JP2018023360A - Nucleic acid amplification reaction method, reagent, and method for using reagent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid amplification reaction method which can suppress nonspecific nucleic acid amplification.SOLUTION: The nucleic acid amplification reaction method comprises: a step of heating a reaction solution containing reverse transcriptase, a polymerase, a primer and a probe to carry out a reverse transcription reaction; and providing a heat cycle step after the heating step to amplify the nucleic acid in the reaction solution. The Tm value of the primer is 65°C to 80°C and the duration of heating in the heating step is 5 seconds to 480 seconds.SELECTED DRAWING: Figure 8

Description

  The present invention relates to a nucleic acid amplification reaction method, a reagent, and a method for using the reagent.

  In recent years, gene-based medical care such as gene diagnosis and gene therapy has attracted attention due to the development of gene utilization technology, and many methods using genes for variety discrimination and variety improvement have been developed in the field of agriculture and livestock. . As a technique for utilizing a gene, a technique such as a PCR (Polymerase Chain Reaction) method is widely used. Today, PCR has become an indispensable technique for elucidating information on biological materials.

  The PCR method is a method for amplifying a target nucleic acid by subjecting a solution (reaction solution) containing a nucleic acid to be amplified (target nucleic acid) and a reagent to thermal cycling. The thermal cycle is a process in which two or more temperatures are periodically applied to the reaction solution. In the PCR method, a method of applying a two-stage or three-stage thermal cycle is common.

  The speeding up of PCR is a technique necessary for shortening the testing time of genetic testing, and is highly expected in the genetic testing industry.

  For example, Patent Document 1 describes a method in which a polymerase is provided at a concentration of at least 0.5 μM and a primer is at least 2 μM, and is completed in a cycle time of less than 20 seconds per cycle.

Special table 2015-520614

  On the other hand, there are two main types of nucleic acids, DNA (Deoxyribo Nucleic Acid) and RNA (Ribo Nucleic Acid). When the template nucleic acid is RNA, RNA is reverse-transcribed into DNA by reverse transcription (Reverse Transcription) before PCR.

  It is possible to use the same primer in the heating step for the reverse transcription reaction as described above and the thermal cycle applying step for applying a thermal cycle for nucleic acid amplification. As a result of intensive studies, the inventor has found that the conditions for suppressing nonspecific amplification in the reverse transcription reaction change when a desired primer is selected in order to speed up the thermal cycle.

  One of the objects according to some embodiments of the present invention is to provide a nucleic acid amplification reaction method capable of suppressing amplification of non-specific nucleic acid. Another object of some embodiments of the present invention is to provide a reagent capable of suppressing amplification of non-specific nucleic acid and a method for using the reagent.

The nucleic acid amplification reaction method according to the present invention comprises:
Heating a reaction solution containing a reverse transcriptase, a polymerase, a primer, and a probe to perform a reverse transcription reaction;
After the heating step, applying a thermal cycle for amplifying nucleic acid to the reaction solution;
Including
The Tm value of the primer is 65 ° C. or more and 80 ° C. or less,
In the heating step,
The time for heating the reaction solution is 5 seconds or more and 480 seconds or less.

  Such a nucleic acid amplification reaction method is a nucleic acid amplification reaction method capable of performing reverse transcription reaction and speeding up the thermal cycle, and can suppress amplification of non-specific nucleic acid (for details) (See “3. Experimental example” below.)

In the nucleic acid amplification reaction method according to the present invention,
In the heating step,
The reaction solution may be heated to 50 ° C. or higher and 70 ° C. or lower.

  In such a nucleic acid amplification reaction method, amplification of non-specific nucleic acid can be suppressed.

In the nucleic acid amplification reaction method according to the present invention,
In the heating step,
The time for heating the reaction solution may be 10 seconds or more and 60 seconds or less.

  In such a nucleic acid amplification reaction method, the fluorescence intensity can be increased in the fluorescence intensity measurement (for details, refer to “3. Experimental example” described later).

In the nucleic acid amplification reaction method according to the present invention,
The amount of the reverse transcriptase contained in the reaction solution may be 30 units or more and 60 units or less.

  In such a nucleic acid amplification reaction method, the fluorescence intensity can be increased in the fluorescence intensity measurement (for details, refer to “3. Experimental example” described later), and the cost can be reduced.

In the nucleic acid amplification reaction method according to the present invention,
In the heating step,
The reaction solution is heated to 60 ° C. or higher and 70 ° C. or lower,
The time for heating the reaction solution may be 10 seconds or more and 30 seconds or less.

  In such a nucleic acid amplification reaction method, the time for the reverse transcription reaction can be shortened while ensuring the fluorescence intensity (for details, refer to “3. Experimental Example” described later).

In the nucleic acid amplification reaction method according to the present invention,
In the heating step,
The reaction solution is heated to 66 ° C. or higher and 70 ° C. or lower,
The reaction solution may be heated for 60 seconds or less.

  In such a nucleic acid amplification reaction method, the fluorescence intensity can be further increased in the fluorescence intensity measurement (for details, refer to “3. Experimental example” described later).

In the nucleic acid amplification reaction method according to the present invention,
In the step of applying the thermal cycle,
The time per one cycle of the thermal cycle may be 9 seconds or less.

  In such a nucleic acid amplification reaction method, the heat cycle can be accelerated.

In the nucleic acid amplification reaction method according to the present invention,
The heating time for the primer annealing reaction may be 6 seconds or less.

  In such a nucleic acid amplification reaction method, the heat cycle can be accelerated.

In the nucleic acid amplification reaction method according to the present invention,
The reaction solution contains a divalent cation,
The concentration of the divalent cation contained in the reaction solution may be 2 mM or more and 7.5 mM or less.

  In such a nucleic acid amplification reaction method, it is possible to suppress the nonspecific amplification and to reduce the yield of the specific amplification product while accelerating the extension reaction by the polymerase and speeding up the PCR.

In the nucleic acid amplification reaction method according to the present invention,
The reaction solution includes MgCl ₂ ;
The divalent cation is derived from MgCl ₂ .
The concentration of MgCl ₂ contained in the reaction solution may be 4 mM or more and 7.5 mM or less.

In such a nucleic acid amplification reaction method, while the elongation reaction by polymerase is accelerated and the thermal cycle is accelerated, the amount of Mg ²⁺ is too much and the yield of specific amplification products is reduced due to the increase of non-specific amplification. Can be suppressed.

In the nucleic acid amplification reaction method according to the present invention,
The reaction solution includes MgSO ₄ ,
The divalent cation is derived from MgSO ₄ ;
The concentration of MgSO ₄ contained in the reaction solution may be 2 mM or more and 3 mM or less.

In such a nucleic acid amplification reaction method, while the elongation reaction by polymerase is accelerated and the thermal cycle is accelerated, the amount of Mg ²⁺ is too much and the yield of specific amplification products is reduced due to the increase of non-specific amplification. Can be suppressed.

  In such a nucleic acid amplification method, the extension reaction is accelerated by the type of divalent cation, the PCR is speeded up, non-specific amplification is suppressed, and the yield of a specific amplification product is reduced. The optimum concentration range is different.

In the nucleic acid amplification reaction method according to the present invention,
The probe may be a hydrolysis probe.

  In such a reagent, when the probe is decomposed by the polymerase, the quenching effect is canceled and the reporter dye emits light, whereby the amount of nucleic acid amplified can be quantified.

In the nucleic acid amplification reaction method according to the present invention,
The probe may include at least one of an artificial nucleic acid and a minor group binder molecule.

  With such a reagent, it is possible to increase the Tm value of the probe while suppressing an increase in the number of bases of the probe.

In the nucleic acid amplification reaction method according to the present invention,
The reverse transcriptase may be derived from mouse Moloney murine Leukemia virus.

  In such a nucleic acid amplification reaction method, amplification of non-specific nucleic acid can be suppressed.

In the nucleic acid amplification reaction method according to the present invention,
The primer may include an artificial nucleic acid.

  In such a nucleic acid amplification reaction method, amplification of non-specific nucleic acid can be suppressed (for details, refer to “3. Experimental example” described later).

In the nucleic acid amplification reaction method according to the present invention,
The primer may be a sequence-specific primer for the target RNA.

  In such a nucleic acid amplification reaction method, since the primer anneals only to a specific base sequence, amplification of non-specific nucleic acid can be suppressed.

The reagent according to the present invention is:
A reagent for performing a reverse transcription reaction and a nucleic acid amplification reaction,
A reverse transcriptase, a polymerase, a primer, a probe, and MgCl ₂ ;
The Tm value of the primer is 65 ° C. or more and 80 ° C. or less,
When the reagent becomes a reaction solution for performing reverse transcription reaction and nucleic acid amplification reaction, the concentration of MgCl ₂ contained in the reaction solution is 4 mM or more and 7.5 mM or less.

  Such a reagent can be used in a nucleic acid amplification reaction method capable of performing a reverse transcription reaction and speeding up the thermal cycle, and can suppress amplification of a non-specific nucleic acid.

The reagent according to the present invention is:
A reagent for performing a reverse transcription reaction and a nucleic acid amplification reaction,
A reverse transcriptase, a polymerase, a primer, a probe, and MgSO ₄ ;
The Tm value of the primer is 65 ° C. or more and 80 ° C. or less,
When the reagent becomes a reaction solution for performing reverse transcription reaction and nucleic acid amplification reaction, the concentration of MgSO ₄ contained in the reaction solution is 2 mM or more and 3 mM or less.

  Such a reagent can be used in a nucleic acid amplification reaction method capable of performing a reverse transcription reaction and speeding up the thermal cycle, and can suppress amplification of a non-specific nucleic acid.

The method of using the reagent according to the present invention includes:
A method of using the reagent according to the present invention, comprising:
The reagent is brought into contact with a template nucleic acid solution containing a template nucleic acid to prepare the reaction solution. After the reaction solution is heated for 5 seconds or more and 480 seconds or less to perform a reverse transcription reaction, the reaction solution is heated. A nucleic acid amplification reaction is performed by applying a cycle.

  Such a reagent can be used in a nucleic acid amplification reaction method capable of performing a reverse transcription reaction and speeding up the thermal cycle, and can suppress amplification of a non-specific nucleic acid.

The graph which shows the relationship between the temperature and relative activity efficiency in Taq polymerase. The flowchart for demonstrating the nucleic acid amplification reaction method which concerns on this embodiment. Sectional drawing which shows typically the thermal cycle apparatus for providing a thermal cycle to the reaction solution which concerns on this embodiment. The graph which shows the relationship between the heating time for reverse transcription reaction, and fluorescence intensity. The graph which shows the relationship between the heating time for reverse transcription reaction, and fluorescence intensity. The graph which shows the relationship between the heating time for reverse transcription reaction, and relative fluorescence intensity. The graph which shows the relationship between the heating time for reverse transcription reaction, and relative fluorescence intensity. Results of electrophoresis. The graph which shows the relationship between the heating time for reverse transcription reaction, and fluorescence intensity. The graph which shows the relationship between the heating time for reverse transcription reaction, and fluorescence intensity. The graph which shows the relationship between the quantity of reverse transcriptase, and fluorescence intensity. The graph which shows the relationship between the heating time for reverse transcription reaction, and fluorescence intensity. The graph which shows the relationship between the heating time for reverse transcription reaction, and fluorescence intensity. The graph which shows the relationship between the heating time for reverse transcription reaction, and fluorescence intensity. The graph which shows the relationship between the reaction time of PCR, and fluorescence intensity. The graph which shows the relationship between the reaction time of PCR, and fluorescence intensity. The graph which shows the relationship between reverse transcription reaction time and fluorescence intensity. Results of electrophoresis. Results of electrophoresis.

  DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. The embodiments described below do not unduly limit the contents of the present invention described in the claims. In addition, not all of the configurations described below are essential constituent requirements of the present invention.

1. Reagent First, the reagent according to the present embodiment will be described. The reagent according to this embodiment is a reagent for performing a reverse transcription reaction and a nucleic acid amplification reaction. For example, the reagent may be in a liquid state or in a freeze-dried state. For example, the freeze-dried reagent is fixed in a container (not shown), and a template nucleic acid solution containing RNA is introduced into the container to bring the template nucleic acid solution into contact with the reagent. The reagent is dissolved in freeze-dried by the moisture of the template nucleic acid solution and taken into the template nucleic acid solution to become a reaction solution. Accordingly, the reaction solution contains the template nucleic acid and the reagent, and serves as a place for proceeding with the nucleic acid amplification reaction.

  The reagent includes a primer, a polymerase, a probe, dNTP, a buffer, and a reverse transcriptase. The reagent according to the present embodiment is, for example, a reagent for one-step type reverse transcription PCR in which reverse transcription reaction and nucleic acid amplification reaction (PCR) are continuously performed.

1.1. Primers Primers are designed to anneal to a template nucleic acid (template). Annealing means that a primer binds to DNA. The reagent is a forward primer (Forward) that anneals to one single-stranded template nucleic acid (single-stranded DNA) after the double-stranded template nucleic acid (double-stranded DNA) is denatured as a primer.
Primer) and a reverse primer (Reverse Primer) that anneals to the other single-stranded DNA. The concentration of the forward primer and the reverse primer contained in the reaction solution is, for example, 0.4 μM or more and 6.4 μM or less, preferably 0.8 μM or more and 3.2 μM or less. The concentration of the forward primer and the concentration of the reverse primer contained in the reaction solution may be the same or different.

  The Tm value of the primer (forward primer and reverse primer) is from 65 ° C. to 80 ° C., preferably from 70 ° C. to 75 ° C. Thereby, the reagent according to the present embodiment can increase the speed (shortening of time) of the thermal cycle (refer to “3. Experimental example” described later for details). The Tm value is an index of the temperature at which the primer anneals to the template nucleic acid, and is the temperature at which 50% of the double-stranded DNA is dissociated into single-stranded DNA, that is, the melting temperature (Melting Temperature). If the temperature is equal to or higher than the Tm value, more than half of the primers anneal to the template nucleic acid. The Tm value of the forward primer and the Tm value of the reverse primer may be the same or different.

  As a calculation method of the Tm value, for example, the Nearest Neighbor method can be cited, and can be calculated by the following formula (1).

Tm = 1000ΔH / (− 10.8 + ΔS + R × ln (Ct / 4)) − 273.15 + 16.6 log [Na ⁺ ] (1)

In Equation (1), ΔH is the sum of nearest enthalpy changes in the hybrid (kcal / mol), ΔS is the sum of nearest neighbor entropy changes in the hybrid (cal / mol / K), and R is , Gas constant (1.987 cal / deg / mol), Ct is the molar concentration of the primer (mol / L), and Na ⁺ is the concentration of monovalent cation contained in the buffer (mol / L). It is.

  When increasing the Tm value of a primer by increasing the number of bases in the primer, the primer is designed to extend inside the amplification region (the primer is located at the 5 ′ end of the template nucleic acid). The Tm value can be increased without increasing the amplification region. As a result, the heat cycle can be speeded up.

  The primer may include an artificial nucleic acid. Thereby, amplification of a non-specific nucleic acid can be suppressed. The artificial nucleic acid will be described later in “1.4. Probe”.

  The primer may be a primer that is sequence specific to the target RNA (RNA that the primer anneals). That is, the primer may be a primer that anneals only to a specific base sequence of the target RNA. Thereby, since a primer anneals only to a specific base sequence, amplification of a non-specific nucleic acid can be suppressed. A primer specific to the target RNA is effective when the sequence of the target RNA is known.

1.2. Polymerase Polymerase is not particularly limited, and includes DNA polymerase. The DNA polymerase polymerizes a nucleotide complementary to the base of the template nucleic acid at the end of the primer annealed to the template nucleic acid having a single-stranded structure (single-stranded DNA). As the DNA polymerase, a heat-resistant enzyme or a PCR enzyme is preferable. For example, there are a large number of commercially available products such as Taq polymerase, KOD polymerase, Tfi polymerase, Tth polymerase, or improved versions thereof. A DNA polymerase that can be used is preferred. The polymerase includes a hydrolyzed form that degrades the probe by hydrolysis, such as Taq polymerase, and a non-hydrolyzed form that does not degrade the probe by hydrolysis, such as KOD polymerase. KOD is Thermococcus kodakarensis KOD1 and is a DNA polymerase of the genus Thermococcus. The amount of polymerase contained in the reaction solution is, for example, 0.5 units (U) or more.

  Here, FIG. 1 is a graph showing the relationship between temperature and relative activity efficiency in Taq polymerase. The vertical axis represents the relative activity efficiency when the value at which the polymerase activity efficiency becomes maximum when the temperature is changed is 100%. The activity efficiency of the polymerase at each temperature is obtained by devising dNTP so that it emits light when dNTP is incorporated into the extension reaction, and measuring the fluorescence intensity (fluorescence luminance) by dNTP after a certain period of time. be able to. The higher the fluorescence intensity, the higher the activity efficiency of the polymerase. In the example shown in FIG. 1, Taq polymerase has the maximum relative activity efficiency at around 70 ° C.

1.3. dNTP
dNTP represents a mixture of four types of deoxyribonucleotide triphosphates. That is, dNTP represents a mixture of dATP, dCTP, dGTP, and dTTP. DNA polymerase joins dATP, dCTP, dGTP, and dTTP to the ends of the annealed primers to form new DNA (extension reaction). The concentration of dNTP contained in the reaction solution is, for example, 0.06 mM or more and 0.75 mM or less, preferably 0.125 mM or more and 0.5 mM or less.

1.4. Probe The probe is a fluorescently labeled probe used for quantifying the amount of nucleic acid amplified. The concentration of the probe contained in the reaction solution is 0.5 μM or more and 2.4 μM or less, preferably 0.5 μM or more and 1.8 μM or less.

  The probe is, for example, a hydrolysis probe containing a reporter dye and a quencher dye. More specifically, the probe is a TaqMan® probe. While the hydrolysis probe is hybridizing to single-stranded DNA to form a double-stranded structure, the reporter dye is emitted by the quencher dye in close proximity to the reporter dye (by the quenching effect). Is suppressed. However, when the probe is degraded by the exonuclease activity by polymerase, the quenching effect is eliminated and the reporter dye emits light. By this luminescence, the amount of nucleic acid amplification can be quantified. Hybridization means that a probe binds to DNA. When a hydrolysis probe is used as the probe, Taq polymerase is used as the polymerase.

  The probe may be a non-hydrolyzed probe other than the hydrolyzed probe. Specifically, the probe may be a Q (Quenching) probe using a fluorescence quenching phenomenon. The Q probe emits light in a state where it is not hybridized to single-stranded DNA, and is quenched when hybridized to single-stranded DNA. The amount of nucleic acid amplification can be quantified based on the difference in emission intensity. When a Q probe is used as the probe, KOD polymerase is used as the polymerase. KOD polymerase has a higher rate of extension reaction than Taq polymerase, and can speed up thermal cycling.

When the probe is a non-hydrolyzed probe, it is not necessary to decompose the probe by an extension reaction, so that it is not necessary to provide an amplification region where the probe anneals inside the forward primer and the reverse primer. This makes it possible to design the amplification region to be narrower than the hydrolysis type system. By narrowing the amplification region, the annealing time can be shortened and the heat cycle can be speeded up.

  The probe may comprise at least one of an artificial nucleic acid and a minor group binder (MGB) molecule. Thereby, Tm value of a probe can be made high, suppressing that the number of bases of a probe increases (suppressing that base length becomes long). When the number of bases of the probe increases, for example, the time for decomposing the probe becomes long, and it may be difficult to increase the speed of PCR.

  An artificial nucleic acid refers to a nucleic acid molecule other than a natural nucleic acid molecule that can bind to a base of DNA or RNA by hydrogen bonding. Examples of the artificial nucleic acid include 2 ′, 4′-BNA (2′-O, 4′-C-methano) in which a 2′-position oxygen atom and a 4′-position carbon atom of a ribose ring of a nucleic acid are bridged with methylene. -Bridged nucleic acid (also known as LNA (Locked Nucleic Acid)). The chemical formula of LNA is shown in the following formula (2).

    In Formula (2), examples of Base (base) include T (thymine), C (cytosine), G (guanine), and A (adenine), but are not particularly limited. The base may be a base modified by methylation or acetylation.

The artificial nucleic acid may be an LNA modified LNA analog, such as 3′amino-2 ′, 4′-BNA, 2 ′, 4′-BNA ^COC , or 2 ′, 4′-BNA. ^NC (N-Me) may be used. The artificial nucleic acid contained in the modified fluorescent probe may be PNA (Peptide Nucleic Acid), GNA (Glycol Nucleic Acid), TNA (Threose Nucleic Acid), or an analog obtained by modifying these. The number of artificial nucleic acids contained in the probe is not particularly limited, and one probe may contain a plurality of artificial nucleic acids.

1.5. Buffer The buffer is, for example, a buffer containing a salt. Examples of the salt contained in the buffer include salts of Tris, Hepes, Pipes, phosphoric acid and the like. With these salts, the pH of the buffer can be adjusted.

The buffer contains a divalent cation. Examples of the divalent cation include Mn, Co, and Mg. When the nucleic acid amplification reaction reagent becomes a reaction solution for performing a nucleic acid amplification reaction, the concentration of the divalent cation contained in the reaction solution is 2 mM or more and 7.5 mM or less. By setting the concentration of the divalent cation to 2 mM or more, it is possible to accelerate the elongation reaction by polymerase and speed up the PCR (specifically, the time per one thermal cycle is 9 seconds or less). be able to). By setting the concentration of the divalent cation to 7.5 mM or less, nonspecific amplification can be suppressed, and a decrease in the yield of a specific amplification product can be suppressed.

Specifically, the buffer includes a divalent cationic compound, KCL, and Tris. More specifically, the buffer includes MgCl ₂ and the divalent cation is derived from MgCl ₂ . That is, the divalent cation is generated by ionizing MgCl ₂ . If divalent cations are derived from MgCl _2, the concentration of ^{Mg 2+} it is due to the activity of the polymerase. When the nucleic acid amplification reaction reagent becomes a reaction solution for performing a nucleic acid amplification reaction, the concentration of MgCl ₂ contained in the reaction solution is 4 mM or more and 7.5 mM or less, preferably 5 mM or more and 7 mM or less. Preferably it is 5 mM. By setting the MgCl ₂ concentration to 4 mM or more, the elongation reaction by the polymerase can be accelerated, and the speed of PCR can be increased. By setting the MgCl ₂ concentration to 7.5 mM or less, nonspecific amplification can be suppressed, and a decrease in the yield of a specific amplification product can be suppressed. When the nucleic acid amplification reaction reagent is lyophilized, the nucleic acid amplification reaction reagent is in a solid state and contains MgCl ₂ , KCl, Tris, an excipient such as trehalose.

The divalent cation compound may be derived from MgSO ₄ . In this case, the buffer contains MgSO ₄ instead of MgCl ₂ , and when the nucleic acid amplification reaction reagent is a reaction solution for performing a nucleic acid amplification reaction, the concentration of MgSO ₄ contained in the reaction solution is 2 mM or more. 3 mM or less, more preferably 2 mM. By setting the concentration of MgSO ₄ to 2 mM or more, the elongation reaction by the polymerase can be accelerated and the speed of PCR can be increased. By setting the concentration of MgSO ₄ to 3 mM or less, nonspecific amplification can be suppressed, and a decrease in the yield of a specific amplification product can be suppressed.

1.6. Reverse transcriptase Reverse transcriptase is an enzyme that synthesizes DNA using an RNA base sequence as a template. The reverse transcription reaction is a reaction in which DNA is synthesized by reverse transcriptase using the base sequence of RNA as a template. In the reverse transcription reaction, complementary DNA can be synthesized by annealing one of the forward primer and reverse primer used in PCR to RNA.

  Examples of the reverse transcriptase include Avian Myeloblast Virus, Ras Associated Virus Type 2 (Ras Associated Virus Type 2), Mouse Moloney Murine Leukemia Virus (Mouse Molly Murine Leukemia Immunodeficiency) Reverse transcriptase derived from virus type 1 (Human Immunodefective Virus type 1) is used.

  The amount of reverse transcriptase contained in the reaction solution is, for example, 5 units or more and 60 units or less, and preferably 30 units or more and 60 units or less. By setting the amount of the reverse transcriptase contained in the reaction solution to 5 units or more, it is possible to prevent the reverse transcription reaction from being performed sufficiently because the amount of the reverse transcriptase is too small. By setting the amount of reverse transcriptase contained in the reaction solution to 60 units or less, the amount of expensive reverse transcriptase can be reduced, so that the cost can be reduced.

1.7. Other Components When the reagent is lyophilized, the reagent (lyophilized reagent) contains a sugar. Examples of the sugar include non-reducing sugars such as sucrose, trehalose, raffinose, and melezitose among disaccharides and trisaccharides. Among disaccharides and trisaccharides, trehalose is particularly preferably used because of its high function as a cryoprotectant. Trehalose can prevent the freeze-dried reagent from coming into contact with water molecules due to its strong hydration power, and can improve the storage stability of the freeze-dried reagent. The lyophilized reagent can be prepared by lyophilizing a mixed reagent solution containing each component of the reagent and a sugar. The temperature at the time of freeze-drying is, for example, about −80 ° C.

1.8. Method of Use In the method of using a reagent according to the present embodiment, a reaction solution is prepared by bringing a reagent into contact with a template nucleic acid solution containing a template nucleic acid, and the reaction solution is heated for 5 seconds to 480 seconds to reverse transcription reaction. After performing, nucleic acid amplification reaction is performed by applying a thermal cycle to the reaction solution.

  The reagent according to the present embodiment has the following features, for example.

The reagent includes reverse transcriptase, polymerase, primer, probe, and MgCl ₂ , and the primer has a Tm value of 65 ° C. or higher and 80 ° C. or lower, and the reagent performs reverse transcription reaction and nucleic acid amplification reaction. Therefore, the concentration of MgCl ₂ contained in the reaction solution is 4 mM or more and 7.5 mM or less. The reagent includes reverse transcriptase, polymerase, primer, probe, and MgSO _{4. The} primer has a Tm value of 65 ° C. or higher and 80 ° C. or lower, and the reagent is reverse transcription reaction and nucleic acid amplification reaction. The concentration of MgSO ₄ contained in the reaction solution is 2 mM or more and 3 mM or less. Therefore, the reagent can be used in a nucleic acid amplification reaction method that can perform a reverse transcription reaction and increase the heat cycle speed, and amplifies non-specific nucleic acid (with primers in regions other than the target region). (Amplification of nucleic acid by annealing) can be suppressed (for details, see “3. Experimental Examples” described later).

  In the reagent, the probe may be a hydrolysis probe. Therefore, when the probe is decomposed by the polymerase in PCR, the quenching effect is canceled and the reporter dye emits light, whereby the amount of nucleic acid amplified can be quantified.

  In the reagent, the probe may include at least one of an artificial nucleic acid and an MGB molecule. Therefore, the reagent can increase the Tm value of the probe while suppressing an increase in the number of bases of the probe.

2. Nucleic Acid Amplification Reaction Method Next, a nucleic acid amplification reaction method according to this embodiment will be described with reference to the drawings. FIG. 2 is a flowchart for explaining the nucleic acid amplification reaction method according to this embodiment.

  First, the reagent according to the present embodiment is brought into contact with the template nucleic acid solution to prepare a reaction solution (step S1). Specifically, a template nucleic acid solution is introduced into a container containing a reagent using a pipette or the like, and the reagent and the template nucleic acid solution are brought into contact with each other to prepare a reaction solution. The reaction solution includes, for example, a template nucleic acid, a primer, a probe, a polymerase, dNTP, a buffer, and a reverse transcriptase.

  The template nucleic acid solution is obtained, for example, as follows. That is, specimens such as cells or viruses derived from organisms such as humans and bacteria are collected using a collection tool such as a cotton swab, and a template nucleic acid is extracted from the specimen using a known extraction technique. Thereafter, the template nucleic acid solution is purified to a predetermined concentration using a known purification method. The solution in the template nucleic acid solution is, for example, water (distilled water, sterilized water) or a Tris-EDTA (ethylenediamine acetetic acid) solution (TE).

Next, the reaction solution is heated to perform a reverse transcription reaction (step S2). In this step, the reaction solution is heated to, for example, 50 ° C. or higher and 70 ° C. or lower, preferably 60 ° C. or higher and 70 ° C. or lower, more preferably 66 ° C. or higher and 70 ° C. or lower. By heating the reaction solution to 50 ° C. or more and 70 ° C. or less, the fluorescence intensity can be increased in the fluorescence intensity measurement after the nucleic acid is amplified (after the nucleic acid amplification reaction). The method of heating the reaction solution is not particularly limited. For example, a beaker (water tank) is placed on a heat block or a hot plate, the liquid (saline solution or oil) in the beaker is warmed, and the reaction solution is placed in the liquid. The method etc. which heat a reaction solution by arrange | positioning the container in which this was accommodated are mentioned.

  In the step of heating to perform the reverse transcription reaction (step S2), the time for heating the reaction solution is 5 seconds or more and 480 seconds or less, preferably 10 seconds or more and 60 seconds or less, more preferably 10 seconds or more. 30 seconds or less. By setting the time for heating the reaction solution to 5 seconds or more and 480 seconds or less, the fluorescence intensity can be increased in the fluorescence intensity measurement after the nucleic acid is amplified (for details, refer to “3. Experimental example” described later). It is possible to detect nucleic acid amplification with high sensitivity. In this step, the “time for heating the reaction solution” is a time that does not include a temperature raising time for achieving a desired temperature for performing the reverse transcription reaction.

  Next, a thermal cycle (for PCR) for amplifying the nucleic acid is applied to the reaction solution (step S3). Here, FIG. 3 is a cross-sectional view schematically showing a thermal cycle apparatus 100 for applying a thermal cycle to the reaction solution 6 according to the present embodiment.

  As shown in FIG. 3, the heat cycle apparatus 100 includes a first hot plate 10, a second hot plate 12, a first beaker 20, a second beaker 22, an arm 30, and a fixing portion 32. .

  The first hot plate 10 heats the liquid 2 contained in the first beaker 20 to the first temperature. The first temperature is a temperature suitable for dissociation (denaturation reaction) of double-stranded DNA, and is, for example, 85 ° C. or higher and 105 ° C. or lower. The liquid 2 is not particularly limited as long as it can be heated to the first temperature by the first hot plate 10, and examples thereof include saline and oil.

  The second hot plate 12 heats the liquid 4 contained in the second beaker 22 to the second temperature. The second temperature is a temperature lower than the first temperature. The second temperature is a temperature suitable for the annealing reaction and the extension reaction, and is, for example, 55 ° C. or higher and 75 ° C. or lower. That is, in this step, an extension reaction is performed in heating for the primer annealing reaction. That is, the annealing reaction and the extension reaction are performed at the same temperature. From FIG. 1 described above, it can be said that the second temperature is optimally about 70 ° C. from the viewpoint of the activity efficiency of the polymerase. The type of the liquid 4 is not particularly limited as long as it can be heated to the second temperature by the second hot plate 12, and examples thereof include saline and oil.

  One end 30a of the arm 30 is fixed by a fixing portion 32, and the other end 30b is a free end. The other end 30b of the arm 30 supports the container 8 in which the reaction solution 6 is accommodated. The arm 30 is reciprocated in a circular arc shape at the other end 30b with one end 30a fixed by a motor (not shown).

  By the reciprocation of the arm 30, the reaction solution 6 is alternately arranged in the liquid 2 heated to the first temperature and in the liquid 4 heated to the second temperature. Thereby, the thermal cycle for PCR can be provided to the reaction solution 6. The number of thermal cycles in this step can be appropriately set by driving and stopping the motor, and is, for example, 20 cycles or more and 60 cycles or less. The moving time of the reaction solution 6 from the liquid 2 to the liquid 4 and the moving time of the reaction solution 6 from the liquid 4 to the liquid 2 are, for example, about 0.5 seconds.

  In the step of applying a thermal cycle (step S3), the heating time for the denaturation reaction per cycle (in the illustrated example, the time during which the reaction solution 6 is placed in the liquid 2) is, for example, 0.3. It is not less than 5 seconds and not more than 5 seconds, preferably not less than 0.5 seconds and not more than 2 seconds. By setting the heating time for the modification reaction to 0.3 seconds or longer, it is possible to prevent the modification reaction from being performed sufficiently because the modification reaction time is too short. By setting the heating time for the modification reaction to 5 seconds or less, the heat cycle can be speeded up.

  In the step of applying the thermal cycle (step S3), the heating time for the annealing reaction and the extension reaction per cycle (in the illustrated example, the time during which the reaction solution 6 is placed in the liquid 4) is, for example, It is 6 seconds or less, preferably 4 seconds or less, more preferably 3 seconds or less, and even more preferably 1 second or more and 1.5 seconds or less. By setting the heating time for the annealing reaction and extension reaction to 6 seconds or less, it is possible to increase the speed of PCR.

  In the step of applying a thermal cycle (step S3), the time per cycle of the thermal cycle is 9 seconds or less, preferably 7 seconds or less, more preferably 6 seconds or less. By setting the time per cycle to 9 seconds or less, the heat cycle can be speeded up. Note that the time per one cycle of the thermal cycle is the time required for the denaturation reaction, annealing reaction, and extension reaction, and the transfer time of the reaction solution for performing these reactions (for example, liquid 2 to liquid 4 of the reaction solution 6). And the movement time from the liquid 4 to the liquid 2).

  Next, the fluorescence intensity of the reaction solution is measured (step S4). For example, the reaction solution to which the thermal cycle has been applied is transferred to a translucent container, and the light intensity is measured by irradiating the translucent container with light. Thereby, the amount of nucleic acid amplification can be quantified.

  The nucleic acid amplification reaction method according to this embodiment has, for example, the following characteristics.

  In the nucleic acid amplification reaction method, the Tm value of the primer is 65 ° C. or more and 80 ° C. or less, and the time for heating the reaction solution is 5 seconds or more and 480 seconds or less in the step of performing the reverse transcription reaction (step S2). Therefore, the nucleic acid amplification reaction method is a nucleic acid amplification reaction method capable of performing reverse transcription reaction and speeding up the heat cycle, and can suppress amplification of non-specific nucleic acid (details will be described later). (See “3. Experimental example”).

  In the nucleic acid amplification reaction method, in the step of heating to perform the reverse transcription reaction (step S2), the reaction solution is heated to 50 ° C. or more and 70 ° C. or less, and the reaction solution is heated for 10 seconds or more and 60 seconds or less. There may be. Therefore, in the nucleic acid amplification reaction method, the fluorescence intensity can be increased in the fluorescence intensity measurement (for details, refer to “3. Experimental example” described later).

  In the nucleic acid amplification reaction method, the amount of reverse transcriptase may be 30 units or more and 60 units or less. Therefore, in the nucleic acid amplification reaction method, the fluorescence intensity can be increased in the fluorescence intensity measurement (for details, refer to “3. Experimental example” described later), and the cost can be reduced.

  In the nucleic acid amplification reaction method, in the step of heating to perform the reverse transcription reaction (step S2), the reaction solution is heated to 60 ° C. or higher and 70 ° C. or lower, and the reaction solution is heated for 10 seconds or longer and 30 seconds or shorter. There may be. Therefore, in the nucleic acid amplification reaction method, the time for the reverse transcription reaction can be shortened while ensuring the fluorescence intensity (for details, refer to “3. Experimental example” described later).

In the nucleic acid amplification reaction method, in the step of heating to perform the reverse transcription reaction (step S2), the reaction solution is heated to 66 ° C. or more and 70 ° C. or less, and the reaction solution is heated for 60 seconds or less. Good. Therefore, in the nucleic acid amplification reaction method, the fluorescence intensity can be further increased in the fluorescence intensity measurement (for details, refer to “3. Experimental example” described later).

  In the nucleic acid amplification reaction method, in the step of applying a thermal cycle (step S3), the time per thermal cycle may be 9 seconds or less. Therefore, in the nucleic acid amplification reaction, the heat cycle can be accelerated.

  In the nucleic acid amplification reaction method, in the step of applying a thermal cycle (step S3), the heating time for the primer annealing reaction may be 6 seconds or less per thermal cycle. Therefore, in the nucleic acid amplification reaction, the heat cycle can be accelerated.

  In the nucleic acid amplification reaction method, the primer may include an artificial nucleic acid. Thereby, amplification of a non-specific nucleic acid can be suppressed (for details, refer to “3. Experimental example” described later).

  In the nucleic acid amplification reaction method, the primer may be a primer specific to the target RNA. Thereby, since a primer anneals only to a specific base sequence, amplification of a non-specific nucleic acid can be suppressed.

3. Experimental Example An experimental example is shown below to describe the present invention more specifically. The present invention is not limited by the following experimental examples.

3.1. First Experimental Example 3.1.1. Preparation of reaction solution and experimental method RNA of influenza B (InfB) virus was used as a template nucleic acid (template RNA). This template nucleic acid was added to the reagent to prepare the following reaction solution.

<Composition of reaction solution>
Platinum Taq polymerase (5 units / μL) 0.4 μL
SuperSCriptIII reverse transcriptase (200 units / μL) 0.04 μL
Buffer 2.0 μL
dNTP (10 mM) 0.25 μL
Influenza detection forward primer (100 μM) 0.32 μL
Reverse primer for detecting influenza (100 μM) 0.32 μL
Fluorescently labeled probe for detection of influenza (10 μM) 0.9 μL
Influenza RNA (100 copies / μL) 1.0 μL
Distilled water 4.77μL

  The fluorescently labeled probe used was a TaqMan (registered trademark) probe manufactured by Sigma Aldrich.

The buffer (buffer solution) includes MgCl ₂ , Tris-HCl (PH 9.0), and KCl. The concentration of MgCl ₂ contained in the reaction solution was 5 mM.

  The Tm values and sequences of the primers and probes are as shown in Table 1 below.

  The Tm values shown in Table 1 were calculated from the above formula (1). In the formula (1), Ct was 500 nM and Na + was 50 mM. This is the same in the experimental examples shown below.

  10 μL of the reaction solution as described above was filled in a container (Light Cycler Capillaries (20 μL) manufactured by Roche), and immersed in a water bath heated to 52 ° C. with a heat block for an arbitrary time to perform a reverse transcription reaction. After the reverse transcription reaction, PCR was performed by reciprocating the high temperature region (90 ° C., 2 seconds) and the low temperature region (66 ° C., 2 seconds) in an apparatus as shown in FIG. The number of thermal cycles was 40 cycles. In order to activate the polymerase, it was first heated in a high temperature region for 20 seconds (hot start). After that, the reaction solution was transferred to another container (MicroAmp Fast Reaction Tubes manufactured by Applied Biosystems), and fluorescence intensity (endpoint fluorescence intensity was measured with Step One Plus Real-time PCR system manufactured by Applied Biosystems). did. The above experiment was performed twice, changing the time for heating the reaction solution.

3.1.2. Results of Fluorescence Intensity Measurement FIGS. 4 and 5 are graphs showing the relationship between the heating time for the reverse transcription reaction and the fluorescence intensity. FIG. 4 shows the results of the first experiment, and FIG. 5 shows the results of the second experiment. FIG. 6 is a graph showing the relative fluorescence intensity when the fluorescence intensity when the heating time is 60 seconds in FIG. 4 is taken as 100%. FIG. 7 is a graph showing the relative fluorescence intensity when the fluorescence intensity when the reverse transcription reaction time is 60 seconds in FIG. 5 is taken as 100%.

  6 and 7, it was found that when the heating time for the reverse transcription reaction is 5 seconds and 480 seconds or less, the relative fluorescence intensity is 60% or more, and the fluorescence intensity can be increased. Furthermore, the relative fluorescence intensity can be set to 70% or more by setting the heating time for the reverse transcription reaction to 10 seconds or more and 480 seconds or less, and the relative fluorescence intensity can be set to 20 seconds or more and 480 seconds or less. 80% or more, and the relative fluorescence intensity could be 90% or more by setting it to 40 seconds or more and 120 seconds or less.

3.1.3. Results of electrophoresis The above reaction solution was analyzed by agarose gel electrophoresis. FIG. 8 shows the result of electrophoresis. In FIG. 8, “M” indicates a molecular weight marker.

  As shown in FIG. 8, when the heating time for the reverse transcription reaction is long, a band (40 bp or more and 80 bp or less) due to amplification of non-specific nucleic acid is confirmed, and the heating time is from 8 minutes (480 seconds). When it became longer, it was remarkably confirmed. In addition, when the heating time for the reverse transcription reaction was long, the band (103 bp) attributed to the target nucleic acid was thinned and became deeper as the heating time was shortened. Note that no band (103 bp) was observed at a heating time of 0 seconds.

From the above, it was found that if the heating time for the reverse transcription reaction is too long, the primer anneals to a region other than the target region to amplify the nucleic acid and inhibit the amplification of the target nucleic acid. This inhibition can be improved by shortening the heating time.

  The reason is presumed as follows. In the case of normal PCR conditions (for example, conditions for annealing reaction and extension reaction longer than 2 seconds), the heating time for reverse transcription reaction is generally 30 minutes to 1 hour. However, under the high-speed thermal cycle conditions (for example, the conditions for the annealing reaction and the extension reaction are 2 seconds or less) as in the first experiment, the Tm value is 65 ° C. or higher and 80 ° C. or higher for speeding up the thermal cycle. Use a primer with a high Tm value and below ℃. For example, if the primer has a large number of bases and a long heating time for the reverse transcription reaction, the primer anneals to other than the target site, and nonspecific nucleic acid amplification is likely to occur. It is done. Therefore, under high-speed heat cycle conditions, it is considered that nonspecific nucleic acid amplification could be suppressed by setting the heating time to 480 seconds or less. This inference is one of the hypotheses.

3.2. Second Experimental Example The fluorescence intensity was measured in the same manner as in the first experimental example, except that the heating temperature and the heating time for the reverse transcription reaction were changed. The heating temperature for the reverse transcription reaction was changed to 52 ° C., 60 ° C., and 70 ° C. FIG. 9 is a graph showing the relationship between the heating time for the reverse transcription reaction and the fluorescence intensity.

  As shown in FIG. 9, by setting the heating temperature to 50 ° C. or more and 70 ° C. or less and the heating time to 10 seconds 60 seconds or less, for example, the fluorescence intensity can be increased as compared with the case where the heating time is 5 seconds or less. all right.

  It was found that when the heating temperature is 60 ° C., the fluorescence intensity is higher than when the heating temperature is 52 ° C., and the heating time can be shortened without reducing the fluorescence intensity. In addition, when the heating temperature is 70 ° C., the fluorescence intensity is reduced when the heating time is 30 seconds or more compared to the case of 52 ° C. and 60 ° C., but when the heating time is 10 seconds, the temperature is 52 ° C. And almost the same fluorescence intensity. In the second experiment, the heating temperature was 60 ° C. and the heating time was 30 seconds. Therefore, it was found that by setting the heating temperature to 60 ° C. or more and 70 ° C. or less and the heating time to 10 seconds or more and 60 seconds or less, the time for the reverse transcription reaction can be shortened while ensuring the fluorescence intensity.

3.3. Third Experimental Example The fluorescence intensity was measured in the same manner as in the first experimental example, except that the heating time for the reverse transcription reaction and the amount of reverse transcriptase were changed. The amount of reverse transcriptase was changed to 8, 30 and 60 units. FIG. 10 is a graph showing the relationship between the heating time for the reverse transcription reaction and the fluorescence intensity.

  As shown in FIG. 10, it was found that by setting the amount of reverse transcriptase to 30 units or more and 60 units or less, the fluorescence intensity can be increased as compared with the case where the amount of reverse transcriptase is 8 units, for example.

3.4. Fourth Experimental Example The fluorescence intensity was measured in the same manner as in the first experimental example, except that the heating temperature and heating time for the reverse transcription reaction and the amount of reverse transcriptase were changed. Specifically, according to the second experimental example, when the heating temperature for the reverse transcription reaction is 60 ° C., the heating time for 30 seconds is the highest in fluorescence intensity, and the heating temperature for the reverse transcription reaction is 70 ° C. In some cases, the fluorescence intensity was highest at a heating time of 10 seconds. Therefore, in the fourth experimental example, reverse transcriptase is obtained when the heating temperature is 60 ° C. and the heating time is 30 seconds (60 ° C./30 seconds), and when the heating temperature is 70 ° C. and the heating time is 10 seconds (70 ° C./10 seconds). The experiment was carried out by changing the amount of 8 units, 30 units and 60 units. FIG. 11 is a graph showing the relationship between the amount of reverse transcriptase and the fluorescence intensity.

  As shown in FIG. 11, by increasing the amount of reverse transcriptase to 30 units or more, the fluorescence intensity can be increased. In the case of “70 ° C./10 seconds”, the time is as short as 10 seconds. A fluorescence intensity almost equivalent to that at “60 ° C./30 seconds” could be obtained.

3.5. Fifth Experimental Example From the fourth experimental example, it was found that if the amount of reverse transcriptase is increased even if the temperature is increased, the fluorescence intensity does not decrease and the reverse transcription reaction can be performed in a short time. Therefore, in the fifth experimental example, at a temperature higher than 60 ° C. (66 ° C., 70 ° C., 74 ° C.), the heating time for the reverse transcription reaction was changed, and the amount of reverse transcriptase was changed to 8 units and 30 units. The experiment was conducted with 60 units. The fluorescence intensity was measured in the same manner as in the first experimental example, except that the heating temperature and heating time for the reverse transcription reaction and the amount of reverse transcriptase were changed. 12 to 14 are graphs showing the relationship between the heating time for the reverse transcription reaction and the fluorescence intensity. 12 shows the case where the heating temperature for the reverse transcription reaction is 66 ° C., FIG. 13 shows the case where it is 70 ° C., and FIG. 14 shows the case where it is 74 ° C.

  As shown in FIGS. 12 to 14, when the heating temperature was 74 ° C., the fluorescence intensity was lower than that at 66 ° C. and 70 ° C. Therefore, it was found that the fluorescence intensity can be increased by setting the heating time to 60 seconds or less and the heating temperature to 66 ° C. or more and 70 ° C. or less. Further, in the fifth experimental example, as in the third experimental example, the fluorescence intensity is higher when the amount of reverse transcriptase is 30 units and 60 units than when the amount of reverse transcriptase is 8 units. It was.

3.6. Sixth experiment 3.6.1. Preparation of reaction solution and experimental method As a template nucleic acid (template DNA), mycoplasma bacteria DNA was used. This template nucleic acid was added to the reagent to prepare the following reaction solution.

<Composition of reaction solution>
Platinum Taq polymerase (5 units / μL) 0.4 μL
Buffer 2.0 μL
dNTP (10 mM) 0.25 μL
Mycoplasmabacterium forward primer (20μM) 1.2μL
Reverse primer for detecting mycoplasma bacteria (20μM) 1.2μL
Fluorescently labeled probe for detecting mycoplasma bacteria (10 μM) 0.9 μL
Mycoplasmabacterium DNA (100 copies / μL) 1.0 μL
Distilled water 3.05μL

  The fluorescently labeled probe used was a TaqMan (registered trademark) probe manufactured by Sigma Aldrich.

The buffer (buffer solution) includes MgCl ₂ , Tris-HCl (PH 9.0), and KCl. The concentration of MgCl ₂ contained in the reaction solution was 5 mM.

  In this experiment, primers having different Tm values were used. Specifically, primers having Tm values of about 60 ° C. (Tm 60), about 70 ° C. (Tm 70), about 75 ° C. (Tm 75), about 80 ° C. (Tm 80), and about 85 ° C. (Tm 85) were used. Tm values of Tm values of about 60 ° C., about 70 ° C., and about 75 ° C. Primers and sequences, and probe sequences are shown in Table 2 below. The Tm value and arrangement of the probe are the same as “No. 1” in Table 2.

10 μL of the reaction solution as described above is placed in a container (Light Cycler Capillaries (20 μL) manufactured by Roche), and PCR is performed by reciprocating the high temperature region and the low temperature region in an apparatus as shown in FIG. It was. The number of thermal cycles was 40 cycles. Then, the reaction solution is transferred to another container (MicroAmp Fast Reaction Tubes manufactured by Applied Biosystems), and Step One Plus Real-time manufactured by Applied Biosystems.
The fluorescence intensity was measured with a PCR system.

  In PCR using each primer (Tm60, Tm70, Tm75, Tm80, and Tm85), the heating temperature (high temperature temperature) for the denaturation reaction was 87 ° C. In PCR using Tm60, Tm70, Tm75, Tm80, and Tm85, the heating temperatures (low temperature temperatures) for the annealing reaction and the extension reaction were 60 ° C, 63 ° C, 66 ° C, 69 ° C, and 72 ° C, respectively. . Table 3 below shows the heating time for the denaturation reaction (high temperature time), the heating time for the annealing reaction and the extension reaction (low temperature time), and the reaction time per cycle in PCR. In order to activate the polymerase, it was first heated at a high temperature for 10 seconds (hot start). In addition to the polymerase activity time, the reaction time is obtained by multiplying the high temperature time and the low temperature time by the number of cycles 40, and adding the movement time of the reaction solution. In Table 3, the time per cycle of the thermal cycle is the sum of the high temperature time and the low temperature time, the movement time from the high temperature region to the low temperature region (0.5 seconds), and the movement from the low temperature region to the high temperature region. Time (0.5 seconds) is added. For example, when the reaction time is 370 seconds, the time per cycle of the thermal cycle is as follows: high temperature time (2 seconds) + high temperature time (6 seconds) + transfer time from high temperature region to low temperature region (0.5 seconds) + low temperature The moving time from the region to the high temperature region (0.5 seconds) = 9 seconds.

3.6.2. Results of Fluorescence Intensity Measurement FIG. 15 is a graph showing the relationship between PCR reaction time and fluorescence intensity. As shown in FIG. 15, when the low temperature time was 6 seconds (when the time per cycle was 9 seconds), amplification was confirmed at Tm70 and Tm75, but when the low temperature time was 4 seconds or less (1 cycle) When the hit time was 7 seconds), amplification of the nucleic acid was not confirmed at Tm60, and amplification was confirmed at Tm70 and Tm75. Therefore, it was found that the nucleic acid can be amplified even by high-speed PCR of 4 seconds or less by setting the Tm value of the primer to 70 ° C. or higher and lower than 80 ° C. This is presumably because the primer with a higher Tm value anneals to the template nucleic acid faster, so that Tm70 and Tm75 are more suitable for higher speed than Tm60. In addition, from FIG. 1 described above, Tm70 and Tm75 have higher polymerase activity efficiency than Tm60 and can accelerate the extension reaction, so that Tm70 and Tm75 are more suitable for higher speed than Tm60. This is probably because of this. In consideration of variations in the apparatus used in this experimental example, it can be said that the nucleic acid is surely amplified when the fluorescence intensity is 35,000 or more. Therefore, when the time per cycle is 9 seconds, it can be said that Tm60 does not reliably amplify the nucleic acid, and Tm70 and Tm75 can surely amplify the nucleic acid.

  Furthermore, FIG. 15 shows that even if the low temperature time is 2 seconds or less, the nucleic acid can be amplified if the Tm value of the primer is 70 ° C. or higher and 75 ° C. or lower. Further, from FIG. 15, nucleic acid amplification was not confirmed at Tm80 and Tm85. This is probably because the Tm value was too high and primer dimers and the like were generated.

  In FIG. 15, the values obtained by subtracting the fluorescence intensity (background) of the unreacted solution not subjected to the thermal cycle from the fluorescence intensity at the end point are plotted. A plot showing a negative fluorescence intensity is considered to be a measurement error.

3.7. Seventh Experimental Example 3.7.1. Preparation of reaction solution and experimental method Bordetella pertussis DNA was used as a template nucleic acid (template DNA). This template nucleic acid was added to the reagent to prepare the following reaction solution.

<Composition of reaction solution>
Platinum Taq polymerase (5 units / μL) 0.4 μL
Buffer 2.0 μL
dNTP (10 mM) 0.25 μL
Forward primer for detection of Bordetella pertussis (100 μM) 0.32 μL
Reverse primer for detection of Bordetella pertussis (100 μM) 0.32 μL
Bordetella pertussis fluorescently labeled probe (10 μM) 0.9 μL
Bordetella pertussis DNA (20 copies or 100 copies / μL) 1.0 μL
Distilled water 4.81 μL

  The fluorescently labeled probe used was a TaqMan (registered trademark) probe manufactured by Sigma Aldrich.

The buffer (buffer solution) includes MgCl ₂ , Tris-HCl (PH 9.0), and KCl. The concentration of MgCl ₂ contained in the reaction solution was 5 mM.

  The Tm value and sequence of the primer and the sequence of the probe are shown in Table 4 below.

  PCR was performed on 10 μL of the reaction solution as described above by performing hot start for 10 seconds as in the sixth experimental example. The high temperature was 90 ° C and the low temperature was 60 ° C. The heating time for the denaturation reaction (high temperature time) and the heating time for the annealing reaction and extension reaction (low temperature time) and the reaction time per cycle are shown in Table 5 below.

3.7.2. Results of Fluorescence Intensity Measurement FIG. 16 is a graph showing the relationship between PCR reaction time and fluorescence intensity. As shown in FIG. 16, even when the Tm value of the primer was about 80 ° C., nucleic acid amplification could be confirmed.

3.8. Eighth Experimental Example 3.8.1. Preparation of reaction solution and experimental method Instead of the reverse primer shown in Table 1, the LNA binding primer (primer with LNA bound (inserted)) shown in Table 6 below was used as the InfB reverse primer, and the reaction solution was reversed. The fluorescence intensity was measured in the same manner as in the first experimental example, except that the photoreaction time was changed to 20 seconds, 120 seconds, and 480 seconds.

  In Table 6, the base to which LNA is bound is underlined. That is, LNA is bonded to “T”, which is the seventh base from the 5 ′ end side, and “C”, which is the thirteenth base from the 5 ′ end side.

3.8.2. Results of Fluorescence Intensity Measurement FIG. 17 is a graph showing the relationship between reverse transcription reaction time and fluorescence intensity. In FIG. 17, the fluorescence of the case where the primer (primer without LNA, that is, the primer to which LNA is not bound) used in the first experimental example and the case of using the primer (LNA binding primer) shown in Table 6 are used. Indicates strength.

  As shown in FIG. 17, it was found that by using an LNA binding primer, the fluorescence intensity did not decrease even when reverse transcription was performed for 480 seconds, and the fluorescence intensity could be kept high.

3.8.3. Results of electrophoresis The above reaction solution was analyzed by agarose gel electrophoresis. FIG. 18 shows the result of electrophoresis in the case of using an LNA binding primer. FIG. 19 shows the result of electrophoresis in the case of using a primer without LNA.

  As shown in FIG. 19, in the primer without LNA, when the heating time for the reverse transcription reaction is long, a band (40 bp or more and 90 bp or less) due to amplification of non-specific nucleic acid is confirmed. On the other hand, as shown in FIG. 18, with the LNA binding primer, non-specific amplification could not be confirmed even when the heating time was longer than 120 seconds. Further, in the primer without LNA, when the heating time for the reverse transcription reaction was long, the band (103 bp) attributed to the target nucleic acid became thin, and became darker as the heating time was shortened. On the other hand, with the LNA binding primer, the band (103 bp) attributed to the purpose became thin even at the reverse transcription time of 480 seconds. A band of 20 bp to 40 bp is caused by the primer.

  As described above, it was found that the use of the LNA binding primer can suppress non-specific amplification due to the heating time for the reverse transcription reaction being too long, and the amplification of the target nucleic acid is not inhibited. Therefore, it was found that the use of an LNA binding primer can improve the robustness of the nucleic acid amplification method and can perform stable RT (Reverse Transfer) -PCR.

  The interpretation of this result is presumed as follows. If the primer is long, the primer is annealed to a region other than the target region to amplify the nucleic acid, thereby inhibiting the amplification of the target nucleic acid. That is, it seems that such a problem can be solved by using a primer having a short chain length. However, in that case, if the primer is simply shortened, the Tm value decreases and high-speed PCR cannot be realized. In this study, LNA was bound to the primer to shorten the chain length of the primer and use a primer having a high Tm value, thereby realizing high-speed PCR while providing robustness to reverse transcription.

  The present invention includes configurations that are substantially the same as the configurations described in the embodiments (for example, configurations that have the same functions, methods, and results, or configurations that have the same objects and effects). In addition, the invention includes a configuration in which a non-essential part of the configuration described in the embodiment is replaced. In addition, the present invention includes a configuration that exhibits the same operational effects as the configuration described in the embodiment or a configuration that can achieve the same object. Further, the invention includes a configuration in which a known technique is added to the configuration described in the embodiment.

2, 4 ... Liquid, 6 ... Reaction solution, 8 ... Container, 10 ... First hot plate, 12 ... Second hot plate, 20 ... First beaker, 22 ... Second beaker, 30 ... Arm, 30a ... One end, 30b ... other end, 32 ... fixed part, 100 ... thermal cycle device

SEQ ID NO: 1 is the sequence of the InfB forward primer.
SEQ ID NO: 2 is the reverse primer sequence of InfB.
SEQ ID NO: 3 is the sequence of an InfB fluorescently labeled probe.
Sequence number 4 is the arrangement | sequence of the forward primer of Mycoplasma bacteria.
SEQ ID NO: 5 is the reverse primer sequence of Mycoplasma bacteria.
Sequence number 6 is the arrangement | sequence of the forward primer of Mycoplasma bacteria.
Sequence number 7 is the arrangement | sequence of the reverse primer of Mycoplasma bacteria.
Sequence number 8 is the arrangement | sequence of the forward primer of Mycoplasma bacteria.
Sequence number 9 is the arrangement | sequence of the reverse primer of Mycoplasma bacteria.
Sequence number 10 is the arrangement | sequence of the forward primer of Mycoplasma bacteria.
Sequence number 11 is the arrangement | sequence of the reverse primer of Mycoplasma bacteria.
Sequence number 12 is the arrangement | sequence of the forward primer of Mycoplasma bacteria.
Sequence number 13 is the arrangement | sequence of the reverse primer of Mycoplasma bacteria.
SEQ ID NO: 14 is the sequence of a fluorescently labeled probe of Mycoplasma bacteria.
Sequence number 15 is the arrangement | sequence of the forward primer of Bordetella pertussis.
SEQ ID NO: 16 is the reverse primer sequence of Bordetella pertussis.
SEQ ID NO: 17 is the sequence of a fluorescent labeled probe of Bordetella pertussis.
SEQ ID NO: 18 is the sequence of InfB reverse primer.

Claims (19)

Heating a reaction solution containing a reverse transcriptase, a polymerase, a primer, and a probe to perform a reverse transcription reaction;
After the heating step, applying a thermal cycle for amplifying nucleic acid to the reaction solution;
Including
The Tm value of the primer is 65 ° C. or more and 80 ° C. or less,
In the heating step,
The nucleic acid amplification reaction method, wherein the time for heating the reaction solution is 5 seconds or more and 480 seconds or less. In claim 1,
In the heating step,
A method for nucleic acid amplification reaction, wherein the reaction solution is heated to 50 ° C or higher and 70 ° C or lower. In claim 2,
In the heating step,
The method for nucleic acid amplification reaction, wherein the reaction solution is heated for 10 seconds to 60 seconds. In any one of Claims 1 thru | or 3,
The method for nucleic acid amplification reaction, wherein the amount of the reverse transcriptase contained in the reaction solution is 30 units or more and 60 units or less. In claim 4,
In the heating step,
The reaction solution is heated to 60 ° C. or higher and 70 ° C. or lower,
The method for nucleic acid amplification reaction, wherein the reaction solution is heated for 10 seconds to 30 seconds. In claim 1,
In the heating step,
The reaction solution is heated to 66 ° C. or higher and 70 ° C. or lower,
The nucleic acid amplification reaction method, wherein the time for heating the reaction solution is 60 seconds or less. In any one of Claims 1 thru | or 6,
In the step of applying the thermal cycle,
The method for nucleic acid amplification reaction, wherein the time per one cycle of the thermal cycle is 9 seconds or less. In claim 7,
The nucleic acid amplification reaction method, wherein the heating time for the primer annealing reaction is 6 seconds or less. In any one of Claims 1 thru | or 8,
The reaction solution contains a divalent cation,
The method for nucleic acid amplification reaction, wherein the concentration of the divalent cation contained in the reaction solution is 2 mM or more and 7.5 mM or less. In claim 9,
The reaction solution includes MgCl ₂ ;
The divalent cation is derived from MgCl ₂ .
The nucleic acid amplification reaction method, wherein the concentration of MgCl ₂ contained in the reaction solution is 4 mM or more and 7.5 mM or less. In claim 9,
The reaction solution includes MgSO ₄ ,
The divalent cation is derived from MgSO ₄ ;
The method for nucleic acid amplification reaction, wherein the concentration of MgSO ₄ contained in the reaction solution is 2 mM or more and 3 mM or less. In any one of Claims 1 thru | or 11,
The nucleic acid amplification reaction method, wherein the probe is a hydrolysis probe. In any one of Claims 1 thru | or 12,
The nucleic acid amplification reaction method, wherein the probe includes at least one of an artificial nucleic acid and a minor group binder molecule. In any one of Claims 1 thru | or 13,
The method for nucleic acid amplification reaction, wherein the reverse transcriptase is derived from mouse Moloney murine Leukemia virus. In any one of Claims 1 thru | or 14,
The nucleic acid amplification reaction method, wherein the primer includes an artificial nucleic acid. In any one of Claims 1 thru | or 15,
The nucleic acid amplification reaction method, wherein the primer is a sequence-specific primer for a target RNA. A reagent for performing a reverse transcription reaction and a nucleic acid amplification reaction,
A reverse transcriptase, a polymerase, a primer, a probe, and MgCl ₂ ;
The Tm value of the primer is 65 ° C. or more and 80 ° C. or less,
The reagent, wherein when the reagent becomes a reaction solution for performing a reverse transcription reaction and a nucleic acid amplification reaction, the concentration of MgCl ₂ contained in the reaction solution is 4 mM or more and 7.5 mM or less. A reagent for performing a reverse transcription reaction and a nucleic acid amplification reaction,
A reverse transcriptase, a polymerase, a primer, a probe, and MgSO ₄ ;
The Tm value of the primer is 65 ° C. or more and 80 ° C. or less,
The reagent, wherein when the reagent becomes a reaction solution for performing a reverse transcription reaction and a nucleic acid amplification reaction, the concentration of MgSO ₄ contained in the reaction solution is 2 mM or more and 3 mM or less. A method of using the reagent according to claim 17 or 18,
The reagent is brought into contact with a template nucleic acid solution containing a template nucleic acid to prepare the reaction solution. After the reaction solution is heated for 5 seconds or more and 480 seconds or less to perform a reverse transcription reaction, the reaction solution is heated. A method of using a reagent in which a nucleic acid amplification reaction is performed by applying a cycle.