JP7565572B2 - Multi-layered premix reagent for nucleic acid amplification reaction and ready-to-use nucleic acid amplification reaction kit - Google Patents

Description

The present invention relates to a multi-layered premix reagent for nucleic acid amplification reactions and a ready-to-use nucleic acid amplification reaction kit containing the same.

The reaction solution for genetic testing, such as PCR (polymerase chain reaction) and LAMP (loop-mediated isothermal amplification), contains primer oligonucleotides, nucleoside triphosphates, reaction buffers, and nucleic acid polymerase. To simplify the reaction procedure, premixed reagents and freeze-dried reagents of these are also sold, but since nucleic acid polymerase is generally relatively unstable, it is preferable to prepare the reagents separately, at least for nucleic acid polymerase and other components. When actually preparing the reaction solution, careful operation is required, such as using a micropipette that can precisely dispense the volume. This is because the amount of polymerase (enzyme) greatly affects the efficiency of the detection reaction, sometimes resulting in false positives or false negatives.

Currently, the RT-PCR method is used to test for infection with COVID-19, which is causing a global pandemic. Although the reaction solution can be prepared relatively easily using a dedicated reagent kit, for the reasons mentioned above, careful operation by a person with some training and physicochemical equipment such as micropipettes that are unfamiliar to the general public are required. This is also the case with other genetic testing methods such as the RT-LAMP method and the SATIC method (Patent Document 1 and Non-Patent Document 1).

WO 2016/152936

Anal. Chem., 2016, 88 (14), pp 7137-7144

The objective of the present invention is to provide a premix reagent for nucleic acid amplification reactions that allows easy nucleic acid amplification reactions to be performed simply by mixing samples and has excellent storage stability, as well as a ready-to-use nucleic acid amplification reaction kit that includes the same.

The present inventors conducted intensive research to solve the above problems. As a result, they succeeded in developing a multi-layered reagent premix that is stored frozen in a layered state and includes a first reagent layer containing a nucleic acid polymerase, a second reagent layer containing a primer oligonucleotide set, and a membrane separating the first and second reagent layers and composed of a substance that is solid below 0°C but dissolves in aqueous solution between 0°C and 35°C. They discovered that by using a nucleic acid amplification reaction kit containing this multi-layered reagent premix in a container, genetic testing based on nucleic acid amplification reaction can be easily performed while maintaining the performance of the reagent, and thus completed the present invention.

The gist of the present invention is as follows.
[1] A kit for nucleic acid amplification reaction comprising a reaction vessel and a nucleic acid amplification reaction reagent contained in the reaction vessel,
The reagent comprises a first reagent layer including a nucleic acid polymerase;
a second reagent layer comprising a primer oligonucleotide;
a membrane separating the first and second reagent layers, the membrane being made of a material that is solid below 0° C. but dissolves in an aqueous solution between 0 and 35° C.;
A kit for nucleic acid amplification reaction, comprising a multi-layered reagent premix stored frozen in a layered state, the multi-layered reagent premix comprising:
[2] The nucleic acid amplification reaction kit according to [1], wherein the second reagent layer further contains a nucleic acid substrate and a reaction buffer.
[3] The kit for nucleic acid amplification reaction according to [1] or [2], wherein the nucleic acid amplification reaction is PCR (polymerase chain reaction).
[4] The kit for nucleic acid amplification reaction according to [1] or [2], wherein the nucleic acid amplification reaction is a loop-mediated isothermal amplification (LAMP) method.
[5] The nucleic acid amplification reaction kit according to [1], wherein the nucleic acid amplification reaction is a SATIC method, the second reagent layer contains a first primer and a second primer, and further includes an intermediate reagent layer between the first reagent layer and the second reagent layer, the intermediate reagent layer containing a first single-stranded circular DNA and a second single-stranded circular DNA, and the intermediate reagent layer is separated from each of the first reagent layer and the second reagent layer by a membrane composed of a substance that is solid below 0°C but dissolves in an aqueous solution between 0 and 35°C.
[6] The nucleic acid amplification reaction kit according to [5], wherein the intermediate reagent layer further contains a nucleic acid substrate and a reaction buffer.
[7] The nucleic acid amplification reaction kit according to [5] or [6], wherein the first primer and the second primer are immobilized on a carrier.
[8] The kit for nucleic acid amplification reaction according to any one of [1] to [7], wherein the substance that is solid below 0°C but dissolves in an aqueous solution between 0 and 35°C is polyethylene glycol, polyvinyl alcohol, a sugar chain compound, a polyamide chain compound, or a water-soluble heterocyclic compound having self-organizing ability.
[9] A method for amplifying a nucleic acid in a nucleic acid-containing sample, comprising the steps of placing a nucleic acid amplification reaction kit according to any one of [1] to [8] in a temperature control device and adding a nucleic acid-containing sample to a reaction vessel.
[10] A step of adding a liquid containing a nucleic acid polymerase to a nucleic acid amplification reaction vessel to form a first reagent layer;
A method for producing a nucleic acid amplification reaction kit, which contains a multi-layered reagent premix in a reaction vessel, includes the steps of: laminating a membrane composed of a substance that is solid below 0°C but dissolves in an aqueous solution between 0 and 35°C on a first reagent layer; and adding a liquid containing a primer oligonucleotide onto the membrane at a temperature below 0°C to form a second reagent layer.
[11] The method for producing a kit for nucleic acid amplification reaction according to [10], wherein the nucleic acid amplification reaction is a PCR or LAMP method.
[12] adding a liquid containing a nucleic acid polymerase to a nucleic acid amplification reaction vessel to form a first reagent layer;
laminating a film on the first reagent layer, the film being composed of a substance that is solid below 0°C but dissolves in an aqueous solution between 0 and 35°C;
adding a liquid containing a first single-stranded circular DNA and a second single-stranded circular DNA onto the membrane at a temperature below 0° C. to form an intermediate reagent layer;
laminating, at a temperature below 0° C., on the intermediate reagent layer a film composed of a material that is solid below 0° C. but dissolves in an aqueous solution between 0 and 35° C.;
A method for producing a kit for SATIC reaction, which contains a multi-layered reagent premix in a reaction vessel, includes a step of adding a liquid containing a first primer and a second primer onto the membrane at a temperature below 0°C to form a second reagent layer.

According to the present invention, nucleic acid amplification reactions can be easily performed simply by adding a sample to a premix, eliminating the need for multiple operations using a micropipette or the like, making it possible for ordinary people without special skills to handle the reaction, and significantly improving the throughput of genetic tests such as PCR tests. Furthermore, because the operation can be significantly simplified, false positives due to contamination and false negatives due to forgetting to add reagents can be significantly reduced. In addition, because the nucleic acid polymerase and primer oligonucleotides are not mixed until immediately before the reaction, there are no problems such as reagent deterioration, and the reaction has excellent storage stability.

FIG. 1 is a schematic diagram of a first embodiment of a multi-layered reagent premix. FIG. 2 is a schematic diagram of a second embodiment of a multi-layered reagent premix (including an intermediate layer). Schematic diagram of the SATIC method. FIG. 1 shows the results of Example 1 (photograph instead of drawing). FIG. 13 is a diagram showing the results of Example 2 (a photograph substituting a drawing). FIG. 13 is a diagram showing the results of Example 3 (a photograph substituting a drawing).

<Nucleic acid amplification reaction kit>
The nucleic acid amplification reaction kit of the present invention comprises:
A kit for nucleic acid amplification reaction comprising a reaction vessel and a nucleic acid amplification reaction reagent contained in the reaction vessel,
The reagent comprises a first reagent layer including a nucleic acid polymerase;
a second reagent layer comprising a primer oligonucleotide;
a membrane separating the first and second reagent layers, the membrane being made of a material that is solid below 0° C. but dissolves in an aqueous solution between 0 and 35° C.;
and storing the multi-layered reagent premix frozen (below 0° C., for example, −10° C. to −30° C.) in a layered state.

A nucleic acid amplification reaction may be any reaction in which a primer is hybridized to a target nucleic acid and a polynucleotide chain is then extended using a nucleic acid polymerase. Examples of such a reaction include, but are not limited to, PCR, LAMP, SATIC, and SmartAmp, and the kit of the present invention can be used for a wide range of nucleic acid amplification reactions.

In the nucleic acid amplification reaction kit of the present invention, the nucleic acid amplification reaction reagent contained in the reaction vessel has a first reagent layer containing a nucleic acid polymerase and a second reagent layer containing a primer oligonucleotide separated by a "membrane composed of a substance that is solid at least below 0°C but dissolves in aqueous solution between 0 and 35°C" (Figure 1A).

By using the above-mentioned substance for this membrane, the nucleic acid polymerase and primer oligonucleotides are separated during frozen storage, and when used, the reagent is thawed. During the temperature rise, the membrane dissolves in the aqueous solution of the first and second reagent layers between 0°C and 35°C and they are mixed (Figure 1A), automatically preparing a reaction solution. The sample (nucleic acid-containing sample) is then added, and the nucleic acid amplification reaction can be carried out simply by setting the reaction vessel to the reaction temperature.

The substance in question may be a substance that is solid at least below 0° C. but dissolves in an aqueous solution between 0 and 35° C. (preferably 10 to 30° C.), and is preferably a substance that does not interfere with the polymerase reaction. Specific examples of the substance include polyethylene glycol having a molecular weight in a predetermined range (600 to 8000, preferably 1000 to 6000) (e.g., PEG1000 or substitutes thereof (PEGs having different molecular weights, branched PEGs, and mixtures thereof), polyvinyl alcohol, sugar chain compounds which may be branched, such as starch, cellulose, agarose, dextran, dextrin, oligosaccharides, and mixtures thereof, polyamide chain compounds which may be branched, such as poly(oligo)lysine, poly(oligo)glutamic acid, collagen, and mixtures thereof, and water-soluble heterocyclic compounds having self-organizing ability, such as melamine and guanine.

The first reagent layer is an aqueous solution containing a nucleic acid polymerase. The nucleic acid polymerase may be a DNA polymerase or an RNA polymerase. The DNA polymerase may be selected arbitrarily depending on the target reaction, and examples include a heat-resistant DNA polymerase (such as Taq or Pfu), a strand-displacing DNA polymerase, and a reverse transcriptase. Examples of strand-displacing DNA polymerases include mesophilic strand-displacing DNA polymerases such as phi29 polymerase, Klenow DNA Polymerase (5'-3', 3'-5' exo minus), Aac DNA Polymerase, Sequenase (registered trademark) Version 2.0 T7 DNA Polymerase (USB), and Bsu DNA Polymerase, Large Fragment (NEB), as well as heat-resistant strand-displacing DNA polymerases such as Bst DNA Polymerase (Large Fragment), Bsm DNA Polymerase, Large Fragment (Fermentas), BcaBEST DNA polymerase (TakaraBio), Vent DNA polymerase (NEB), Deep Vent DNA polymerase (NEB), and DisplaceAce (registered trademark) DNA Polymerase (Epicentre).

For example, a heat-stable DNA polymerase can be used for PCR, and a strand-displacing DNA polymerase can be used for the LAMP method or the SATIC method.
The content of the nucleic acid polymerase is not particularly limited as long as it is the amount necessary for the nucleic acid amplification reaction, and may be contained in an amount required depending on the specific activity of the enzyme, etc. Two or more types of nucleic acid polymerase may be used in combination.
The first reagent layer may include a stabilizing agent, such as glycerol, to stabilize the nucleic acid polymerase.

The second reagent layer is an aqueous solution containing a primer oligonucleotide. The primer oligonucleotide may be any number of primer oligonucleotides required depending on the type of nucleic acid amplification reaction. For example, for PCR, a set of forward and reverse primers for amplifying the target site of the target nucleic acid is sufficient, but for nested PCR, three or more types of primer oligonucleotides may be contained.
In the case of the LAMP method (Reference: Principles and Applications of the LAMP Method, Modern Media, Vol. 60, No. 7, 2014, p. 211-213), four types of primers may be used: FIP, BIP, F3 primer, and B3 primer; four types of primers may be used: FIP, BIP, Loop primer B, and Loop primer F; or six types of primers may be used: FIP, BIP, F3 primer, B3 primer, Loop primer B, and Loop primer F.
In addition, in the SmartAmp method (reference: PLoS ONE, 2012, doi:10.1371/journal.pone.0030236), four types of TP, FP, OP, and BP may be used.
In the SATIC method, two types of primers, a first primer and a second primer, as described below, may be used.
In the case of an RNA polymerase reaction or a reverse transcription reaction, only one type of primer may be used.

The second reagent layer may further contain a nucleic acid substrate (such as deoxynucleoside triphosphate) and a reaction buffer. The reaction buffer can be appropriately selected depending on the enzyme.
The second reagent layer may further contain a salt, a surfactant, or a detection reagent such as a color-developing reagent or a luminescent reagent.
The concentrations of primers, nucleic acid substrates, etc. may be appropriately adjusted taking into consideration the final volume of the reaction solution.
In addition, in the case where an intermediate reagent layer is provided in the premix for the SATIC method as described below, the intermediate reagent layer may contain a nucleic acid substrate (e.g., deoxynucleoside triphosphate) and a reaction buffer, as well as a salt, a surfactant, and a detection reagent such as a color-developing reagent or a luminescent reagent.

Whichever of the first and second reagent layers may be the upper layer, but it is preferable for the first reagent layer to be the lower layer.

The SATIC method will be explained below.
The SATIC method is a method using a single-stranded circular DNA and a primer disclosed in Patent Document 1, in which a ternary complex of a target (template) nucleic acid, a single-stranded circular DNA, and a primer is formed, and the nucleic acid is amplified isothermally (37° C.), and preferably means a method using first and second single-stranded circular DNAs and first and second primers described below.

In a preferred embodiment of the SATIC method,
(i) a sequence of 10 to 30 bases that is complementary to a first site of a target nucleic acid;
a first primer binding sequence of 7 to 8 bases adjacent to the 5' side of the sequence;
a second single-stranded circular DNA binding sequence;
A first single-stranded circular DNA comprising:
(ii) a sequence of 8 to 15 bases that is complementary to a second site adjacent to the 3' side of the first site of the target nucleic acid;
a 7-8 base sequence adjacent to the 3' side of the sequence, which is complementary to the first primer binding site of the first single-stranded circular DNA;
A first oligonucleotide primer comprising:
(iii) a sequence identical to the second single-stranded circular DNA binding sequence of the first single-stranded circular DNA;
a second primer binding sequence adjacent to the 5' side of the sequence;
A second single-stranded circular DNA comprising:
(iv) a sequence identical to a site adjacent to the 5' side of the second single-stranded circular DNA binding sequence of the first single-stranded circular DNA;
a sequence complementary to a second primer binding sequence of a second single-stranded circular DNA adjacent to the 3' side of the sequence;
A second oligonucleotide primer comprising:
is used,
More preferably,
the first oligonucleotide primer is attached via its 5' end to a support;
The second oligonucleotide primer is bound via its 5' end to the support to which the first oligonucleotide primer is bound.

<First single-stranded circular DNA>
The first single-stranded circular DNA comprises a 10-30 base sequence complementary to a first site of a target nucleic acid;
a primer binding sequence of 7 to 8 bases adjacent to the 5' side of the sequence;
a second single-stranded circular DNA binding sequence;
Includes.

The SATIC method will be described with reference to Figure 2. However, in the case of single-stranded circular DNA, the clockwise direction is 5' to 3'. For convenience, the carrier is not shown in Figure 2.
The first single-stranded circular DNA 20 comprises a sequence 201 complementary to a first site 211 of the target nucleic acid 21 , a primer binding sequence 202 linked to the 5′ side thereof, and a second single-stranded circular DNA binding sequence 203 .
The length of sequence 201 is usually 10 to 30 bases, preferably 15 to 25 bases, and the GC content is preferably 30 to 70%. The length of sequence 202 is 7 or 8 bases, and the sequence is not particularly limited, but the GC content is preferably 30 to 70%. The entire length of the first single-stranded circular DNA 20 is preferably 35 to 100 bases.

The first single-stranded circular DNA 20 can be obtained by circularizing single-stranded DNA (ssDNA). Circularization of single-stranded DNA can be performed by any means, for example, using CircLigase (registered trademark), CircLigase II (registered trademark), ssDNA Ligase (Epicentre), or ThermoPhage ligase (registered trademark) single-stranded DNA (Prokzyme).

<First Oligonucleotide Primer>
The first oligonucleotide primer 22 comprises an 8-15 base sequence 221 that is complementary to a second site 212 adjacent to the 3' side of the first site 211 of the target nucleic acid 21, and a 7-8 base sequence 222 that is complementary to the primer binding site 202 of the first single-stranded circular DNA 20 linked to the 3' side thereof.

A first oligonucleotide primer 22 is attached via its 5' end to the carrier.
Examples of the 5' end of the first oligonucleotide primer 22 include those modified with biotin, an amino group, an aldehyde group, or an SH group. Examples of the carrier include carriers that can bind to each of these, such as a carrier to which avidin (including its derivatives, i.e., streptavidin, neutravidin, etc.) is fixed, and carriers that have been surface-treated with a silane coupling agent having an amino group, an aldehyde group, an epoxy group, etc. The immobilization may be performed according to a conventional method.

The carrier is preferably one that can immobilize the first oligonucleotide primer 22 and the second oligonucleotide primer 25 described below in close proximity. By having the two in close proximity, the step of amplifying the first amplification product 23 from the first oligonucleotide primer 22 and the step of amplifying the second amplification product 26 from the second oligonucleotide primer 25 are carried out more efficiently than when two types of primers are used that are free in solution, resulting in a significant improvement in detection sensitivity.

Preferred carriers include, for example, beads and planar carriers such as substrates used in sensors. Beads are particulate insoluble carriers, and their average particle size is, for example, 10 nm to 100 μm, preferably 30 nm to 10 μm, more preferably 30 nm to 1 μm, and even more preferably 30 nm to 500 nm. There are no particular limitations on the material of the beads. Examples include magnetic materials (magnetic materials such as iron oxides such as ferrite and magnetite, chromium oxide, and cobalt), silica, agarose, and sepharose. Magnetic beads are sometimes called "magnetic beads." Metal colloid particles such as gold colloids can also be used.

<Second single-stranded circular DNA>
The second single-stranded circular DNA 24 has a sequence 241 identical to the second single-stranded circular DNA binding sequence 203 of the first single-stranded circular DNA 20,
a second primer binding sequence 242 adjacent to the 5' side of the sequence;
Includes.
The length of sequence 203 is usually 10 to 30 bases, preferably 15 to 25 bases, and the GC content is preferably 30 to 70%. The length of sequence 242 is 7 or 8 bases, and the sequence is not particularly limited, but the GC content is preferably 30 to 70%. The length of sequence 242 is 7 or 8 bases, and the sequence is not particularly limited, but the GC content is preferably 30 to 70%. The entire length of the second single-stranded circular DNA 24 is preferably 35 to 100 bases. The second single-stranded circular DNA 24 can be obtained by circularizing a single-stranded DNA (ssDNA) by the method described above.

The second single-stranded circular DNA 24 preferably contains a sequence complementary to a detection reagent binding sequence, such as a G-quadruplex forming sequence.
The G-quadruplex-forming sequence includes the sequence described in Nat Rev Drug Discov. 2011 Apr; 10(4): 261-275., which is represented by G ₃ N _1-10 G ₃ N _1-10 G ₃ N _1-10 G ₃ , and specifically includes the sequence described in Patent Document 1. Thus, the sequence 243 complementary to the G-quadruplex-forming sequence is exemplified by C ₃ N _1-10 C ₃ N _1-10 C ₃ N _1-10 C _3. That is, it is a sequence in which three consecutive Cs are repeated four times with a spacer consisting of 1 to 10 (preferably 1 to 5) arbitrary bases (N=A, T, G, or C).

In FIG. 2, the second single-stranded circular DNA 24 includes a sequence 243 complementary to the G-quadruplex-forming sequence. However, the second single-stranded circular DNA 24 may also be configured to have an aptamer sequence or a molecular beacon (a hairpin-shaped oligonucleotide having a fluorescent group (donor) and a quencher group (acceptor) that cause FRET) binding sequence as the detection reagent binding sequence, and may be detected using an aptamer-binding chromogenic molecule or a molecular beacon as the detection reagent (ChemBioChem 2007, 8, 1795-1803; J. Am. Chem. Soc. 2013, 135, 7430-7433).

<Second Oligonucleotide Primer>
The second oligonucleotide primer 25 comprises a sequence 251 (preferably a sequence of 8 to 15 bases) identical to the site 204 adjacent to the 5' side of the second single-stranded circular DNA binding sequence 203 of the first single-stranded circular DNA 20;
a sequence 252 (preferably a sequence of 7 to 8 bases) complementary to the second primer binding sequence 242 of the second single-stranded circular DNA 24 adjacent to the 3' side of the sequence;
Includes.

A second oligonucleotide primer 25 is bound via its 5' end to the carrier to which the first oligonucleotide primer 22 is bound.
That is, it is preferable that the first oligonucleotide primer 22 is modified at its 5' end with biotin and is bound via the biotin to the support on which avidin is immobilized, and the second oligonucleotide primer 25 is modified at its 5' end with biotin and is bound via the biotin to the support to which the first oligonucleotide primer 22 is bound.

The molar ratio of the first oligonucleotide primer 22 to the second oligonucleotide primer 25 immobilized on the carrier is preferably 1:10 to 1:100, more preferably 1:10 to 1:30, and even more preferably 1:10 to 1:25.

The concentration of the first oligonucleotide primer 22 during the amplification reaction (when used) is, in molar ratio, preferably 0.0025 pmol/μL or more, more preferably 0.005 pmol/μL or more, while it is preferably 0.04 pmol/μL or less, more preferably 0.02 pmol/μL or less.
In addition, the concentration of the second oligonucleotide primer 25 during the amplification reaction (when used) is, in molar ratio, preferably 0.0125 pmol/μL or more, more preferably 0.025 pmol/μL or more, while being 0.8 pmol/μL or less, more preferably 0.4 pmol/μL or less.

The molar ratio of the first single-stranded circular DNA 20 to the second single-stranded circular DNA 24 during the amplification reaction (use) is preferably 1:2 to 1:1000, more preferably 1:3 to 1:500, and even more preferably 1:4 to 1:400.
The lower limit of the concentration of the first single-stranded circular DNA 20 during the amplification reaction (during use) is, for example, 0.1 nM or more, 1 nM or more, 10 nM or more, or 50 nM or more, while the upper limit is, for example, 500 nM or less, 200 nM or less.
The lower limit of the concentration of the second single-stranded circular DNA 24 during the amplification reaction (when in use) is, for example, 20 nM or more, 40 nM or more, 100 nM or more, or 200 nM or more, while the upper limit is, for example, 1000 nM or less, or 500 nM or less.

<Amplification method>
As shown in FIG. 2, first, a first single-stranded circular DNA 20 and a primer 22 are hybridized to a target nucleic acid 21 to form a complex of the three, and then a nucleic acid amplification reaction based on the target nucleic acid 21 is carried out by the rolling circle amplification (RCA) method.

A person skilled in the art can set the hybridization conditions appropriately by considering the combination of single-stranded circular DNA 20, target nucleic acid 21, and primers.

The RCA method is described in Lizardi et al., Nature Genet. 19: 225-232 (1998); U.S. Patent Nos. 5,854,033 and 6,143,495; PCT Application WO 97/19193, etc. The RCA method can be carried out using, for example, a strand-displacing DNA polymerase such as the above-mentioned phi29 DNA polymerase.

The DNA extension reaction by RCA is carried out at a constant temperature, for example, in the range of 25°C to 65°C. The reaction temperature is set appropriately by standard procedures based on the optimal temperature of the enzyme and the denaturation temperature (the temperature range at which the primer binds (anneals)/dissociates from DNA) based on the strand length of the primer. Furthermore, it is also carried out at a constant, relatively low temperature. For example, when phi29 DNA polymerase is used as the strand-displacing DNA synthesis enzyme, the reaction is preferably carried out at 25°C to 42°C, more preferably about 30°C to 37°C.

By RCA, a first amplification product 23 is amplified from the primer 22 along the first single-stranded circular DNA 20 in a target nucleic acid 21-dependent manner.

Since the amplification product 23 contains a sequence 233 complementary to the second single-stranded circular DNA binding sequence 203 of the first single-stranded circular DNA 20, the second single-stranded circular DNA 24 containing a sequence 241 identical to this sequence 203 hybridizes to the sequence 233 of the first amplification product 23 via the sequence 241.
A second oligonucleotide primer 25 hybridizes to the complex of the first amplification product 23 and the second single-stranded circular DNA 24 thus formed, forming a three-component complex.

In other words, the second oligonucleotide primer 25 has a sequence 251 identical to the site 204 adjacent to the 5' side of the second single-stranded circular DNA binding sequence 203 of the first single-stranded circular DNA 20, and therefore hybridizes via sequence 251 to the region 234 of the first amplification product 23 that is complementary to the site 204 of the first single-stranded circular DNA 20.

In addition, the second oligonucleotide primer 25 has a sequence 252 on the 3' side of sequence 251 that is complementary to the second primer binding sequence 242 of the second single-stranded circular DNA 24, and therefore hybridizes to the second single-stranded circular DNA 24 via sequence 252.

A second amplification product 26 is amplified by RCA from this ternary complex of the first amplification product 23, the second single-stranded circular DNA 24, and the second oligonucleotide primer 25. The second amplification product 26 contains, for example, a sequence 261 that contains a G-quadruplex, and is detected by a G-quadruplex detection reagent 262. The second single-stranded circular DNA 24 hybridizes to each of the regions 231 contained in the first amplification product 23, causing an RCA reaction.

By immobilizing the first oligonucleotide primer 22 and the second oligonucleotide primer 25 on the same support, the step of amplifying the first amplification product 23 from the first oligonucleotide primer 22 and the step of amplifying the second amplification product 26 from the second oligonucleotide primer 25 are carried out in close proximity to each other, thereby achieving a significant improvement in detection sensitivity.

Since the second single-stranded circular DNA 24 contains a sequence complementary to the detection reagent binding sequence, the second amplification product 26 obtained by RCA contains the detection reagent binding sequence. If the detection reagent binding sequence is a G-quadruplex forming sequence or the like, the amplification product obtained by RCA can be detected using a G-quadruplex binding reagent. Examples of the G-quadruplex binding reagent include thioflavin T (ThT) or a derivative thereof disclosed in Patent Document 1.

In addition, a ThT derivative containing the following PEG chain (ThT-PEG) can also be used.
Here, ^R5 is an amino group, a hydroxyl group, an alkyl group, or a carboxyl group, and n is an integer from 4 to 50, preferably an integer from 5 to 20, more preferably an integer from 8 to 15, and particularly preferably 11. As ThT-PEG, a compound in which ^R5 is an amino group is more preferable.

It is also possible to use a ThT derivative in which the following ThT is linked with a PEG chain (ThT-PEG-ThT).
Here, n is an integer of 4 to 50, preferably an integer of 5 to 20, more preferably an integer of 8 to 15, and particularly preferably 11. In addition, the PEG chain of ThT-PEG-ThT may be substituted with a spermine linker.

For example, the G-quadruplex structure in the test DNA can be detected by contacting a ThT derivative with a sample containing an RCA product and detecting the ThT derivative bound to the G-quadruplex structure based on the fluorescence emitted by the ThT derivative. It is preferable that the ThT derivative is added to the reaction solution in advance.

When ThT-PEG or ThT-PEG-ThT is used as the G-quadruplex binding reagent, specific aggregation occurs when ThT-PEG or ThT-PEG-ThT binds to the RCA product, and the presence or absence of RCA amplification can be easily confirmed by visually observing the aggregation without using a fluorescence detection device. ThT-PEG and ThT-PEG-ThT may be used simultaneously. The concentration of ThT-PEG or ThT-PEG-ThT is, for example, 5 to 50 μM, preferably 5 to 20 μM.

The reagent premix for the SATIC method may contain a nucleic acid polymerase such as phi29 DNA polymerase in the first reagent layer, and other reagents, i.e., the first and second primers, the first and second circular DNAs, as well as dNTPs, buffers, and detection reagents (such as ThT-PEG) in the second reagent layer. When the second reagent layer contains the first and second primers, an intermediate reagent layer containing the first and second circular DNAs, as well as dNTPs, buffers, and detection reagents (such as ThT-PEG) may be provided between the first and second reagent layers, separated by a membrane composed of a substance such as PEG that is solid at least below 0°C but dissolves in an aqueous solution between 0 and 35°C (Figure 1B). The first and second primers contained in the second reagent layer may be immobilized on the same carrier.

<Nucleic acid amplification method>
To carry out a nucleic acid amplification reaction using the nucleic acid amplification reaction kit of the present invention, a step of placing the nucleic acid amplification reaction kit in a temperature control device and a step of adding a nucleic acid-containing sample to a reaction vessel are performed.
The step of placing the reaction vessel in a temperature control device and the step of adding a nucleic acid-containing sample into the reaction vessel may be performed in any order.
As the temperature control device, in the case of PCR, a thermal cycler can be mentioned, and in the case of isothermal amplification methods such as the LAMP method and the SATIC method, a constant temperature device (bath) can be mentioned.

<Method of manufacturing the nucleic acid amplification reaction kit>
The nucleic acid amplification reaction kit of the present invention includes the steps of: adding a liquid (aqueous solution) containing a nucleic acid polymerase to a nucleic acid amplification reaction vessel to form a first reagent layer;
laminating a film on the first reagent layer, the film being composed of a substance that is solid at temperatures below 0° C. and dissolves in an aqueous solution at temperatures between 0 and 35° C.;
The second reagent layer can be produced by adding a liquid (aqueous solution) containing a primer oligonucleotide or the like onto the film of the substance at a temperature below 0°C, i.e., in a state in which the film does not dissolve, to form a second reagent layer.
When the first reagent layer is disposed on the upper layer, the second reagent layer may be formed first, a membrane may be laminated thereon, and then the second reagent layer may be formed.

The reaction vessel may be a reaction tube (microtube) such as an Eppendorf tube, or a vessel such as a multi-well plate.

To laminate a film on the first reagent layer that is composed of a substance that is solid below 0°C and dissolves in an aqueous solution at 0 to 35°C, the substance can be dissolved in an appropriate solvent, the solution is laminated on the first reagent layer, and the solvent is volatilized by vacuum drying or the like. The solvent can be selected appropriately depending on the type of substance, but an organic solvent is preferred. There are no particular limitations on the lamination method, but a spray method is preferred for forming a thin film. The concentration of the substance is preferably such that when the film is dissolved, the final concentration in the nucleic acid amplification reaction system is 3 to 20 w/v%, and more preferably 5 to 12 w/v%.

Then, the reaction vessel is placed at a temperature at which the membrane does not dissolve in water, preferably below 0°C (e.g., on ice), and an aqueous solution containing a primer oligonucleotide, etc., is added onto the membrane to form a second reagent layer.

Then, the resulting nucleic acid amplification reaction kit containing the multilayered premixed reagent can be stored frozen at a low temperature at which the membrane does not dissolve, preferably below 0°C (e.g., -10 to -30°C), until the time of use.

When manufacturing a multi-layered premixed reagent for the SATIC method, which includes a first reagent layer containing a nucleic acid polymerase, a second reagent layer containing first and second primers, and an intermediate reagent layer containing a polynucleotide primer or the like, which is located between the first and second reagent layers and is arranged with a membrane of the above-mentioned material between the first and second reagent layers, the multi-layered premixed reagent can be manufactured by carrying out the steps described below.

A step of adding a liquid containing a nucleic acid polymerase to a nucleic acid amplification reaction vessel to form a first reagent layer, and a step of laminating a film composed of a substance that is solid below 0°C but dissolves in an aqueous solution between 0 and 35°C on the first reagent layer;
adding a liquid containing a first single-stranded circular DNA and a second single-stranded circular DNA onto the membrane at a temperature below 0° C. to form an intermediate reagent layer;
A step of laminating a film composed of a substance that is solid below 0°C but dissolves in an aqueous solution between 0 and 35°C on the intermediate reagent layer at a temperature below 0°C, and a step of adding a liquid containing a first primer and a second primer onto the film at a temperature below 0°C to form a second reagent layer.

The process of laminating each film can be the same as that described above.
The method of laminating the intermediate reagent layer and the second reagent layer can be the same as that described above. When the first primer and the second primer are immobilized on a carrier, a suspension containing the carrier may be laminated.

The present invention will be specifically described below by way of examples, but the present invention is not limited to the aspects of the following examples.

Example 1. Preparation and verification of multi-layered premix (SATIC method)
1-1) Preparation of primer-immobilized nanoparticles
a) Preparation of biotinylated primer Biotinylated primer (P ₁ -1 (corresponding to target region 1)) and biotinylated primer (P ₂ ) were dissolved in 1×φ29 DNA polymerase reaction buffer to prepare a P ₁ /P ₂ mixed solution of P ₁ (1 μM) and P ₂ (20 μM).
b) Preparation and cleaning of nanoparticles before use
FG beads (streptavidin) were thoroughly vortexed and stirred until they became uniform particles, and 40 μL was scooped out and placed in a 1.5 mL Eppendorf tube.
The tube was placed on a magnetic rack (a tube holder with a magnet) and the magnetic beads and the supernatant were separated (5 minutes). The supernatant was removed and 40 μL of 1×φ29 DNA polymerase reaction buffer was added and pipetted.
The above procedure was repeated two more times.
c) Immobilization and washing of primers on nanoparticles The tubes were placed on a magnetic rack (a tube holder with a magnet) to separate the magnetic beads from the supernatant (5 minutes). The supernatant was then removed.
To the washed FG beads, 4 μL of the above _P1 · _P2 mixed solution was added.
The mixture was incubated at 25°C for 30 minutes, with vortexing every 5 minutes.
The tube was placed on a magnetic rack (a tube holder with a magnet) and the magnetic beads and the supernatant were separated (5 minutes). The supernatant was removed and 40 μL of 1×φ29 DNA polymerase reaction buffer was added and pipetted.
The above procedure was repeated two more times.
The tube was placed on a magnetic rack (a tube holder with a magnet) to separate the magnetic beads from the supernatant (5 minutes). The supernatant was removed, and 40 μL of water was added and pipetted.
The above procedure was repeated two more times, and the mixture was stored in a refrigerator until use.
When preparing the premix, the tube was placed on a magnetic rack (a tube holder with a magnet) to separate the magnetic beads from the supernatant (5 minutes). The supernatant was removed. Then, 20 μL of PEG1000 and 20 μL of 10 μg/μL BSA that had been dissolved by heating were added (liquid 2).

1-2) Preparation of multi-layered premix
i. 1 μL of 10 U/μL φ29 DNA polymerase was added to the bottom of the tube.
ii. 0.5g/mL PEG 1000 dichloromethane solution was sprayed onto the top of the tube using a spray bottle. (This creates a layer of PEG 1000 on top of the polymerase.)
iii. The tube was cooled (0°C) and vacuum dried (5 minutes).
iv. The tube was cooled to -21°C. While the tube was cooled on ice, chilled Liquid 1 was added onto the tube in ii. 43 μL was added.
v. The tube was cooled to -21°C (freezing liquid 1).
vi. While cooling the tube (0°C), spray 0.5g/mL PEG 1000 dichloromethane solution onto the top of the tube with a spray bottle. (PEG 1000 is coated on the frozen liquid 1 to create a layer.)
vii. 1 μL of liquid 2 was carefully added onto the PEG1000 layer.
viii. This was used as the SATIC premix reagent and stored frozen.

1-3) Reaction using primer-immobilized nanoparticles
To the SATIC premix reagent, 5 μL of target DNA (purified 1 kbp DNA fragment (10 pM) or cell lysate) was further added.
The reaction was carried out at 37°C with shaking at 1500 rpm for 20 minutes.

1-4) Visual Observation The changes in the nanoparticles were observed visually.

result
When the frozen premixed reagent was used in the SATIC reaction, the target DNA was specifically detected (Figure 3).

Example 2. Preparation and validation of multi-layered premix (for PCR)
2-1) Preparation of multi-layered premix
i. Add 1 μL of 5 U/μL Taq DNA polymerase to the bottom of the tube.
ii. 0.5g/mL PEG 1000 dichloromethane solution was sprayed onto the top of the tube using a spray bottle. (This creates a layer of PEG 1000 on top of the polymerase.)
iii. The tube was cooled (0°C) and vacuum dried (5 minutes).
iv. The tube was cooled to -21°C. While the tube was cooled on ice, the cooled liquid 3 was added onto the tube in ii. 44 μL was added. The tube was cooled to -21°C (liquid 3 was frozen).
v. This was used as a PCR premix reagent and stored frozen.

2-2) PCR reaction
5 μL of target DNA (purified 1 kbp DNA fragment (10 pM) or cell lysate) was added to the PCR premix reagent (5 μL of water was added as a negative control).
PCR cycles were performed
1. 95℃ 5min
2. 95℃ 0.5min
3. 62℃ 0.5min
4. 72℃ 2min
28 cycles of 2→4
5. 72℃ 5min

2-3) Agarose electrophoresis PCR products were observed by agarose electrophoresis.

result:
When the frozen premixed reagent was used, a PCR product of 1 kbp was obtained (Figure 4).

Example 3. Preparation and verification of multi-layered premix (LAMP method)
3-1) Preparation of multi-layered premix
i. Add 1 μL of 1.6 U/μL Bst3.0 DNA polymerase to the bottom of the tube.
ii. 0.5g/mL PEG 1000 dichloromethane solution was sprayed onto the top of the tube using a spray bottle. (PEG1000 was coated on top of the polymerase to create a layer.)
iii. The tube was cooled (0°C) and vacuum dried (5 min).
iv. The tube was cooled to -21°C. While the tube was cooled on ice, the cooled liquid 4 was added onto the tube in ii. 44 μL was added. The tube was cooled to -21°C (liquid 4 was frozen).
v. This was used as the LAMP premix reagent and stored frozen.

3-2) LAMP reaction
5 μL of target DNA (purified 1 kbp DNA fragment (10 pM) or cell lysate) was added to the LAMP premix reagent.
The reaction was carried out at 60°C for 30 minutes.

3-3) Visual Observation
The color change of HNB was observed visually.

result:
When the frozen premixed reagent was used in the LAMP reaction, the target DNA was specifically detected (Figure 5).

Sequences of template DNA and primers used: Purified 1 kbp DNA fragment:
(SEQ ID NO: 1) Length: 1001bp
cT ₁ (CYP2C19*3):CAA AAA ACA GGG GGT GCT TAC AAT CCT AGG ATT GTA TAG AAG CTG TTG TAT TGT TGT CGA AGA AGA AAA GTC C (SEQ ID NO: 2) Length: 73base
cT2 CCC AAC CCT ACC CAC CCT CAA GAA AAA AAA GTG ATA ATT GTT GTC GAA GAA GAA AAA AAA TT (SEQ ID NO: 3) Length: 62base
_P1 (CYP2C19*3): TTA CCT GGA TCT TTT TTG (SEQ ID NO: 4) Length: 18 bases
_P2GAA GCT GTT GTT ATC ACT (SEQ ID NO: 5) Length: 18 bases
PCR-FP (CYP2C19*3): TTGTCTACAGACTTTGCAGACTGATGTGAT (SEQ ID NO: 6) Length: 30 bases
PCR-RP (CYP2C19*3): GAGACATCAAACACCTCTGCCCTATTCAGT (SEQ ID NO: 7) Length: 30 bases
FIP (CYP2C19*3): TCCAGGGGTCTTAACTTGATGGAAAAAT (SEQ ID NO: 8) Length: 28 bases
BIP (CYP2C19*3): GGATCCAGGCCCAGAAAAAAAGACTGT (SEQ ID NO: 9) Length: 28 bases
F3 (CYP2C19*3): TCCAGAAACGTTTCG (SEQ ID NO: 10) Length: 15 base
B3 (CYP2C19*3): AGGGCTTGGTCAATAT (SEQ ID NO: 11) Length: 16 base
LoopF (CYP2C19*3): GCTTACAATCCTGATGTT (SEQ ID NO: 12) Length: 18 bases
Loop B (CYP2C19*3): GTAAGGCCAAGTTTTTTG (SEQ ID NO: 13) Length: 18 bases

[Synthesis Example]
Synthesis of ThT derivatives: ThT-PEG-ThT was synthesized according to the following scheme. The synthesis method of ThT-AE was as described in the reference (Kataoka, Y.; Fujita, H.; Afanaseva, A.; Nagao, C.; Mizuguchi, K.; Kasahara, Y.; Obika, S.; Kuwahara, M. Biochemistry, 2019, 58, 493.).

[Synthesis of compound T1]
Dry dichloromethane ( _{CH2Cl2 )} (0.30 mL) was added to ThT-AE (32 mg, 0.10 mmol) and stirred. Triethylamine (TEA) (85 μL, 0.61 mmol) was then added, followed by succinic anhydride (11 mg, 0.11 mmol), and the mixture was stirred at room temperature for 30 minutes. The reaction mixture was evaporated under reduced pressure, and the residue was suspended in water and washed _{with CH2Cl2} . Compound T1 was quantitatively obtained by evaporating the aqueous layer under reduced pressure. ESI-MS (positive ion mode) m/z, found =412.15, calculated for [M ⁺ ] =412.17.

[Synthesis of ThT-PEG-ThT]
ThT-PEG (10 mg, 10 μmol) was added to dry DMF (0.3 mL) and stirred, and then HOBt.H ₂ O (4.
To the mixture, compound T1 (5.6 mg, 13 μmol) dissolved in dry DMF (0.2 mL) was added, and the mixture was stirred at room temperature for 5 hours. The reaction mixture was evaporated under reduced pressure, and the residue was dissolved in CH ₂ Cl ₂ and washed with water. The organic layer was evaporated under reduced pressure, and the mixture was extracted with diethyl ether and purified using HPLC to obtain ThT-PEG-ThT.
Yield: 0.82 mg Yield: 6.1%

¹ H NMR (500 MHz, Deuterium oxide) δ 8.01 (2H, d) 7.97 (2H, s) 7.75 (6H, t) 6.95
(4H, d) 5.12 (4H, t) 3.86 (4H, s) 3.68 (44H, q) 3.61 (4H, q) 3.42-3.35 (4H, m) 3.10 (12H, s) 2.65 (4H, t) 2.57 (3H, s) 2.54 (3H, t); ESI-MS (positive ion mode)
m/z, found = 1348.51, calculated for [H ⁺ ]= 1348.67.

20...single-stranded circular DNA, 21...target nucleic acid, 22...first oligonucleotide primer, 23...first amplification product, 24...second single-stranded circular DNA, 25...second oligonucleotide primer, 26...second amplification product, 201...sequence complementary to first site, 202...first primer binding sequence, 203...second single-stranded circular DNA binding sequence, 204...site adjacent to the 5' side of 203, 211...first site, 212...second site, 221...sequence complementary to second site, 222...second Sequence complementary to the 1 primer binding site, 231..., complementary region of 203, 232...region complementary to site 204, 233...sequence complementary to sequence 203, 241...sequence identical to the second single-stranded circular DNA binding sequence 203, 242...second primer binding sequence, 243...sequence complementary to the G-quadruplex forming sequence, 251...sequence identical to site 204, 252...sequence complementary to the second primer binding sequence 242 of the second single-stranded circular DNA, 261...sequence containing G-quadruplex, 262...G-quadruplex detection reagent

Claims (6)

A kit for nucleic acid amplification reaction comprising a reaction vessel and a nucleic acid amplification reaction reagent contained in the reaction vessel,
The nucleic acid amplification reaction is a SATIC method,
The reagent comprises a first reagent layer including a nucleic acid polymerase;
a second reagent layer comprising a primer oligonucleotide ;
an intermediate reagent layer between the first reagent layer and the second reagent layer, the intermediate reagent layer including a first single-stranded circular DNA and a second single-stranded circular DNA;
Including,
the intermediate reagent layer is separated from the first reagent layer and the second reagent layer by a first membrane and a second membrane, respectively, each of which is composed of a substance that is solid below 0° C. but dissolves in aqueous solution between 0 and 35° C.;
the second reagent layer includes a first primer and a second primer;
A kit for nucleic acid amplification reaction, characterized in that each of the layers and each of the membranes is a multilayered reagent premix that is frozen and stored on the first reagent layer in a layered state in the following order, viewed from the first reagent layer: first membrane, intermediate reagent layer, second membrane, and second reagent layer . 2. The nucleic acid amplification reaction kit according to claim 1 , wherein the intermediate reagent layer further comprises a nucleic acid substrate and a reaction buffer. 3. The nucleic acid amplification reaction kit according to claim 1 , wherein the first primer and the second primer are immobilized on a carrier. The nucleic acid amplification reaction kit according to any one of claims 1 to 3, wherein the substance that is solid below 0°C but dissolves in an aqueous solution between 0 and 35 °C is polyethylene glycol, polyvinyl alcohol, a sugar chain compound, a polyamide chain compound, or a water-soluble heterocyclic compound having a self-organizing ability. A method for amplifying a nucleic acid in a nucleic acid-containing sample, comprising the steps of: placing the nucleic acid amplification reaction kit according to any one of claims 1 to 4 in a temperature control device; and adding the nucleic acid-containing sample to a reaction vessel. A step of adding a liquid containing a nucleic acid polymerase to a nucleic acid amplification reaction vessel to form a first reagent layer, and a step of laminating a film composed of a substance that is solid below 0°C but dissolves in an aqueous solution between 0 and 35°C on the first reagent layer;
adding a liquid containing a first single-stranded circular DNA and a second single-stranded circular DNA onto the membrane at a temperature below 0° C. to form an intermediate reagent layer;
A method for producing a SATIC reaction kit, which contains a multi-layered reagent premix in a reaction vessel, includes the steps of: laminating, on an intermediate reagent layer, a film composed of a substance that is solid at temperatures below 0°C but dissolves in an aqueous solution between 0 and 35°C, at a temperature below 0°C; and adding, on the film, a liquid containing a first primer and a second primer, at a temperature below 0°C, to form a second reagent layer.

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