, and they are inexpensive and easily available. One of these tetramethylammonium salts may be used alone, or the effect of the present invention is exerted even when two or more are used in any combination.

In the present invention, when a tetramethylammonium salt is used, the concentration of the tetramethylammonium salt in the reaction solution during the nucleic acid amplification reaction is not particularly limited as long as the effects of the present invention are exerted. It is preferably at least 50 mM, more preferably at least 75 mM, further preferably at least 90 mM, particularly preferably at least 100 mM. On the other hand, the upper limit of the concentration of the tetramethylammonium salt is not particularly limited as long as the effects of the present invention are exerted, but as an example, it is preferably 200 mM or less based on the whole reaction solution. Therefore, the nucleic acid amplification reaction solution used in the present invention is preferably prepared such that the concentration of the tetramethylammonium salt in the reaction solution during the nucleic acid amplification reaction is within the above range.

In the present invention, when the nucleic acid amplification reaction solution is prepared as, for example, a nucleic acid amplification reaction reagent in a concentrated state for storage, as described above, the nucleic acid amplification reaction solution can be in a 2 to 10-fold concentrated state. In such a case, the tetramethylammonium salt may be contained at a concentration of 2 to 10 times the final concentration in the reaction solution so that the final concentration in the reaction solution of the nucleic acid amplification reaction becomes a preferable concentration.

For the detection and concentration measurement of the tetramethylammonium salt in the solution or solid, NMR (nuclear magnetic resonance) analysis, a method by liquid chromatography, and the like are generally used. Preferably, detection and concentration measurement of the diol compound can be performed by liquid chromatography.

In the present invention, when a tetramethylammonium salt is used, the mixing ratio of at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol to the tetramethylammonium salt is as follows. Although not particularly limited as long as the effects of the present invention are exerted, as an example, when the tetramethylammonium salt is 100 mM, the diol compound is preferably 1 to 20% by volume, more preferably 3 to 10% by volume. . By including both components at such a ratio, the nucleic acid amplification efficiency can be more effectively improved, and particularly, the nucleic acid amplification efficiency when amplifying a long-chain target nucleic acid can be improved.

In order to carry out a nucleic acid amplification reaction, the nucleic acid amplification reaction solution (composition for nucleic acid amplification reaction) used in the present invention contains, in addition to the diol compound and the tetramethylammonium salt, a nucleic acid, a DNA polymerase, and a primer that serve as templates. As necessary, depending on the nucleic acid amplification method used, such as an oligonucleotide, a dideoxynucleoside triphosphate (dNTPs), a reaction buffer, a metal ion (eg, magnesium ion). As an example, in a nucleic acid amplification method using a PCR method, it is preferable that a DNA polymerase is further included, and a template nucleic acid, a DNA polymerase, an oligonucleotide, and dNTPs are more preferably included, and a template nucleic acid, a DNA polymerase, an oligonucleotide, More preferably, it contains dNTPs and a reaction buffer.

The DNA polymerase used in the nucleic acid amplification reaction of the present invention is not particularly limited, but it is preferable to use a DNA polymerase derived from Thermus thermophilus. Here, the DNA polymerase derived from Thermus thermophilus is not limited to a DNA polymerase obtained directly from Thermus thermophilus, and may be a DNA polymerase obtained directly from a mutant strain of Thermus thermophilus, or may be a Thermus thermophilus or a mutant thereof. A DNA polymerase which has been modified by genetic engineering or chemical methods based on the sequence information of a DNA polymerase obtained from the body, and which has substantially the same activity as a DNA polymerase derived from Thermus thermophilus may be used. The DNA polymerase derived from Thermus thermophilus may be a DNA polymerase directly extracted from Thermus thermophilus, or may be a recombinantly produced product thereof in an appropriate expression system.

As used herein, a DNA polymerase mutant refers to, for example, 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more, based on the amino acid sequence of the wild-type DNA polymerase from which it is derived. % Or more, preferably 99% or more, and has the activity of amplifying DNA similarly to wild-type DNA polymerase. Here, the method of calculating the amino acid sequence identity can be performed by any means known in the art. For example, it can be calculated using an analysis tool that is commercially available or available through a telecommunication line (Internet). For example, the homology algorithm BLAST (Basic local alignment search tool) http of the National Center for Biotechnology Information (NCBI) can be used. /// www. ncbi. nlm. nih. It is possible to calculate amino acid sequence identity by using default (initial setting) parameters in gov / BLAST /. In addition, the mutant that can be used in the present invention is obtained by substituting, deleting, inserting, and / or adding one or several amino acids in the amino acid sequence of the wild-type DNA polymerase from which the mutant is derived. It may be a polypeptide comprising an amino acid sequence having a "mutated" amino acid sequence and having the activity of amplifying DNA similarly to a wild-type DNA polymerase. The term “one or several” as used herein means, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, more preferably 1 to 5, and particularly preferably 1 to 3. Although possible, there is no particular limitation.

When used for performing real-time PCR, the nucleic acid amplification reaction solution used in the present invention preferably further contains a fluorescent dye or a fluorescently labeled probe in order to confirm nucleic acid amplification over time. Such a fluorescent dye or a fluorescent-labeled probe is not particularly limited as long as the effect of the present invention is exerted, and examples thereof include fluorescent dyes such as SYBR GreenI (registered trademark), Eva Green (registered trademark), and Pico Green (registered trademark). It is preferable to include a fluorescently labeled probe.

When the nucleic acid amplification reaction solution used in the present invention is used for performing RT-PCR or real-time RT-PCR, reverse transcriptase is used to efficiently convert RNA to be detected into DNA. It is preferable to further include.

When used for performing a hot start PCR method, the nucleic acid amplification reaction solution used in the present invention preferably contains an anti-DNA polymerase antibody. The anti-DNA polymerase antibody here may be a single kind of monoclonal antibody, a combination of a plurality of monoclonal antibodies, or a polyclonal antibody.

A further aspect of the present invention provides a nucleic acid amplification reaction comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol, and a tetramethylammonium salt. A composition. Here, the composition for a nucleic acid amplification reaction may be provided as, for example, a reagent for a nucleic acid amplification reaction. The nucleic acid amplification reaction composition may be in the form of a reagent which is prepared in advance and can be used as it is during the nucleic acid amplification reaction, or may be a reagent which is prepared before use before the nucleic acid amplification reaction. . For example, the nucleic acid amplification reaction composition of the present invention can be provided in the form of a PCR reagent, a real-time PCR reagent, an RT-PCR reagent, a real-time RT-PCR reagent, and the like.

The composition for nucleic acid amplification reaction according to the present invention, comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol, and a tetramethylammonium salt. The concentration of the component is not particularly limited as long as the effects of the present invention are exhibited. For example, the concentration (final concentration) of the diol compound in the reaction solution at the time of the nucleic acid amplification reaction is adjusted to 1 to 20% by volume, preferably 3 to 10% by volume, based on the entire reaction solution. The concentration may be adjusted as follows. Further, for example, the concentration (final concentration) of the tetramethylammonium salt in the reaction solution at the time of the nucleic acid amplification reaction is adjusted to 25 to 400 mM, preferably 50 to 200 mM, based on the entire reaction solution. (For example, a concentration adjusted to be 50 to 100 mM). The type of the tetramethylammonium salt used in the present embodiment is not particularly limited as long as the effects of the present invention are exhibited. For example, a tetramethylammonium salt as described in the method for amplifying the target nucleic acid can be used. . Preferably, the tetramethylammonium salt is at least one selected from the group consisting of tetramethylammonium acetate, tetramethylammonium chloride, and tetramethylammonium hydroxide. The composition of the present embodiment can further contain optional components, and preferably further contains a DNA polymerase derived from Thermus thermophilus.

The composition for nucleic acid amplification of this embodiment is particularly useful when amplifying a long-chain target nucleic acid, which has tended to be difficult to amplify by conventional methods. For example, amplifying a target nucleic acid having an amplified chain length of 200 bp or more Used to The amplification chain length of the target nucleic acid of interest is not particularly limited, but may be preferably 300 bp or more, more preferably 400 bp or more (eg, 500 bp or more, 600 bp or more). The upper limit of the amplified chain length of the target nucleic acid targeted by the present invention is not particularly limited as long as the effects of the present invention are exerted. For example, the upper limit may be 1000 bp or less, or 800 bp or less. May be 500 bp or less.

Another aspect of the present invention is to amplify a target nucleic acid having an amplification chain length of 200 bp or more, comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol. Reagent. A further embodiment of the present invention is a kit including at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol, and preferably has an amplification chain length of 200 bp or more. This is a kit used for amplifying a target nucleic acid. Such a reagent or kit preferably further includes a tetramethylammonium salt in combination with the diol compound. One preferred embodiment of the present invention provides a nucleic acid amplification reaction comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol, and a tetramethylammonium salt. Kit. For example, when the kit of the present invention further contains a tetramethylammonium salt, such a kit for a nucleic acid amplification reaction of the present invention is selected from the group consisting of 1,2-propanediol and 1,2-ethanediol. At least one diol compound and the tetramethylammonium salt may be provided in the same container, or may be provided in separate containers, and may be used by mixing before use. Good. When provided in a separate container, other components that may optionally be included (eg, a DNA polymerase derived from Thermus thermophilus) may be included in the same container as the diol compound, or may be included in the tetramer. The diol compound and the tetramethylammonium salt may be contained in the same container as the methyl ammonium salt, or may be contained in another container. It is preferable that the diol compound and the tetramethylammonium salt are contained in the same container from the viewpoint that the diol compound and the tetramethylammonium salt can be used more easily. It is more preferable to include other components that may be included (for example, a DNA polymerase derived from Thermus thermophilus). The concentration and type of the diol compound and the tetramethylammonium salt used in the above-described kit of the present invention can be the same as those described in the nucleic acid amplification method.

The nucleic acid amplification method of the present invention can provide the diol compound in a form such as a reagent or a kit, and optionally a tetramethylammonium salt, for example, in a state containing both, or can be prepared in-house. And can be provided in any form.

The concentration or type of the diol compound or tetramethylammonium salt used in the method of the present invention as described above may be the same as that described in the nucleic acid amplification method or the reagent or kit for the nucleic acid amplification reaction. .

In a specific embodiment, a technique for evaluating PCR efficiency (PCR amplification efficiency) is used as an index for evaluating the efficiency of amplification in the real-time PCR method. The PCR efficiency is a value obtained from the slope of the calibration curve, and it is known that when the initial template concentration (Log10) is taken on the X-axis and the Ct value is taken on the Y-axis, the following equation is used. PCR efficiency E = 10 ^{[−1 / slope]} −1 This PCR efficiency is ideally closer to 100%, but varies depending on the design of PCR inhibitors and primers. For this reason, 80% to 120% is said to be an appropriate value, and if less than that, it is said that it is usually necessary to review the experimental system. According to the present invention, the PCR efficiency can be increased. For example, the PCR efficiency can be 80% to 120%, preferably 85% to 115%, more preferably 90%. １１０110%.

In the real-time PCR method, a calibration curve obtained by a dilution series of a standard sample is used as an absolute quantification method of an unknown sample. In this case, it must be noted that the PCR efficiencies of the standard sample and the unknown sample must match, and if they do not match, incorrect results may be given.

According to the present invention, the Ct value and the PCR efficiency can be improved even when a long target nucleic acid is amplified. Therefore, for example, a multiplex PCR method that amplifies a plurality of target nucleic acids having different amplification product lengths (for example, a multiplex using both long target nucleic acids of 400 bp or more and short target nucleic acids of less than 200 bp as target nucleic acids) In the PCR method or the like), the variation in amplification efficiency due to the difference in the length of the amplification product can be improved, accurate results can be obtained, and a decrease in detection sensitivity of the target DNA can be suppressed.

In a preferred embodiment, the nucleic acid amplification method, reagent, kit and the like of the present invention are used for amplifying a target nucleic acid having an amplified chain length of 200 bp or more. In the present embodiment, the amplified chain length of the target nucleic acid of interest is not particularly limited as long as it is 200 bp or more, but may be preferably 300 bp or more, more preferably 400 bp or more (eg, 500 bp or more, 600 bp or more). The upper limit of the amplified chain length of the target nucleic acid targeted by the present invention is not particularly limited as long as the effects of the present invention are exhibited, but may be, for example, 1000 bp or less, or may be 800 bp or less, In certain embodiments, it may further be, for example, 500 bp or less.

Hereinafter, the present invention will be described in more detail based on examples. The present invention is not particularly limited to the examples.

Reference Example 1 Evaluation of general real-time PCR reagent

(1) DNA sample

The artificially synthesized DNA described in SEQ ID NO: 1 was used as a DNA sample. The artificially synthesized DNA was serially diluted 1/10 from 10,000,000 copies to 10 copies and used as a template.

Then, primers were designed as in SEQ ID NOs: 2 to 6 so that the lengths of the amplified chain lengths were 200 bp, 300 bp, 400 bp, and 500 bp, and the PCR efficiency was measured by a real-time PCR reaction. SEQ ID Nos. 2 and 3 amplify the amplified chain length of 200 bp, SEQ ID Nos. 2 and 4 amplify the amplified chain length of 300 bp, SEQ ID Nos. 2 and 5 amplify the amplified chain length of 400 bp, and SEQ ID Nos. 2 and 6 amplify the amplified chain length of 500 bp. Respectively corresponding to the forward and reverse primers.

(2) Real-time PCR reaction

In order to evaluate the influence on the Ct value and PCR efficiency due to the difference in the amplified chain length in the real-time PCR reaction, a real-time PCR reaction was performed using the following reagents, and the PCR efficiency was evaluated. Here, the concentration conditions of the primer added to the PCR reaction solution were in accordance with the description of the package insert of each product.

・ PowerUpSYBR Green Master Mix (ThermoFisher SCIENTIFIC)

・ SYBR Premix Ex Taq ^™ II (Tli RNaseH Plus) (TaKaRa)

For the real-time PCR reaction, a CFX96 Touch Deep Well real-time PCR analysis system was used, and the reaction conditions were 40 cycles of initial denaturation at 95 ° C for 30 seconds and a PCR cycle of 95 ° C for 5 seconds to 60 ° C for 30 seconds. Subsequently, melting curve analysis was performed.

(3) Result

The result is shown in FIG. FIG. 1 shows the average Ct value performed twice under each condition and the value of the PCR efficiency (%) obtained by the PCR efficiency E = 10 ^{[−1 / slope]} −1. Here, the smaller the Ct value, the higher the amplification efficiency, and the closer the PCR efficiency value is to 100%, the higher the PCR efficiency. Note that ND is not detected in the drawing. In any of the reagents, the PCR efficiency was high under the condition that the amplification chain length was up to 200 bp, but the Ct value and the PCR efficiency decreased as the amplification chain length increased. From these results, it can be seen that the efficiency of the nucleic acid amplification reaction generally decreases when the amplified chain length is 300 bp or more.

Example 1 Evaluation of 1,2-propanediol

To verify the effect of 1,2-propanediol on the real-time PCR reaction, a real-time PCR reaction was performed without adding 1,2-propanediol or 7% by volume, and the PCR efficiency was evaluated.

・ Preparation of reagent for PCR reaction

The reagent for PCR reaction used in this example was prepared according to the following formulation. The same forward primer and reverse primer as those used in Reference Example 1 were used.

10 mM Tris-HCl (pH 7.5)

40 mM potassium acetate

3 mM magnesium sulfate

0.3 mM dNTPs

0.3μM forward primer

0.3μM reverse primer

0.05 U / μL rTth DNA polymerase (TOYOBO)

0.4 μg anti-Tth antibody (TOYOBO)

1/20000 SYBR Green I (registered trademark)

100 mM tetramethylammonium acetate

The conditions of the real-time PCR reaction were 40 cycles of initial denaturation at 95 ° C. for 30 seconds and PCR cycles of 95 ° C. for 5 seconds to 60 ° C. for 30 seconds. Subsequently, melting curve analysis was performed.

(3) Result

The result is shown in FIG. Under the condition without addition of 1,2-propanediol, amplification was possible when the amplification chain length was 200 bp or 300 bp, and the PCR efficiency was 85% or more. On the other hand, when the amplified chain length was 400 bp or 500 bp, the Ct value was reduced, and it was clear that the amplification efficiency was reduced. Under conditions where 1,2-propanediol was added in an amount of 7% by volume, it was confirmed that the PCR efficiency was improved when the amplified chain length was 200 bp and 300 bp. In addition, even when the amplification chain length was as long as 400 bp or 500 bp, the Ct value and the PCR efficiency did not decrease, and high amplification efficiency could be maintained. That is, even when the amplified chain length is 200 to 300 bp, 1,2-propanediol shows an excellent effect, but it becomes clear that the longer the amplified chain length, the more preferable effects can be obtained.

Example 2 Evaluation of diol compound in nucleic acid amplification

To evaluate the effect of the diol compound on nucleic acid amplification, real-time PCR was performed using various diol compounds, and the nucleic acid amplification sensitivity and PCR efficiency were evaluated. Specifically, as the diol compound, 1,2-propanediol (1,2-propanediol), 1,3-propanediol (1,3-propanediol), 1,2-ethanediol (1,2-ethanediol) , Glycerol and a concentration of 7% by volume each was added to the reaction solution. The primer set of SEQ ID NO: 2 and SEQ ID NO: 6 having an amplified chain length of 500 bp was used, and the other reaction conditions were the same as in Example 1.

The result is shown in FIG. When glycerol was added, an increase in Ct value (a decrease in amplification efficiency) and a decrease in PCR efficiency were observed. When 1,3-propanediol was added, the fluorescence intensity reached tended to decrease as the amount of template decreased, and detection of 100 copies or less was undetectable. On the other hand, when 1,2-ethanediol and / or 1,2-propanediol was used, the Ct value and the PCR efficiency were improved. Surprisingly, similar compounds have different effects, and 1,2-propanediol and 1,2-ethanediol have particularly remarkable improvement effects, especially when amplifying long-chain targets.

Example 3 Evaluation of 1,2-propanediol concentration in nucleic acid amplification

To evaluate the effect of 1,2-propanediol concentration on nucleic acid amplification, real-time PCR was performed under various conditions in which 1,2-propanediol was added at various concentrations, and nucleic acid amplification sensitivity and PCR efficiency were evaluated. . The primer set of SEQ ID NO: 2 and SEQ ID NO: 6 having an amplified chain length of 500 bp was used, and the other reaction conditions were the same as in Example 1.

FIG. 4 shows the results. When 1,2-propanediol was added in an amount of 3% by volume or more, a remarkable effect of improving the Ct value and the PCR efficiency could be obtained.

Example 4 Evaluation of tetramethylammonium acetate in nucleic acid amplification

In order to evaluate the effect of tetramethylammonium salt on nucleic acid amplification, real-time PCR reaction was performed under the conditions of tetramethylammonium acetate (TMAA) no addition or 50, 100 mM TMAA addition, and Ct value and PCR efficiency were evaluated. The tetramethylammonium acetate concentration was changed to 0, 50, and 100 mM under the condition that 7% by volume of 1,2-propanediol was added, and the condition that neither 1,2-propanediol nor tetramethylammonium acetate was contained. Under these conditions, a primer set of SEQ ID NO: 2 and SEQ ID NO: 6 having an amplified chain length of 500 bp was used, and the other reaction conditions were the same as in Example 1. .

The result is shown in FIG. When neither 1,2-propanediol nor tetramethylammonium acetate was contained, the result was that the nucleic acid amplification reaction did not proceed under the test conditions. When a nucleic acid amplification reaction is carried out under a condition containing 1,2-propanediol at 7% by volume, the absence of tetramethylammonium acetate allows the PCR reaction to proceed when the amount of template is large, but results in low PCR efficiency. became. On the other hand, under the condition where the amount of tetramethylammonium acetate was added at 50 mM, the effect of improving the Ct value was observed, and under the condition where the amount of tetramethylammonium acetate was added at 100 mM, the PCR efficiency was improved in addition to the improvement of the Ct value. It became clear. From these results, although 1,2-propanediol alone has the effect of improving the efficiency of the nucleic acid amplification reaction, more favorable results can be obtained by using tetramethylammonium acetate in combination with 1,2-propanediol. You can see that it can be done. The composition for nucleic acid amplification reaction containing tetramethylammonium acetate and 1,2-propanediol prepared in this example is stable even when stored at room temperature for a long period (3 months). Could be used as well.

The embodiments and examples disclosed above are all illustrative and not restrictive. Embodiments and examples in which the contents disclosed in the embodiments and examples are combined are also included in the scope of the present invention. The technical scope of the present invention is valid according to the appended claims, and includes all changes, modifications, replacements, and the like within the meaning and scope equivalent to the description of the appended claims.

The present invention can enhance the amplification efficiency and detection sensitivity of a nucleic acid amplification reaction even when, for example, amplifying a long target nucleic acid having a length of 200 bp or more. The nucleic acid can be amplified. Therefore, it is useful not only in the field of life science research, but also in the medical field such as genetic diagnosis and clinical testing, and in microbiological testing of foods and the environment.

Claims

A method for amplifying a target nucleic acid having an amplification chain length of 200 bp or more, wherein the nucleic acid is contained in a reaction solution containing at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol A nucleic acid amplification method comprising performing an amplification reaction.
The method according to claim 1, wherein the reaction solution further comprises a tetramethylammonium salt.
The method according to claim 1 or 2, wherein the tetramethylammonium salt is at least one selected from the group consisting of tetramethylammonium acetate, tetramethylammonium chloride, and tetramethylammonium hydroxide.
The method according to any one of claims 1 to 3, wherein the amplified chain length is 200 bp or more and 1000 bp or less.
The method according to any one of claims 1 to 4, wherein the amplified chain length is 400 bp or more and 500 bp or less.
The method according to any one of claims 1 to 5, wherein the nucleic acid amplification reaction is real-time PCR.
The method according to any one of claims 1 to 6, wherein the extension time in the nucleic acid amplification reaction is 60 seconds or less.
The method according to any one of claims 1 to 7, wherein the elongation time in the nucleic acid amplification reaction is 30 seconds or less.
The method according to any one of claims 1 to 8, wherein the reaction solution further comprises a DNA polymerase derived from Thermus thermophilus.
A composition for a nucleic acid amplification reaction, comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol, and a tetramethylammonium salt.
11. The composition for a nucleic acid amplification reaction according to claim 10, wherein the concentration of the diol compound in the reaction solution at the time of the nucleic acid amplification reaction is adjusted to 1 to 20% by volume based on the entire reaction solution.
12. The nucleic acid amplification reaction composition according to claim 10, wherein the concentration of the diol compound in the reaction solution at the time of the nucleic acid amplification reaction is adjusted to 3 to 10% by volume based on the entire reaction solution.
13. The nucleic acid amplification reaction composition according to claim 10, wherein the tetramethylammonium salt is at least one selected from the group consisting of tetramethylammonium acetate, tetramethylammonium chloride, and tetramethylammonium hydroxide. Stuff.
14. The nucleic acid amplification reaction according to claim 10, wherein the concentration of the tetramethylammonium salt in the reaction solution at the time of the nucleic acid amplification reaction is adjusted to 50 to 200 mM with respect to the entire reaction solution. Composition.
15. The composition for a nucleic acid amplification reaction according to claim 10, further comprising a DNA polymerase derived from Thermus thermophilus.
The composition for a nucleic acid amplification reaction according to any one of claims 10 to 15, wherein the nucleic acid amplification reaction is a real-time PCR reaction.
The nucleic acid amplification reaction composition according to any one of claims 10 to 16, which is used for amplifying a target nucleic acid having an amplification chain length of 200 bp or more.
A reagent for amplifying a target nucleic acid having an amplification chain length of 200 bp or more, comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol.
A kit for amplifying a target nucleic acid having an amplification chain length of 200 bp or more, comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol.
A nucleic acid amplification reaction kit comprising at least one diol compound selected from the group consisting of 1,2-propanediol and 1,2-ethanediol, and a tetramethylammonium salt.
The nucleic acid amplification reaction kit according to claim 20, which is used for amplifying a target nucleic acid having an amplification chain length of 200 bp or more.