Primer set and method for detecting target nucleic acid using same

Abstract

One or more embodiments of the present invention solve the problem that the reaction efficiency is lowered in a nucleic acid amplification reaction using a primer, said primer having a polynucleotide tag added thereto, to prepare a target nucleic acid amplification product which is detectable on a solid phase support.　One or more embodiments of the present invention pertain to a primer set comprising: a first primer containing a first polynucleotide, which contains, at the 3'-end thereof, polynucleotide A hybridizable with a complementary strand of partial polynucleotide A' at the 5'-end of a target nucleic acid, and first polynucleotide tag which is a polynucleotide independent from nucleic acid amplification reaction; a second primer containing a second polynucleotide which contains, at the 3'-end thereof, polynucleotide B hybridizable with partial polynucleotide B' at the 3'-end of the target nucleic acid; and a third primer containing a third polynucleotide, which hybridizes with the complementary strand of the target nucleic acid competitively with polynucleotide A of the first primer, but containing no polynucleotide which is independent from the nucleic acid amplification reaction.

Description

[Technical field]

[0001] One or more embodiments of the present invention relate to a primer set for preparing an amplification product of a target nucleic acid by a nucleic acid amplification reaction, the amplification product being detectable on a solid support. One or more embodiments of the present invention also relate to a kit for detecting a target nucleic acid comprising the primer set. One or more embodiments of the present invention also relate to a method for detecting a target nucleic acid, comprising carrying out a nucleic acid amplification reaction using the primer set. One or more embodiments of the present invention also relate to a multiplex primer set comprising two or more of the above primer sets. [Background technology]

[0002] In recent years, a gene detection method based on chromatography has been attracting attention as a method for detecting a target nucleic acid that is easy to operate, rapid, and simple (see, for example, Patent Document 1). The outline of this method is as follows. A target nucleic acid is amplified by a nucleic acid amplification reaction using a polynucleotide primer to which a polynucleotide tag independent of the nucleic acid amplification reaction is added, to generate a target nucleic acid fragment to which a polynucleotide tag is added. Meanwhile, a probe that binds to the polynucleotide tag (for example, a complementary polynucleotide to the polynucleotide tag) is immobilized in advance on a test strip for chromatography. Then, a sample containing a tagged target nucleic acid fragment is applied to this test strip, and the sample is developed in the test strip by utilizing capillary action, and the target nucleic acid fragment is captured by the probe and detected.

[0003] On the other hand, as a nucleic acid amplification method, an isothermal nucleic acid amplification method has been developed that amplifies the target gene isothermally, unlike the PCR method, which requires a thermal cycler to change the temperature of the sample. Isothermal nucleic acid amplification has characteristics suitable for application to POC (Point of Care) testing, such as being able to be performed with simple equipment, short reaction time, and high detection sensitivity. Known isothermal nucleic acid amplification methods include the RPA method (Recombinase Polymerase Amplification), TRIAmp method (Tandem Repeat-mediated Isothermal Amplification), LAMP method (Loop-mediated isothermal amplification), and HDA method (Helicase-dependent amplification).

[0004] Prior art documents disclosing the use of a polynucleotide primer having a polynucleotide tag attached thereto in an isothermal nucleic acid amplification method include Patent Documents 2 and 3.

[0005] Patent Document 2 describes the addition of a tag sequence and a label-binding substance to two specific primers out of six primers used in the LAMP method, and the capture and detection of the amplified product on a solid phase carrier using the same. Patent Document 3 describes an isothermal nucleic acid amplification method based on a new principle in which a tag that is independent of the nucleic acid amplification reaction is added to a primer. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] JP 2016-73312 A [Patent Document 2] WO2017 / 43114 [Patent Document 3] WO2018 / 038232 [Patent Document 4] Patent No. 5688702 [Non-patent literature]

[0007] [Non-Patent Document 1] (Frontiers in Microbiology.vol.8, Article 192) Summary of the Invention [Problem to be solved by the invention]

[0008] The present inventors have found that in nucleic acid amplification reactions such as PCR and isothermal nucleic acid amplification, the efficiency of the nucleic acid amplification reaction may be reduced when a primer having a polynucleotide tag is used, compared to when a primer without a tag is used. In addition, the present inventors have found that in isothermal amplification reactions using strand displacement polymerase, the reduction in the efficiency of the nucleic acid amplification reaction due to the use of a primer having a polynucleotide tag may be particularly significant.

[0009] Therefore, one or more embodiments of the present invention address the problem of reduced reaction efficiency in nucleic acid amplification reactions using primers tagged with polynucleotides to prepare amplification products of target nucleic acids that can be detected on a solid phase carrier. [Means for solving the problem]

[0010] This specification discloses the following invention as a means for solving the above problems. (1) A primer set for preparing an amplification product of a target nucleic acid detectable on a solid phase carrier by a nucleic acid amplification reaction, comprising: the primer set includes a first primer, a second primer, and a third primer, The first primer comprises a first polynucleotide comprising, at its 3' end, a polynucleotide A capable of hybridizing to a complementary strand of a partial polynucleotide A' at the 5' end of a target nucleic acid, and a first polynucleotide tag which is a polynucleotide independent of a nucleic acid amplification reaction and is linked to the 5' end of the first polynucleotide; the second primer comprises a second polynucleotide comprising a polynucleotide B at its 3' end capable of hybridizing to a partial polynucleotide B' at the 3' end of the target nucleic acid; the third primer comprises a third polynucleotide comprising, at its 3' end, a polynucleotide C capable of competitively hybridizing to the polynucleotide A of the first primer with respect to a complementary strand of the target nucleic acid, and does not comprise a polynucleotide independent of a nucleic acid amplification reaction; The primer set. (2) The primer set described in (1), wherein the polynucleotide C of the third primer is capable of hybridizing with a polynucleotide having 50% or more of the number of bases contained in the complementary strand of the partial polynucleotide A' of the target nucleic acid. (3) The primer set according to (1) or (2), wherein the ratio of the third primer to the total amount of the first primer and the third primer is 1 mol % or more and 90 mol % or less. (4) The primer set according to (3), wherein a ratio of the third primer to the total amount of the first primer and the third primer is 2.5 mol % or more and 75 mol % or less. (5) The primer set according to any one of (1) to (4), wherein in the first primer, the 3' end of the first polynucleotide tag and the 5' end of the first polynucleotide are linked via an inhibitor that inhibits a nucleic acid amplification reaction. (6) The primer set according to any one of (1) to (5), wherein the second primer further comprises a labeling substance linked to the second polynucleotide. (7) The primer set according to (6), wherein the labeling substance is biotin, a fluorescent dye or a hapten. (8) the primer set further comprises a fourth primer, The second primer further comprises a second polynucleotide tag linked to the 5' end of the second polynucleotide, the second polynucleotide tag being a polynucleotide independent of a nucleic acid amplification reaction; The primer set according to any one of (1) to (5), wherein the fourth primer comprises a fourth polynucleotide having, at its 3' end, a polynucleotide D that is capable of competitively hybridizing to the polynucleotide B of the second primer for the target nucleic acid, and does not comprise a polynucleotide that is independent of a nucleic acid amplification reaction. (9) The primer set according to (8), wherein the polynucleotide D of the fourth primer is capable of hybridizing with a polynucleotide having 50% or more of the number of bases contained in the partial polynucleotide B' of the target nucleic acid. (10) The primer set according to (8) or (9), wherein the ratio of the fourth primer to the total amount of the second primer and the fourth primer is 1 mol % or more and 90 mol % or less. (11) The primer set according to (10), wherein a ratio of the fourth primer to the total amount of the second primer and the fourth primer is 2.5 mol % or more and 75 mol % or less. (12) The primer set according to any one of (8) to (11), wherein in the second primer, the 3' end of the second polynucleotide tag and the 5' end of the second polynucleotide are linked via an inhibitor that inhibits a nucleic acid amplification reaction.

[0011] (13) A nucleic acid detection device comprising the primer set according to any one of (1) to (7) and a solid phase carrier, The solid phase carrier includes a capture substance holder that includes a capture substance capable of binding to the first polynucleotide tag. A kit for detecting a target nucleic acid. (14) A nucleic acid detection device comprising the primer set according to any one of (8) to (12) and a solid phase carrier, The solid phase carrier includes a capture substance holder including a capture substance capable of binding to the first polynucleotide tag, and a labeling substance holder including a labeling substance capable of binding to the second polynucleotide tag. A kit for detecting a target nucleic acid. (15) The kit according to (13) or (14), wherein the nucleic acid detection device further comprises a sample receiving portion for receiving a sample containing a nucleic acid amplification product.

[0012] (16) A nucleic acid amplification step comprising subjecting a test substance that may contain a target nucleic acid to a nucleic acid amplification reaction using the primer set according to any one of (1) to (12); and A detection step of detecting a target nucleic acid in the reaction product obtained in the nucleic acid amplification step. A method for detecting a target nucleic acid. (17) The method according to (16), wherein in the detection step, the target nucleic acid is detected using a solid phase carrier. (18) The method according to (16) or (17), wherein the nucleic acid amplification reaction in the nucleic acid amplification step is carried out using a strand displacement polymerase. (19) The method according to (18), wherein the nucleic acid amplification reaction in the nucleic acid amplification step is carried out by an RPA method, a TRIamp method, a LAMP method, or an HDA method. (20) The method according to (16) or (17), wherein the nucleic acid amplification reaction in the nucleic acid amplification step is carried out by a PCR method.

[0013] (21) The method includes the step of: the target nucleic acids amplified by the nucleic acid amplification reaction using the two or more primer sets are different from each other; Multiplex primer set.

[0014] This specification includes the disclosures of Japanese Patent Application No. 2019-239045, which is the priority basis of this application. Effect of the Invention

[0015] By carrying out a nucleic acid amplification reaction using a primer set according to one or more embodiments of the present invention, it is possible to efficiently prepare an amplification product of a target nucleic acid to which a polynucleotide tag has been added, which can be detected on a solid phase support. [Brief description of the drawings]

[0016] [Figure 1-1] Fig. 1-1(a) shows an example of a test nucleic acid 3 including a target nucleic acid 1 and a complementary strand 2 of the target nucleic acid 1, a first primer 10, a second primer 20, and a third primer 30. Fig. 1-1(b1) shows an isothermal nucleic acid amplification reaction using the test nucleic acid 3 as a template and the first primer 10 and the second primer 20. Fig. 1-1(b2) shows an isothermal nucleic acid amplification reaction using the test nucleic acid 3 as a template and the third primer 30 and the second primer 20. [Figure 1-2] Fig. 1-2(c1) shows a reaction in which a first primer 10 and a second primer 20 hybridize to a double strand of a target nucleic acid 1 and a complementary strand 2, which is an amplified product of a previous nucleic acid amplification reaction, to initiate a polymerase extension reaction. Fig. 1-2(c2) shows a schematic diagram of an isothermal nucleic acid amplification reaction using a third primer 30 and a second primer 20, with the double strand of the target nucleic acid 1 and the complementary strand 2 as a template. Fig. 1-2(d) shows a detection nucleic acid 7, which is a double-stranded nucleic acid consisting of a target nucleic acid 1 linked to a first polynucleotide tag 12 at its 5' end and a complementary strand 2 linked to a labeling substance 23 at its 5' end. [Diagram 2] Fig. 2(a) shows an example of a test nucleic acid 3 including a target nucleic acid 1 and a complementary strand 2 of the target nucleic acid 1, a first primer 10, a second primer 20, a third primer 30, and a fourth primer 40. Fig. 2(b) shows a detection nucleic acid 8 which is a double-stranded nucleic acid consisting of a target nucleic acid 1 linked to a first polynucleotide tag 12 at its 5' end and a complementary strand 2 linked to a second polynucleotide tag 22 at its 5' end. [Diagram 3] 3 is a schematic diagram of a lateral flow type nucleic acid detection device 700. 71: solid phase carrier, 72: conjugate pad (labeled substance holding portion), 73: sample pad (sample receiving portion), 74: absorption pad, 75: substrate, 76: capture substance holding portion. [Figure 4] FIG. 4 shows the results of nucleic acid amplification by the TRIAmp method using primers with different ratios of DNA-tagged primers and non-tagged primers as forward and reverse primers in Example 2, and serially diluted target nucleic acids at different concentrations. [Diagram 5] FIG. 5 shows the results of nucleic acid amplification reactions of target nucleic acids by the TRIAmp method measured with a real-time PCR device using primer mix 1 of a forward primer and a reverse primer without a DNA tag attached, primer mix 2 of a forward primer with a DNA tag attached and a biotin-added reverse primer, and primer mix 3 of a forward primer with a DNA tag attached, a forward primer without a DNA tag attached, and a biotin-added reverse primer in Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS One or more embodiments of the invention are described in detail below.

[0018] <Terminology> In one or more embodiments of the present invention, the terms nucleic acid and polynucleotide refer to DNA or RNA, typically DNA. The terms nucleic acid and polynucleotide also include oligonucleotides without any particular limitation on the number of bases. In one or more embodiments of the present invention, the target nucleic acid serving as a template for a nucleic acid amplification reaction, the complementary strand of the target nucleic acid, and the polynucleotide are typically polymers of natural nucleotides. A natural nucleotide is a nucleotide composed of natural adenine, thymine, guanine, cytosine, and uracil bases, a sugar moiety of deoxyribose or ribose, and a phosphate group, and each part is a nucleotide that has not been artificially modified. Natural nucleotides are usually D-type nucleotides. A D-type nucleotide refers to a nucleotide whose sugar moiety is composed of D-type deoxyribose or ribose.

[0019] A polynucleotide may be a partial polynucleotide contained in a part of a relatively long polynucleotide. When "polynucleotide P2 containing polynucleotide P1" or "polynucleotide P2 contains polynucleotide P2", it includes both the case where polynucleotide P1 is the whole of polynucleotide P2 (i.e., polynucleotide P1 and polynucleotide P2 are identical) and the case where polynucleotide P1 is a partial polynucleotide of polynucleotide P2.

[0020] In one or more embodiments of the present invention, the term "target nucleic acid" refers to a nucleic acid containing a base sequence to be detected and / or amplified, or a nucleic acid containing a complementary base sequence of a base sequence to be detected and / or amplified. The target nucleic acid may exist as a double strand together with its complementary strand. One strand of a target nucleic acid existing as a double strand may be referred to as the "target nucleic acid". Either the nucleic acid to be detected or its complementary strand may be referred to as the "target nucleic acid". That is, in one or more embodiments of the present invention, "detecting a target nucleic acid" or "amplifying a target nucleic acid" includes both detecting or amplifying a target nucleic acid for the purpose of detecting or amplifying the target nucleic acid itself, and detecting or amplifying a target nucleic acid, a complementary strand of a target nucleic acid, or a double-stranded nucleic acid of a target nucleic acid and a complementary strand for the purpose of detecting or amplifying the complementary strand of the target nucleic acid or the double-stranded nucleic acid of the target nucleic acid and the complementary strand.

[0021] In one or more embodiments of the present invention, the total length of the target nucleic acid is not particularly limited, and is usually 20 bases or more, 40 bases or more, or 100 bases or more in length. The upper limit of the total length of the target nucleic acid is not particularly limited, and is usually 1000 bases or less, 500 bases or less, or 400 bases or less in length.

[0022] In one or more embodiments of the present invention, the nucleic acid serving as a template for a nucleic acid amplification reaction may be DNA or RNA as long as it contains a target nucleic acid and / or its complementary strand in a portion thereof, but is preferably DNA. Usually, the nucleic acid serving as a template for a nucleic acid amplification reaction is a double-stranded DNA consisting of a polynucleotide strand containing at least a portion of the target nucleic acid and a complementary strand of the polynucleotide strand.

[0023] The template nucleic acid may be natural or artificially synthesized. For example, it may be natural nucleic acid extracted from a biological sample, amplified by PCR or the like, or cDNA synthesized by reverse transcription reaction.

[0024] In one or more embodiments of the present invention, polynucleotide X "hybridizes" to polynucleotide Y means that polynucleotide X (particularly DNA) hybridizes to polynucleotide Y (particularly DNA) under stringent conditions and does not hybridize to polynucleotides that do not have the base sequence of polynucleotide Y. In other words, hybridization refers to specific hybridization. Here, "stringent conditions" refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed, and can be appropriately determined by referring to, for example, Green and Sambrook, Molecular Cloning, 4th Ed (2012), Cold Spring Harbor Laboratory Press. Specifically, stringent conditions can be set by the temperature and salt concentration contained in the solution during Southern hybridization, and the temperature and salt concentration contained in the solution during the washing step of Southern hybridization. More specifically, stringent conditions include, for example, a sodium concentration of 25 to 500 mM, preferably 25 to 300 mM, and a temperature of 40 to 68° C., preferably 40 to 65° C. in the hybridization step. More specifically, hybridization can be performed in 1 to 7×SSC (saline-sodium citrate buffer), 0.02 to 3% SDS, and at a temperature of 40 to 60° C. In addition, a washing step may be performed after hybridization, and the washing step can be performed, for example, in 0.1 to 2×SSC, 0.1 to 0.3% SDS, and at a temperature of 50 to 65° C. However, hybridization between a first polynucleotide tag (fixing tag) described later and a solid support to which a polynucleotide has been added, and hybridization between a second polynucleotide tag (labeling tag) described later and a labeling substance to which a polynucleotide has been added do not need to be performed under the stringent conditions listed here, and can be performed under the conditions described later.

[0025] When polynucleotide X hybridizes to polynucleotide Y, polynucleotide X (particularly DNA) and polynucleotide Y (particularly DNA) need only be a combination capable of forming sufficient hydrogen bonds to hybridize and form a stable double strand under the annealing conditions of a nucleic acid amplification reaction, and do not need to have completely complementary base sequences. For example, there may be some mismatches between the base sequence of polynucleotide X (hereinafter referred to as "base sequence X") and the base sequence of polynucleotide Y (hereinafter referred to as "base sequence Y"), such as one mismatch or less in 10 bases, one mismatch or less in 20 bases, or one mismatch or less in 30 bases. When polynucleotide X hybridizes to polynucleotide Y, it usually satisfies one or more of the following relationships (A) to (C).

[0026] (A) The complementary base sequence of base sequence X is identical to base sequence Y. When one of the complementary base sequence of base sequence X and base sequence Y is a DNA base sequence and the other is an RNA base sequence, thymine in one and uracil in the other are considered to be the same base. (B) The base sequence Y is a base sequence in which one or several bases have been deleted, substituted, added and / or inserted in the complementary sequence of the base sequence X. (C) Base sequence Y has an identity of 80% or more with the complementary sequence of base sequence X.

[0027] In the above (B), "one or several" preferably refers to one to five, more preferably one to four, more preferably one to three, particularly preferably one or two, and most preferably one.

[0028] In (C), the identity value indicates a value calculated by using software (e.g., FASTA, DANASYS, and BLAST) that calculates identity between multiple base sequences with default settings. The identity value of base sequences is calculated by calculating the number of matching bases when a pair of base sequences are aligned to maximize the degree of identity, and is calculated as a ratio of the number of matching bases to the total number of bases of the compared base sequences. Here, when there is a gap, the total number of bases is the number of bases counted with one gap as one base. For details of the method of determining identity, see, for example, Altschul et al., Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al., J. Mol. Biol. 215, 403-410, 1990.

[0029] In the above (C), the identity is more preferably 90% or more, more preferably 95% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, and more preferably 99% or more. Among the above (A) to (C), the above (A) is particularly preferred. The expression "polynucleotide X hybridizes to polynucleotide Y" is used synonymously with the expression "base sequence X hybridizes to base sequence Y."

[0030] In one or more embodiments of the present invention, the method for producing the polynucleotides constituting the primer set is not particularly limited, and the polynucleotides may be produced using a polynucleotide synthesizer or a custom synthesis service.

[0031] In this specification, a polynucleotide that is "independent of a nucleic acid amplification reaction" can be rephrased as a polynucleotide that is "not involved in a nucleic acid amplification reaction." A polynucleotide that is "independent of a nucleic acid amplification reaction" is a polynucleotide that is not used as a template for a nucleic acid amplification reaction and is not formed into double strands by a nucleic acid amplification reaction.

[0032] <Nucleic acid amplification reaction> The nucleic acid amplification reaction using the primer set according to one or more embodiments of the present invention may be a nucleic acid amplification reaction using a thermostable polymerase or a nucleic acid amplification reaction using a strand displacement polymerase. The polymerase refers to a nucleic acid polymerase, which is a DNA polymerase or an RNA polymerase, and is preferably a DNA polymerase.

[0033] An example of a nucleic acid amplification reaction using a heat-resistant polymerase is the polymerase chain reaction (PCR). As the heat-resistant polymerase, a commercially available DNA polymerase can be used, and for example, TaKaRa Ex Taq (registered trademark) can be preferably used. In addition, the temperature, time, buffer composition, etc. can be appropriately selected depending on the DNA polymerase used and the concentration of each primer. Each condition of the denaturation, annealing, and extension steps in the PCR method, such as time, temperature, buffer composition, substrate nucleotide concentration, and cycle number, can be appropriately set in consideration of factors such as the selected DNA polymerase, primer sequence, number of bases of the target nucleic acid, and template concentration.

[0034] Strand displacement polymerases are enzymes that synthesize new DNA strands while dissociating hydrogen bonds of double-stranded nucleic acids including the target nucleic acid, and examples of such polymerases include φ29 DNA polymerase, Bst DNA polymerase, Klenow fragment of DNA polymerase I, Vent DNA polymerase, Vent (Exo-) DNA polymerase, DeepVent DNA polymerase, DeepVent (Exo-) DNA polymerase, 96-7 DNA polymerase, Aac DNA polymerase, and Csa DNA polymerase. Strand displacement polymerases do not require thermal denaturation for dissociation of the double strand, so they are capable of isothermal nucleic acid amplification.

[0035] As a nucleic acid amplification reaction using a strand displacement polymerase, an isothermal nucleic acid amplification method is preferable, and examples of the method include the RPA method (Recombinase Polymerase Amplification), the TRIAmp method (Tandem Repeat-mediated Isothermal Amplification), the LAMP method (Loop-mediated isothermal amplification), and the HDA method (Helicase-dependent amplification).

[0036] In the RPA method and the TRIAmp method, at least one pair of primer sets is used to amplify a target nucleic acid. In this case, as one of the pair of primer sets, the first and third primers of the primer set may be used in one or more embodiments of the present invention, and as another primer, the second primer or the second and fourth primers of the primer set may be used in one or more embodiments of the present invention.

[0037] In the LAMP method, four or more primers are used to amplify one target nucleic acid. As one of the four or more primers in the LAMP method, the first and third primers of a primer set can be used in one or more embodiments of the present invention, and as another primer, the second primer or the second and fourth primers of the primer set can be used in one or more embodiments of the present invention. Preferably, one or both of the one primer and the other primer are loop primers.

[0038] The isothermal nucleic acid amplification method using a strand displacement polymerase proceeds by incubating a template nucleic acid containing a target nucleic acid, a primer, a strand displacement polymerase, and substrate nucleotides, as well as other enzymes as necessary (for example, in the RPA method, recombinase and single-stranded DNA binding protein (SSB) are used as other enzymes) at a temperature at which the amplification primer can form stable base pairs with the template nucleic acid and at which the enzyme activity can be exerted. Each condition of the nucleic acid amplification reaction by the isothermal nucleic acid amplification method, such as temperature, buffer composition, substrate nucleotide concentration, and reaction time, can be appropriately set in consideration of factors such as the selected strand displacement polymerase, primer sequence, number of bases in the target nucleic acid, and template nucleic acid concentration. Examples of recombinases used in the RPA method include UvsX, RecA, and analogs thereof.

[0039] <Primer set> The primer set of one or more embodiments of the present invention, which includes a first primer, a second primer, and a third primer, is a primer set that, when a test substance to be analyzed contains a nucleic acid including a base sequence of a target nucleic acid, can prepare a detection nucleic acid including a target nucleic acid having a first polynucleotide tag linked to one end by a nucleic acid amplification reaction using the target nucleic acid in the test substance as a template. The structure of each primer will be described below, followed by a description of a preferred embodiment of the primer set.

[0040] (First primer) The first primer comprises a first polynucleotide comprising, at its 3' end, a polynucleotide A capable of hybridizing to a complementary strand of partial polynucleotide A' at the 5' end of the target nucleic acid, and a first polynucleotide tag linked to the 5' end of the first polynucleotide, which is a polynucleotide independent of the nucleic acid amplification reaction.

[0041] The partial polynucleotide A' is a partial polynucleotide of a target nucleic acid, which is composed of a plurality of consecutive bases including the base at the 5' end of the target nucleic acid. The number of bases of the partial polynucleotide A' is not particularly limited, and may be, for example, 8 bases or more, 10 bases or more, 12 bases or more, 15 bases or more, 17 bases or more, or 20 bases or more, or 40 bases or less, 30 bases or less, 27 bases or less, or 25 bases or less.

[0042] The polynucleotide A may be any polynucleotide capable of hybridizing with the complementary strand of the partial polynucleotide A'. In a preferred embodiment of the polynucleotide A, one or more consecutive bases, preferably two or more bases, preferably three or more bases, more preferably five or more bases, and most preferably the entire base sequence at the 3' end of the polynucleotide A are identical to the base sequence of the corresponding portion of the partial polynucleotide A'. The number of bases of the polynucleotide A is not particularly limited and may be appropriately determined depending on the number of bases of the partial polynucleotide A', and may be, for example, 8 or more bases, 10 or more bases, 12 or more bases, 15 or more bases, 17 or more bases, or 20 or more bases, or 40 or less bases, 30 or less bases, 27 or less bases, or 25 or less bases.

[0043] The base sequence of the first polynucleotide may contain the base sequence of polynucleotide A at its 3'-end, and may further include another base sequence added to the 5'-end side of the base sequence of polynucleotide A, or may consist of only the base sequence of polynucleotide A. That is, polynucleotide A may be a partial polynucleotide including the 3'-end of the first polynucleotide, or may be the entire first polynucleotide. The number of bases of the first polynucleotide may be appropriately determined depending on the number of bases of polynucleotide A, and may be, for example, 8 bases or more, 10 bases or more, 12 bases or more, 15 bases or more, 17 bases or more, or 20 bases or more, or 40 bases or less, 30 bases or less, 27 bases or less, or 25 bases or less.

[0044] The first primer comprises a first polynucleotide and a first polynucleotide tag linked to its 5' end, the first polynucleotide tag being a polynucleotide independent of the nucleic acid amplification reaction. The first polynucleotide tag may be referred to as a "first tag" or an "immobilization tag". The polynucleotide contained in the first polynucleotide tag is not particularly limited as long as it does not substantially interfere with the nucleic acid amplification reaction by the primer set of one or more embodiments of the present invention, but is a polynucleotide having, for example, 5 to 50 bases, preferably 10 to 35 bases, and for example, a polynucleotide containing the base sequence described in Anal. Biochem. 364 (2007) 78-85 can be exemplified as a preferred example.

[0045] The first polynucleotide tag is capable of binding to a capture substance contained in a solid phase carrier described below. A detection nucleic acid containing a target nucleic acid to which the first polynucleotide tag has been added can be immobilized on the capture substance holding portion of the solid phase carrier and detected.

[0046] The 5' end of the first polynucleotide and the first tag are linked such that the first tag is not double-stranded with the first polynucleotide functioning as a primer by extension in a nucleic acid amplification reaction. The 5' end of the first polynucleotide and the 3' end of the first tag may be linked, or the 5' end of the first polynucleotide and the 5' end of the first tag may be linked, but preferably the 5' end of the first polynucleotide and the 3' end of the first tag are linked.

[0047] In one or more embodiments, the 5' end of the first polynucleotide and the first tag are linked via an inhibitor (hereinafter sometimes referred to as a "spacer") that inhibits a nucleic acid amplification reaction.

[0048] In another embodiment, the first tag can be a polynucleotide that does not serve as a template for a polymerase reaction and is not double-stranded by extension in a nucleic acid amplification reaction, such as a polynucleotide containing a modified nucleic acid such as LNA (Locked Nucleic Acid), an L-type nucleic acid, or a 2'-O-methylated nucleotide. In this case, the first polynucleotide and the first tag may be directly linked without the spacer.

[0049] The spacer may be any that can suppress or stop the progress of the polymerase reaction in the nucleic acid amplification reaction and prevent the first tag from being double-stranded. Examples of the spacer include, but are not limited to, a nucleic acid sequence having a strong hairpin structure or pseudoknot structure, an L-type nucleic acid, a peptide nucleic acid (PNA), a bridged nucleic acid (Bridged Nucleic Acid (BNA) or Locked Nucleic Acid (LNA)), fluorescein, Cy3, Cy5, a divalent group containing an azobenzene structure represented by the following formula I, an aliphatic chain (alkylene chain or polyoxyalkylene chain), a divalent group containing an inverted sequence structure such as a 5'-5' bond or a 3'-3' bond, etc. Such a spacer allows the first polynucleotide and the first tag to be linked in the same direction. That is, the 3' end of the first tag and the 5' end of the first polynucleotide can be linked via the spacer.

[0050] [ka]

[0051] When two polynucleotide molecules are connected via a divalent group represented by formula I, the phosphate group at the 3' end of one of the divalent groups refers to the phosphate group of the 5'-terminal nucleotide of one polynucleotide molecule, and the oxygen atom at the 5' end of the other forms a phosphate ester bond with the phosphate group of the 3'-terminal nucleotide of the other polynucleotide molecule. An example of the aliphatic chain spacer is a spacer represented by the following formula (IV). 5'-OC m H 2m-O-3' Formula (IV) (In the formula, 5' represents an oxygen atom of a phosphodiester bond on the 5' side, 3' represents an oxygen atom of a phosphodiester bond on the 3' side, and m represents an integer of 1 to 40. H may be substituted with a substituent.)

[0052] In formula (IV), m is preferably 2 or more and 36 or less, more preferably 3 or more and 16 or less. H in formula (IV) may be substituted with a substituent, and typical examples of the substituent include an alkyl group, an alkoxy group, and a hydroxyl group. The number of carbon atoms in the alkyl group and alkoxy group as the substituent is preferably 1 to 8, more preferably 1 to 4. When the compound has two or more substituents, the substituents may be the same or different. Furthermore, it is also preferable that the compound has no substituent.

[0053] Another example of the spacer is a spacer represented by the following formula (V): 5'-(OC n H 2n ) L -O-3' Formula (V) (In the formula, 5' represents an oxygen atom of a phosphodiester bond on the 5' side, 3' represents an oxygen atom of a phosphodiester bond on the 3' side, n represents an integer of 2 or more and 4 or less, and L represents an integer of 1 or more such that (n+1)×L is 40 or less. H may be substituted with a substituent.)

[0054] In formula (V), (n+1)×L is preferably 2 or more and 36 or less, more preferably 3 or more and 16 or less. The same embodiments as those of the substituents in formula (IV) are applied to the substituents of H in formula (V). Furthermore, when two polynucleotide molecules are linked 5'-5', an aliphatic chain spacer represented by the above formula (IV) or formula (V) can be used.

[0055] Other aliphatic chain spacers include, for example, the following divalent groups:

[0056] [ka]

[0057] When two polynucleotide molecules are connected via these divalent groups, the phosphate group at one end of each divalent group points to the phosphate group of the 3'- or 5'-terminal nucleotide of one polynucleotide molecule, and the oxygen atom at the other end forms a phosphate ester bond with the phosphate group of the 5'- or 3'-terminal nucleotide of the other polynucleotide molecule.

[0058] (Second primer) The second primer comprises a second polynucleotide comprising, at its 3' end, a polynucleotide B capable of hybridizing to a partial polynucleotide B' at the 3' end of the target nucleic acid.

[0059] The partial polynucleotide B' is a partial polynucleotide of the target nucleic acid, which is composed of a plurality of consecutive bases including the base at the 3' end of the target nucleic acid. The number of bases of the partial polynucleotide B' is not particularly limited, and may be, for example, 8 bases or more, 10 bases or more, 12 bases or more, 15 bases or more, 17 bases or more, or 20 bases or more, or 40 bases or less, 30 bases or less, 27 bases or less, or 25 bases or less.

[0060] Polynucleotide B may be any polynucleotide capable of hybridizing with partial polynucleotide B'. In a preferred embodiment of polynucleotide B, one or more consecutive bases, preferably two or more bases, preferably three or more bases, more preferably five or more bases, and most preferably the entire base sequence at the 3' end of polynucleotide B is identical to the base sequence of the corresponding portion of the complementary strand of partial polynucleotide B'. The number of bases of polynucleotide B is not particularly limited and may be appropriately determined depending on the number of bases of partial polynucleotide B', and may be, for example, 8 or more bases, 10 or more bases, 12 or more bases, 15 or more bases, 17 or more bases, or 20 or more bases, or 40 or less bases, 30 or less bases, 27 or less bases, or 25 or less bases.

[0061] The base sequence of the second polynucleotide may contain the base sequence of polynucleotide B at its 3'-end, and may further include another base sequence added to the 5'-end side of the base sequence of polynucleotide B, or may consist of only the base sequence of polynucleotide B. That is, polynucleotide B may be a partial polynucleotide including the 3'-end of the second polynucleotide, or may be the entire second polynucleotide. The number of bases of the second polynucleotide may be appropriately determined depending on the number of bases of polynucleotide B, and may be, for example, 8 bases or more, 10 bases or more, 12 bases or more, 15 bases or more, 17 bases or more, or 20 bases or more, or 40 bases or less, 30 bases or less, 27 bases or less, or 25 bases or less.

[0062] In one or more embodiments of the present invention, the second primer may include a second polynucleotide and a labeling substance linked to the second polynucleotide. A detection nucleic acid, which is an amplification product of a target nucleic acid by a primer set including the second primer according to this embodiment, includes a first polynucleotide tag at one end and a labeling substance at the other end. This detection nucleic acid can be captured on a solid phase carrier having a capture substance holding portion including a capture substance capable of binding to the first polynucleotide tag, and detected using the labeling substance as an index.

[0063] The labeling substance used herein may be any labeling substance that does not inhibit the nucleic acid amplification reaction, and may be a labeling substance that can be detected by itself or a labeling substance that can bind to another detectable substance through a secondary reaction.

[0064] Labeling substances that do not inhibit nucleic acid amplification reactions include biotin, dyes, haptens, radioisotope-containing substances, etc. Examples of dyes include fluorescent dyes (fluorescein such as fluorescein isothiocyanate (FITC), cyanines, etc.). Examples of haptens include digoxigenin (DIG) and fluorescein isothiocyanate (FITC).

[0065] Biotin can be detected by a secondary reaction with a detectable substance linked to avidin or streptavidin. Hapten can be detected by a secondary reaction with a detectable substance linked to an antibody. The detectable substance referred to here may be a dye, a radioactive substance, a colored particle, an enzyme (peroxidase, alkaline phosphatase, luciferase, etc.), etc., and is preferably a colored particle. The "colored particle" may be a metal (e.g., gold, silver, copper, platinum, etc.) particle, a metal rod, a colored latex particle, a silica nanoparticle containing a dye, etc.

[0066] The binding position of the labeling substance on the second polynucleotide is not particularly limited as long as it does not inhibit the nucleic acid amplification reaction, but is usually the 5' end of the second polynucleotide.

[0067] In one or more other embodiments of the present invention, the second primer comprises a second polynucleotide and a second polynucleotide tag, which is a polynucleotide independent of the nucleic acid amplification reaction and is linked to the 5' end of the second polynucleotide. The detection nucleic acid, which is an amplification product of the target nucleic acid by the primer set including the second primer according to this embodiment, comprises a first polynucleotide tag at one end and a second polynucleotide tag at the other end. The detection nucleic acid can be captured on a solid support having a capture substance holding section including a capture substance capable of binding to the first polynucleotide tag, and can be detected by binding to a labeling substance capable of binding to the second polynucleotide tag, using the labeling substance as an index. It is preferable that the labeling substance capable of binding to the second polynucleotide tag is contained in the labeling substance holding section of the solid support in advance, and when a sample containing the detection nucleic acid applied to the solid support comes into contact with the labeling substance holding section, the second polynucleotide tag of the detection nucleic acid is bound to the labeling substance.

[0068] The second polynucleotide tag may be referred to as a “second tag” or a “labeling tag.” The polynucleotide contained in the second polynucleotide tag is not particularly limited as long as it does not substantially interfere with the nucleic acid amplification reaction using the primer set of one or more embodiments of the present invention, and is, for example, a polynucleotide having a base number of 5 to 50, preferably 10 to 35, and for example, a polynucleotide containing the base sequence described in Anal. Biochem. 364 (2007) 78-85 can be exemplified as a preferred example.

[0069] The 5'-end of the second polynucleotide and the second tag are linked so that the second tag is not double-stranded together with the second polynucleotide functioning as a primer by extension in a nucleic acid amplification reaction. The 5'-end of the second polynucleotide and the 3'-end of the second tag may be linked, or the 5'-end of the second polynucleotide and the 5'-end of the second tag may be linked, but preferably the 5'-end of the second polynucleotide and the 3'-end of the second tag are linked.

[0070] In one or more embodiments, the 5' end of the second polynucleotide and the second tag are linked via an inhibitor (spacer) that inhibits a nucleic acid amplification reaction. The spacer linking the 5' end of the second polynucleotide and the second tag can be selected from the same range as those described for the spacer linking the 5' end of the first polynucleotide and the first tag.

[0071] In another embodiment, the second tag can be a polynucleotide that does not serve as a template for a polymerase reaction and is not double-stranded by extension in a nucleic acid amplification reaction, such as a polynucleotide containing a modified nucleic acid such as LNA (Locked Nucleic Acid), an L-type nucleic acid, or a 2'-O-methylated nucleotide. In this case, the second polynucleotide and the second tag may be directly linked without the spacer.

[0072] The labeling substance capable of binding to the second tag is preferably a labeling substance to which a polynucleotide capable of hybridizing to the second tag (for example, a polynucleotide having a sequence complementary to the base sequence of the second tag) is linked. The conditions for hybridization between the detection nucleic acid containing the second tag and the labeling substance to which a polynucleotide capable of hybridizing to the second tag is linked are not particularly limited as long as hybridization occurs, and can be performed, for example, by reacting them at 20°C to 40°C in a buffer solution (pH 6 to 7) containing 10 mM to 50 mM phosphate. In order to increase hybridization efficiency, the buffer solution can further contain a salt such as sodium chloride.

[0073] The labeling substance to which the polynucleotide capable of hybridizing to the second tag is linked may be, in addition to the above-mentioned labeling substances linked to the second polynucleotide, colored particles, enzymes (peroxidase, alkaline phosphatase, luciferase, etc.), etc., and is preferably colored particles. The term "colored particles" includes metal (e.g., gold, silver, copper, platinum, etc.) particles, metal rods, colored latex particles, silica nanoparticles containing dyes, etc.

[0074] (Third primer) The third primer comprises a third polynucleotide having at its 3' end a polynucleotide C that is capable of competitively hybridizing to polynucleotide A of the first primer with respect to the complementary strand of the target nucleic acid, and does not comprise a polynucleotide that is independent of the nucleic acid amplification reaction. The third primer preferably consists solely of the third polynucleotide.

[0075] "Polynucleotide C capable of competitively hybridizing with polynucleotide A of the first primer to a complementary strand of a target nucleic acid" means that when either polynucleotide A of the first primer or polynucleotide C of the third primer hybridizes to a complementary strand of a target nucleic acid, the other cannot hybridize to a complementary strand of the same target nucleic acid. For example, when polynucleotide C of the third primer can hybridize to polynucleotides with 50% or more, 60% or more, 80% or more, 90% or more, 95% or more, or 100% of the number of bases contained in the complementary strand of partial polynucleotide A' of the target nucleic acid (part capable of hybridizing with polynucleotide A of the first primer), polynucleotide C can competitively hybridize with polynucleotide A of the first primer to a complementary strand of a target nucleic acid.

[0076] In a preferred embodiment of polynucleotide C, one or more consecutive bases, preferably two or more bases, preferably three or more bases, more preferably five or more bases, and most preferably the entire base sequence at the 3' end of polynucleotide C are identical to the base sequence of the corresponding portion of partial polynucleotide A'. The number of bases of polynucleotide C is not particularly limited and can be appropriately determined depending on the number of bases of partial polynucleotide A', and can be, for example, 8 or more bases, 10 or more bases, 12 or more bases, 15 or more bases, 17 or more bases, or 20 or more bases, and can be 40 or less bases, 30 or less bases, 27 or less bases, or 25 or less bases. Polynucleotide C is particularly preferably identical to polynucleotide A.

[0077] The base sequence of the third polynucleotide may contain the base sequence of polynucleotide C at its 3' end, and may further include another base sequence added to the 5' end side of the base sequence of polynucleotide C, or may consist of only the base sequence of polynucleotide C. That is, polynucleotide C may be a partial polynucleotide including the 3' end of the third polynucleotide, or may be the entire third polynucleotide. The number of bases of the third polynucleotide may be appropriately determined according to the number of bases of polynucleotide C, and may be, for example, 8 bases or more, 10 bases or more, 12 bases or more, 15 bases or more, 17 bases or more, or 20 bases or more, and may be 40 bases or less, 30 bases or less, 27 bases or less, or 25 bases or less. Particularly preferably, the third polynucleotide is the same as the first polynucleotide.

[0078] (Mechanism of nucleic acid amplification reaction using primer sets) In one example of a reaction for amplifying a target nucleic acid by an isothermal nucleic acid amplification method using a primer set according to one or more embodiments of the present invention, the significance of using a third primer together with the first primer as a forward primer for the target nucleic acid will be described with reference to Figures 1-1 and 1-2.

[0079] FIG. 1-1(a) shows an example of a test nucleic acid 3 including a target nucleic acid 1 and a complementary strand 2 of the target nucleic acid 1, a first primer 10, a second primer 20, and a third primer 30. In FIG.

[0080] The test nucleic acid 3 is a double-stranded DNA, and part of it contains a target nucleic acid 1 and a complementary strand 2, which are hybridized to each other. The target nucleic acid 1 contains a partial polynucleotide A'4 at its 5' end and a partial polynucleotide B'5 at its 3' end. The complementary strand 2 of the target nucleic acid 1 contains a complementary strand 6 of the partial polynucleotide A'4 at its 3' end.

[0081] The first primer 10 comprises a first polynucleotide 11 having at its 3' end a polynucleotide A capable of hybridizing to the complementary strand 6 of the partial polynucleotide A'4, and a first polynucleotide tag 12 linked to the 5' end of the first polynucleotide 11 and independent of the nucleic acid amplification reaction.

[0082] The second primer 20 comprises a second polynucleotide 21 comprising, at its 3′ end, a polynucleotide B capable of hybridizing to the partial polynucleotide B′ 5 , and a labeling substance 23 .

[0083] The third primer 30 comprises a third polynucleotide 31 having at its 3' end a polynucleotide C that is capable of competitively hybridizing to polynucleotide A contained in the first polynucleotide 11 of the first primer 11 with respect to the complementary strand 2 of the target nucleic acid 1, and does not comprise a polynucleotide independent of the nucleic acid amplification reaction.

[0084] When the target nucleic acid 1 in the test nucleic acid 3 is amplified by an isothermal nucleic acid amplification method using the above-mentioned primer set and strand displacement polymerase, it is presumed that the initial reactions that occur are as shown in Figure 1-1(b1) in which the first primer 10 and the second primer 20 hybridize to the test nucleic acid 3 to initiate a polymerase extension reaction, and as shown in Figure 1-1(b2) in which the third primer 30 and the second primer 20 hybridize to the test nucleic acid 3 to initiate a polymerase extension reaction.

[0085] Since the first primer 10 contains the first polynucleotide tag 12 that is not involved in the nucleic acid amplification reaction, it is considered that the steric hindrance is large when the first primer 10 hybridizes with the complementary strand 2 of the target nucleic acid 1 in the test nucleic acid 3 as shown in FIG. 1-1(b1). Moreover, since the nucleic acid amplification reaction using the strand displacement polymerase does not include a step of dissociating the hydrogen bonds of the double-stranded test nucleic acid 3 under high temperature conditions, it is considered that the influence of the above steric hindrance is greater than that in the PCR method. In contrast, since the third primer 30 does not contain a polynucleotide that is not involved in the nucleic acid amplification reaction, it is considered that the third primer 30 easily hybridizes with the complementary strand 2 of the target nucleic acid 1 in the test nucleic acid 3 as shown in FIG. 1-1(b2). Therefore, in the initial stage of the nucleic acid amplification reaction, it is considered that the amplification reaction of the target nucleic acid 1 by the third primer 30 and the second primer 20 as shown in FIG. 1-1(b2) easily proceeds, and the amplification reaction involving the first primer 10 as shown in FIG. 1-1(b1) does not easily proceed.

[0086] As the nucleic acid amplification reaction proceeds, as shown in FIG. 1-2(c1) and FIG. 1-2(c2), the amplification reaction using the fragmented target nucleic acid 1, which is the amplification product of the previous nucleic acid amplification reaction, and its complementary strand 2 as templates becomes dominant. Since the steric hindrance of the first primer 10 has little effect on the double strand of the fragmented target nucleic acid 1, which is the amplification product, and its complementary strand 2, both the reaction shown in FIG. 1-2(c1) using the first primer 10 as a forward primer and the reaction shown in FIG. 1-2(c2) using the third primer 30 proceed. Although not shown, an amplification reaction using a fragment of the target nucleic acid 1 linked to the first polynucleotide tag 12 at the 5' end as a template also proceeds.

[0087] The final amplification product includes a detection nucleic acid 7, which is a double-stranded nucleic acid consisting of a target nucleic acid 1 linked to a first polynucleotide tag 12 at its 5' end and a complementary strand 2 linked to a labeling substance 23 at its 5' end, as shown in FIG. 1-2(d). This detection nucleic acid 7 can be immobilized on a solid support including a capture substance holder that holds a capture substance that binds to the first polynucleotide tag 12, and can be detected using the labeling substance 23 as an indicator. Although not shown, the amplification product also includes a double-stranded nucleic acid consisting of a target nucleic acid 1 not linked to the first polynucleotide tag 12 at its 5' end and a complementary strand 2 linked to a labeling substance 20 at its 5' end.

[0088] In the experiments described herein, the present inventors found that the reaction efficiency was low in the amplification of the target nucleic acid 1 by the PCR method and the isothermal nucleic acid amplification method using only the first primer 10 without using the third primer 30 as a forward primer and the second primer 10 as a reverse primer, whereas the reaction efficiency was significantly improved when the first primer 10 and the third primer 30 were used in combination as forward primers. This tendency was particularly remarkable in the isothermal nucleic acid amplification method using a strand displacement polymerase, rather than in the PCR method. The experimental results supported the validity of the above presumption that the amplification reaction efficiency is low when only the first primer 10 is used as a forward primer, since the amplification reaction involving the first primer 10, which has a large steric hindrance, is difficult to proceed in the early stage of the nucleic acid amplification reaction. In contrast, in the present embodiment in which the first primer 10 and the third primer 30 are used in combination, the amplification reaction of the target nucleic acid 1 mainly proceeds by the third primer 30 and the second primer 20 in the early stage of the nucleic acid amplification reaction, and as the production of the target nucleic acid 1 progresses, the amplification reaction of the target nucleic acid 1 also proceeds by the first primer 10 and the second primer 20, so that the detection nucleic acid 7 that can be detected on a solid phase carrier can be efficiently obtained.

[0089] (Ratio of the amount of the first primer to the third primer) In the primer set according to one or more embodiments of the present invention, the ratio of the amount of the first primer to the third primer is not particularly limited and can be adjusted appropriately. For example, the ratio of the third primer to the total amount of the first primer and the third primer is preferably 1 mol% or more, more preferably 2.5 mol% or more, more preferably 10 mol% or more, more preferably 25 mol% or more, most preferably 40 mol% or more, and preferably 90 mol% or less, more preferably 75 mol% or less.

[0090] (Embodiments of the primer set further comprising a fourth primer) As described above, the second primer may further contain, in addition to the second polynucleotide, a second polynucleotide tag that is a polynucleotide independent of the nucleic acid amplification reaction and linked to the 5' end of the second polynucleotide. When only the second primer containing the second polynucleotide tag is used as a reverse primer, the efficiency of the initial nucleic acid amplification reaction tends to be reduced due to the influence of the three-dimensional structure of the second primer containing the second polynucleotide tag, as in the case where only the first primer is used as a forward primer.

[0091] Therefore, when a second primer containing a second polynucleotide tag is used as a reverse primer, it is preferable to use in combination with a fourth primer that further contains a fourth polynucleotide that contains, at its 3' end, a polynucleotide D that is capable of competitively hybridizing to polynucleotide B of the second primer with respect to the target nucleic acid, and does not contain a polynucleotide that is independent of the nucleic acid amplification reaction.

[0092] The fourth primer preferably consists solely of the fourth polynucleotide.

[0093] "Polynucleotide D capable of hybridizing competitively with polynucleotide B of the second primer to a target nucleic acid" means that when either polynucleotide B of the second primer or polynucleotide D of the fourth primer hybridizes to a target nucleic acid, the other cannot hybridize to the same target nucleic acid. For example, when polynucleotide D of the fourth primer can hybridize to polynucleotides with 50% or more, 60% or more, 80% or more, 90% or more, 95% or more, or 100% of the number of bases contained in partial polynucleotide B' (part capable of hybridizing with polynucleotide B of the second primer) of the target nucleic acid, polynucleotide D can hybridize competitively with polynucleotide B of the second primer to a target nucleic acid.

[0094] In a preferred embodiment of polynucleotide D, one or more consecutive bases, preferably two or more, preferably three or more, more preferably five or more, and most preferably the entire base sequence at the 3' end of polynucleotide D is identical to the base sequence of the corresponding portion of the complementary strand of partial polynucleotide B'. The number of bases of polynucleotide D is not particularly limited and can be appropriately determined depending on the number of bases of partial polynucleotide B', and can be, for example, 8 or more, 10 or more, 12 or more, 15 or more, 17 or more, or 20 or more, and can be 40 or less, 30 or less, 27 or less, or 25 or less. Polynucleotide D is particularly preferably identical to polynucleotide B.

[0095] The base sequence of the fourth polynucleotide may contain the base sequence of polynucleotide D at its 3' end, and may further include another base sequence added to the 5' end side of the base sequence of polynucleotide D, or may consist of only the base sequence of polynucleotide D. That is, polynucleotide D may be a partial polynucleotide including the 3' end of the fourth polynucleotide, or may be the entire fourth polynucleotide. The number of bases of the fourth polynucleotide may be appropriately determined according to the number of bases of polynucleotide D, and may be, for example, 8 bases or more, 10 bases or more, 12 bases or more, 15 bases or more, 17 bases or more, or 20 bases or more, and may be 40 bases or less, 30 bases or less, 27 bases or less, or 25 bases or less. The fourth polynucleotide is particularly preferably the same as the second polynucleotide.

[0096] In this embodiment, the ratio of the amount of the second primer to the amount of the fourth primer is not particularly limited and can be appropriately adjusted. For example, the ratio of the amount of the fourth primer to the total amount of the second primer and the fourth primer is preferably 1 mol% or more, more preferably 2.5 mol% or more, more preferably 10 mol% or more, more preferably 25 mol% or more, most preferably 40 mol% or more, and is preferably 90 mol% or less, more preferably 75 mol% or less.

[0097] Fig. 2(a) shows an example of a test nucleic acid 3 including a target nucleic acid 1 and a complementary strand 2 of the target nucleic acid 1, a first primer 10, a second primer 20, a third primer 30, and a fourth primer 40. The characteristics of the target nucleic acid 1, the complementary strand 2, the test nucleic acid 3, the first primer 10, and the third primer 30 in the example shown in Fig. 2 are the same as those shown in Fig. 1-1 and Fig. 1-2, so a description thereof will be omitted.

[0098] In this embodiment, the second primer 20 comprises a second polynucleotide 21 having a polynucleotide B at its 3' end that is hybridizable to partial polynucleotide B'5, and a second polynucleotide tag 22 that is independent of the nucleic acid amplification reaction and is linked to the 5' end of the second polynucleotide 21.

[0099] The fourth primer 40 comprises a fourth polynucleotide 41 having at its 3' end a polynucleotide D that is capable of competitively hybridizing to polynucleotide B contained in the second polynucleotide 21 of the second primer 20 for the target nucleic acid 1, and does not comprise a polynucleotide independent of the nucleic acid amplification reaction.

[0100] The final amplification product prepared by carrying out a nucleic acid amplification reaction using the first primer 10, the second primer 20, the third primer 30, and the fourth primer 40 of the embodiment shown in Fig. 2(a) with the test nucleic acid 3 as a template contains a detection nucleic acid 8 which is a double-stranded nucleic acid consisting of a target nucleic acid 1 linked to a first polynucleotide tag 12 at its 5' end and a complementary strand 2 linked to a second polynucleotide tag 22 at its 5' end, as shown in Fig. 2(b). This detection nucleic acid 8 can be captured on a solid phase carrier having a capture substance holder containing a capture substance capable of binding to the first polynucleotide tag 12, and can be detected by binding to a labeling substance capable of binding to the second polynucleotide tag 22, using the labeling substance as an index.

[0101] <Detection of target nucleic acid amplification products> One or more embodiments of the present invention also include A nucleic acid amplification step including subjecting a test substance that may contain a target nucleic acid to a nucleic acid amplification reaction using a primer set according to one or more embodiments of the present invention; A detection step of detecting a target nucleic acid in the reaction product obtained in the nucleic acid amplification step. The present invention relates to a method for detecting a target nucleic acid.

[0102] In one or more embodiments of the present invention, the sample to be analyzed containing the test substance is not particularly limited, but typically includes samples containing food and drink, parts of living organisms such as animals and plants (organs, tissues, cells, blood, body fluids, etc.), feces, microorganisms, viruses, etc.

[0103] A test substance obtained from a sample to be analyzed can be used in a nucleic acid amplification reaction. The test substance may be a nucleic acid extracted and purified from the sample to be analyzed, or may be a nucleic acid extracted and roughly purified from the sample to be analyzed. When the test substance is a nucleic acid, it may be called a test nucleic acid. Alternatively, if the sample contains a test nucleic acid at a relatively high concentration, such as a part of a cell or tissue, the test sample itself can be directly subjected to a nucleic acid amplification reaction. cDNA prepared from the sample to be analyzed can also be used as a template. The test substance used as a template is preferably DNA. Hereinafter, preferred embodiments of the nucleic acid amplification step, the detection step, and the labeling step that is performed as necessary will be described.

[0104] (1. Nucleic Acid Amplification Step) A preferred embodiment of the nucleic acid amplification reaction in the nucleic acid amplification step is as described above.

[0105] By using a primer set according to one or more embodiments of the present invention in the nucleic acid amplification process, when a target nucleic acid is contained in a test substance, a detection nucleic acid containing a target nucleic acid having a first polynucleotide tag linked to one end can be obtained.

[0106] When detecting two or more different target nucleic acids, a multiplex primer set, which is a combination of two or more of the primer sets designed to amplify two or more target nucleic acids, can be used to carry out a nucleic acid amplification reaction on a test substance obtained from a sample to be analyzed. The multiplex primer set will be described separately below.

[0107] (2. Detection process) The detection step is a step of detecting a target nucleic acid in the reaction product obtained in the nucleic acid amplification step, and is preferably a step of contacting the reaction product with a solid phase carrier including a capture substance holding portion that contains a capture substance capable of binding to the first polynucleotide tag, and detecting the detection nucleic acid in the capture substance holding portion of the solid phase carrier.

[0108] The reaction product obtained in the nucleic acid amplification process refers to the reaction solution of the nucleic acid amplification reaction, which may contain the detection nucleic acid as an amplified product, or a sample in which the amplified product has been further concentrated from the reaction solution.

[0109] In an embodiment in which the primer set used in the nucleic acid amplification step includes a second primer to which a labeling substance is linked, the detection nucleic acid is linked to a labeling substance, and therefore the detection nucleic acid can be detected using the labeling substance as an indicator.

[0110] In an embodiment in which the primer set used in the nucleic acid amplification process includes a second primer linked to a second polynucleotide tag, since the detection nucleic acid is linked to a second polynucleotide tag, a labeling process can be further carried out in which a labeling substance is bound to the second polynucleotide tag.

[0111] The solid phase carrier is not particularly limited, and may be made of resin, metal, polysaccharide, mineral, etc., and may be in the form of a membrane, film, nonwoven fabric, plate, gel, etc. Preferably, the solid phase carrier has a porous structure so that the amplification product or labeling substance in the solution can be developed. Usable solid phase carriers include, for example, filter paper, nitrocellulose membrane, polyethersulfone membrane, nylon membrane, various dried gels (silica gel, agarose gel, dextran gel, gelatin gel), silicon, glass, plastic, etc. The size and shape of the solid phase carrier may be appropriately selected from those suitable for various operations and detections.

[0112] The capture substance held in the capture substance holding portion of the solid phase carrier is not particularly limited as long as it can bind to the first polynucleotide tag, but is preferably a polynucleotide that can hybridize with the first polynucleotide tag. The polynucleotide that can hybridize with the first polynucleotide tag is particularly preferably a polynucleotide that contains a complementary base sequence of the first polynucleotide tag. The base sequence of the first polynucleotide tag and the base sequence of the polynucleotide used as the capture substance can be easily modified, so that it is easy to control the binding specificity. For example, when a sample containing a plurality of detection nucleic acids containing different target nucleic acids is developed and detected on one solid phase carrier, the first primer is designed so that a first polynucleotide tag with a different base sequence is added to each of the plurality of detection nucleic acids, and a capture substance holding portion that holds a polynucleotide that can specifically hybridize with the first polynucleotide tag of each detection nucleic acid can be provided at a plurality of different positions on the solid phase carrier.

[0113] The capture substance may be held in the capture substance holding portion of the solid phase carrier via chemical bonding, or may be held by impregnation into the capture substance holding portion of the porous solid phase carrier.

[0114] The reaction product may be brought into contact with the capture substance holding portion of the solid phase carrier under appropriately adjusted conditions (e.g., conditions under which the above-mentioned specific hybridization is formed, or buffer conditions of about pH 5 to 9) so that the first polynucleotide tag of the detection nucleic acid can bind to the capture substance holding portion if the detection nucleic acid is contained in the reaction product.

[0115] When the detection nucleic acid captured in the capture substance holding portion of the solid phase carrier contains a labeling substance, detection of the detection nucleic acid containing the target nucleic acid can be carried out by detecting the labeling substance, preferably by visual detection.

[0116] (3. Labeling process) The labeling step is a step performed when the second primer of the primer set contains a second polynucleotide tag, or when the second primer of the primer set contains a labeling substance that can bind to another detectable substance by a secondary reaction. In this case, the detection nucleic acid containing the target nucleic acid is added with a second polynucleotide tag or a labeling substance. In the labeling step, the reaction product is brought into contact with the labeling substance or the detectable substance, thereby binding the labeling substance or the detectable substance to the second polynucleotide tag in the detection nucleic acid containing the target nucleic acid. The labeling step may be performed before or after the reaction product is brought into contact with the solid phase support, or may be performed simultaneously with the contact.

[0117] Specific examples of the labeling substance capable of binding to the second polynucleotide tag are as explained in the description of the second primer.

[0118] Kits of one or more embodiments of the present invention One or more embodiments of the invention also provide a kit for detecting a target nucleic acid, comprising: A nucleic acid detection device including a primer set according to one or more embodiments of the present invention and a solid phase carrier, The solid phase carrier includes a capture substance holder that includes a capture substance capable of binding to the first polynucleotide tag. The present invention relates to a kit for detecting a target nucleic acid.

[0119] In an embodiment in which the primer set includes a second primer to which a second polynucleotide tag is linked, it is preferable that the solid phase carrier further includes, in addition to the capture substance holding portion, a labeling substance holding portion containing a labeling substance capable of binding to the second polynucleotide tag.

[0120] The kit of one or more embodiments of the present invention may further include a PCR buffer, dNTPs, a DNA polymerase, a nucleic acid chromatography developing solution, and the like. The characteristics of the solid phase carrier are as described above.

[0121] By using a nucleic acid detection device containing the solid phase carrier, the presence or absence of the detection nucleic acid in a reaction product can be detected and determined, and results can be obtained simply and quickly.

[0122] A schematic diagram of one embodiment of a nucleic acid detection device that can be used in one or more embodiments of the present invention is shown in Fig. 3, but the nucleic acid detection device is not limited to this embodiment. In the following description, the reference numerals given to the respective members correspond to the reference numerals shown in Fig. 3.

[0123] The nucleic acid detection device 700 in FIG. 3 is formed by arranging a sample pad 73, which is a sample receiving portion for receiving the reaction product of the nucleic acid amplification step, a conjugate pad 72, which is a labeling substance holding portion for holding a labeling substance, a porous solid phase carrier 71 including a capture substance holding portion 76 in which a capture substance capable of binding to a first polynucleotide contained in the detection nucleic acid is held, and an absorbent pad 74, in this order on a base material 75 so as to be in contact with each other. The capture substance holding portion 76 of the solid phase carrier 71 is a portion where the capture substance is locally arranged and immobilized. The sample pad 73, the conjugate pad 72, the solid phase carrier 71, and the absorbent pad 74 can be made of a material having a porous structure that can be used as the solid phase carrier. These may be made of the same material or different materials. The base material 75 may be any material that can support various members arranged thereon and can facilitate the operation of the nucleic acid detection device 700, and may be made of, for example, paper, resin, metal, mineral, etc. When a labeling substance is mixed in the developing solution, or when detecting an amplification product using a primer set of an embodiment including a second primer to which a labeling substance has been added in advance, the conjugate pad 72 can be omitted. A part of the nucleic acid detection device 700 may be covered with a film such as polyester to prevent contamination and to prevent the liquid from volatilizing from the surface of the solid phase carrier during the test.

[0124] A sample containing the reaction product of the nucleic acid amplification reaction obtained by the nucleic acid amplification step is added to the sample pad 73. The reaction product of the nucleic acid amplification reaction may be added as is as the sample, or may be added together with an appropriate developing solution (e.g., phosphate buffer, Tris buffer, Good's buffer, SSC buffer). The developing solution may further contain a surfactant, salt, protein, nucleic acid, etc., as necessary. The sample added to the sample pad 73 develops by capillary phenomenon from upstream to downstream in the direction shown by the arrow in FIG. 3.

[0125] In another embodiment, development can also be performed by immersing the sample pad 73 of the nucleic acid detection device 700 in the detection sample and / or development solution contained in a container (e.g., a PCR tube, an Eppendorf tube, a 96-well plate, etc.).

[0126] In addition, in a nucleic acid detection device that is used by immersing in a container that holds a sample containing a reaction product of a nucleic acid amplification reaction and / or a developing solution, the sample pad 73 can be omitted from the nucleic acid detection device 700 shown in Fig. 3. Furthermore, the end of the solid phase carrier 71 on the base material 75 on the side where the absorption pad 74 is not arranged may be directly immersed in the sample and / or the developing solution.

[0127] When detecting an amplification product using a primer set of an embodiment including a second primer having a second polynucleotide tag attached thereto, the detection nucleic acid is labeled by binding to the labeling substance as it passes through conjugate pad 72, which is a labeling substance holding section that holds a labeling substance capable of binding to the second polynucleotide tag. Next, when the detecting nucleic acid passes through the solid phase carrier 71 , it comes into contact with the capture substance in the capture substance holding section 76 , and is captured and bound to the capture substance holding section 76 .

[0128] When the detection nucleic acid is present in the reaction product, the labeling substance bound to the detection nucleic acid captured and bound to the capture substance holding section 76 of the solid phase carrier 71 is detected. If the labeling substance can be visually confirmed, the capture substance holding section 76 will develop a color due to the labeling substance. The presence or absence of the detection nucleic acid can be determined using the presence or absence of the detection (coloration) of the labeling substance as an indicator, and based on that, the presence or absence of the target nucleic acid in the test substance can be determined.

[0129] <Multiplex primer set> One or more further embodiments of the present invention include The primer set includes two or more primer sets according to one or more embodiments of the present invention described above, the target nucleic acids amplified by the nucleic acid amplification reaction using the two or more primer sets are different from each other; Concerning multiplex primer sets.

[0130] In this embodiment, it is preferable that the first polynucleotide tags of the first primers included in two or more primer sets have different base sequences and bind to different capture substances.

[0131] A solid phase carrier for detecting a reaction product from a nucleic acid amplification reaction using a multiplex primer set preferably has two or more capture substance holding portions holding different capture substances at different positions on the solid phase carrier. In this embodiment, each capture substance is preferably a polynucleotide capable of hybridizing to one of the first polynucleotide tags of the first primers included in the two or more primer sets. EXAMPLES

[0132] <Example 1> (1) Primer design The primers used in the TRIAmp reaction were prepared with reference to the forward primer DRa21 (SEQ ID NO: 1) and reverse primer DRb19 (SEQ ID NO: 2) described in Patent Document 4, which target a repetitive sequence of Mycobacterium tuberculosis as the target nucleic acid.

[0133] DRa21-Tag1 was prepared by linking the 3'-end of a DNA tag sequence (Tag1) (sequence number 3) for membrane capture in nucleic acid chromatography to the 5'-end of the forward primer DRa21 via a divalent group represented by the above formula I containing an azobenzene structure as a polymerase inhibitor. Tag1: 5'-ATGCTACCGTATGCCCAGTG-3' (SEQ ID NO: 3)

[0134] DRb19-Tag2 was prepared by linking the 3'-end of a DNA tag sequence (Tag2) (SEQ ID NO: 4) for gold colloid labeling to the 5'-end of the reverse primer DRb19 via a divalent group represented by the above formula I containing an azobenzene structure as a polymerase inhibitor. Tag2: 5'-GACAACGGAGACAGAGCCAA-3' (SEQ ID NO: 4) The primers used are shown in Table 1.

[0135] [Table 1]

[0136] (2) Preparation of oligonucleotide-bound gold colloids Gold Colloid (40 nm, 9.0 × 10 10 (particle number / ml, British Biocell International) was mixed with the thiol group-containing oligonucleotide represented by SEQ ID NO:5 below and incubated at 50°C for 16 hours. The mixture was centrifuged at 6000 rpm for 15 minutes, the supernatant was removed, and 0.05 M sodium chloride and 5 mM phosphate buffer (pH 7) were added and mixed, and then incubated again at 50°C for 40 hours.

[0137] After incubation, the mixture was centrifuged (6000 rpm, 15 minutes), the supernatant was removed, and 5 mM phosphate buffer (pH 7) was added. This buffer replacement was carried out again. The prepared gold colloid solution was added uniformly to a glass fiber pad, and then dried in a vacuum dryer to obtain a conjugate pad. (Thiol group-containing oligonucleotide): 5'-TTGGCTCTGTCTCCGTTGTC-SH-3' (SEQ ID NO: 5)

[0138] The thiol group-containing oligonucleotide shown in SEQ ID NO:5 has a phosphate group at the 3' position of the cytosine nucleotide at the 3' end and HO-(CH2) m It is bonded to the hydroxyl group of a compound represented by -SH (m is 6) through a phosphate ester bond.

[0139] (3) Preparation of membrane for holding tag capture substance A solution containing an oligonucleotide probe represented by SEQ ID NO:6 as a tag capture substance was applied to a nitrocellulose membrane (Hi-Flow180) manufactured by Merck Millipore Co., Ltd. in a line shape of 1 mm width perpendicular to the direction of development using a dispenser. The membrane was then dried at 40°C for 30 minutes to obtain a membrane equipped with a tag capture substance holding portion. (Oligonucleotide probe): 5'-CACTGGGCATACGGTAGCAT-3' (SEQ ID NO: 6)

[0140] (4) Preparation of a lateral flow nucleic acid detection device The lateral flow type nucleic acid detection device was produced in accordance with the detection device shown in the schematic diagram of FIG.

[0141] That is, a polypropylene backing sheet (Lohmann) was used as the substrate 75, a conjugate pad prepared in (2) above was used as the conjugate pad 72, a membrane equipped with a tag capture substance holding portion prepared in (3) above was used as the solid phase carrier 71 equipped with a capture substance holding portion 76, a glass fiber sample pad was used as the sample pad 73, and a cellulose absorbent pad was used as the absorbent pad 74 were overlapped and bonded together as shown in Figure 7 to prepare a lateral flow nucleic acid detection device 700.

[0142] (5) TRIAMp reaction The primer mix shown in Table 2 was prepared using the primers described in (1) above.

[0143] [Table 2]

[0144] Next, a 2xTRIAmp buffer mix was prepared consisting of 40 mM Tris buffer (pH 8.8), 20 mM potassium chloride, 20 mM ammonium sulfate, 0.2% Tween 20, 1.8 M betaine, 1.6 mM deoxynucleotide triphosphate, and 12 mM magnesium sulfate.

[0145] Next, 2 μL of DNA sample was added to 12.5 μL of TRIAmp buffer mix, 2 μL of each primer mix, 8 units of Bst DNA polymerase, and 5 μL of 10,000-fold diluted SYBR Green I solution to prepare a total volume of 25 μL of TRIAmp reaction solution. The DNA sample was a DNA solution (100 pg / μL) extracted and purified from Mycobacterium bovis BCG strain.

[0146] The reaction was carried out at 68° C. for 1 hour using a Roche real-time PCR device (LightCycler96), and fluorescence was measured over time.

[0147] After the reaction, 5 μL of the reaction solution was added to the sample pad of a lateral flow type nucleic acid detection device, 80 μL of developing solution was added, and the colored line was visually judged. If very strong coloring was observed, it was marked as "+++", if coloring was observed, it was marked as "++", if weak coloring was observed, it was marked as "+", and if no coloring was observed, it was marked as "-". The results are shown in Table 3.

[0148] [Table 3]

[0149] When primer mix 5, which used primers with DNA tags attached to all of the primers, was used, the TRIAMp reaction proceeded, but the reaction rate was significantly reduced compared to primer mix 1, which did not contain primers with DNA tags attached. In contrast, primer mixes 2 to 4, in which some of the primers in primer mix 5 were replaced with primers without DNA tags attached, showed a faster reaction rate compared to primer mix 5. In particular, primer mixes 3 and 2, in which more than half of the total amount was replaced, required approximately half the time for a positive determination compared to primer mix 5. With any primer mix containing primers with DNA tags attached, detection was possible using the colored lines of a lateral flow type nucleic acid detection device.

[0150] These results show that the TRIAMp reaction is inhibited when a DNA-tagged primer is used, but that this inhibition can be significantly improved by adding a primer without a DNA tag.

[0151] <Example 2> In Example 1, it was shown that the reaction rate of the TRIAmp reaction decreases when a primer with a DNA tag is used. Furthermore, it was shown that the rate decrease can be suppressed by replacing a part of the primer with a DNA tag with a primer without a DNA tag. However, since the amount of the amplified product is measured by the fluorescence of SYBR GreenI in Example 1, it cannot be confirmed whether the amount of the amplified product with a DNA tag that can be detected by a nucleic acid detection device actually increases.

[0152] Therefore, primers with DNA tags attached thereto and primers without DNA tags attached thereto were mixed in various ratios, and a detection limit test was performed using serial dilutions of DNA extracted from Mycobacterium bovis BCG strain. The same primers as those used in Example 1 were used. An experiment in which the DNA sample containing the target nucleic acid was not added was used as a negative control experiment (Nega). The composition of the primer mix is ​​shown in Table 4. As a comparative example, a primer mix consisting entirely of primers with DNA tags attached thereto (Comparative Example 1) was prepared.

[0153] [Table 4]

[0154] The TRIAmp reaction was carried out in the same manner as in Example 1, except that SYBR GreenI was not added. After the reaction, 5 μL of the reaction solution was added to the sample pad of a lateral flow type nucleic acid detection device, 80 μL of developing solution was added, and the colored line was visually judged. If very strong coloring was observed, it was marked as "+++", if coloring was observed, it was marked as "++", if weak coloring was observed, it was marked as "+", and if no coloring was observed, it was marked as "-".

[0155] The results are shown in FIG. 4 and Table 5. In the comparative example using a primer mix consisting of only primers with DNA tags, detection was possible up to 50 pg, whereas primer mix 1 containing primers without DNA tags was able to detect up to 5 pg, primer mix 2 was able to detect up to 500 fg, and primer mixes 3 to 5 were able to detect up to 5 fg. Primer mixes 4 and 5, which had a tag addition rate of 25% or less, tended to produce fainter colored lines. Primer mix 3, which had a tag addition rate of 50%, showed the best performance in terms of the lower limit of detection and the density of the colored line.

[0156] [Table 5]

[0157] <Comparative Example 2> A primer mix consisting of forward primer DRa21 (SEQ ID NO: 1) and reverse primer DRb19 (SEQ ID NO: 2) without DNA tag was prepared, and reaction and fluorescence measurement were carried out using a real-time PCR device in the same manner as in Example 1. A serial dilution of DNA extracted from Mycobacterium bovis BCG strain was used as the DNA sample. An experiment in which the DNA sample containing the target nucleic acid was not added was used as a negative control experiment (Nega).

[0158] The results are shown in Table 6. The lower limit of detection for primers without DNA tags was 100 fg. The lower limit of detection for primer mixes 3 to 5 in Example 2 above was 5 fg. It was shown that by using a primer mix having the configuration of one or more embodiments of the present invention, more sensitive detection than conventional methods is possible without using a real-time PCR device.

[0159] [Table 6]

[0160] <Example 3> The same procedure as in Example 2 was carried out, except that the primer mix in Table 7 was used.

[0161] [Table 7]

[0162] The results are shown in Table 8. Primer mix 1 with a tag content (proportion of primers with DNA tags attached to the total amount of primers) of 99% showed the same lower detection limit as Comparative Example 3, but the line coloring intensity of 5p, which is the lower detection limit concentration, was improved. Primer mixes with tag contents of 97.5% and 95% were able to detect 500 fg, indicating improved detection limit performance.

[0163] [Table 8]

[0164] <Example 4> Instead of DRb19-Tag2, DRb19-biotin was used, which was modified with biotin as a labeling substance at the 5' end of the DRb19 primer by a standard method, to prepare the primer mix shown in Table 9. In addition, streptavidin-bound gold colloid was used as a detectable substance that binds to biotin to prepare the conjugate pad of the lateral flow type nucleic acid detection device. The rest of the procedure was the same as in Example 1.

[0165] [Table 9]

[0166] The results of fluorescence measurement using a real-time PCR device are shown in Figure 5. Compared to primer mix 1, which contains a forward primer without a DNA tag, primer mix 2, in which all of the forward primers were replaced with primers with DNA tags, showed a significant delay in the start of amplification. Primer mix 3, in which half of the forward primers were replaced with primers without DNA tags, showed a significant improvement in the amplification rate.

[0167] <Example 5> Using primer mixes 2 and 3 of Example 4, a detection limit test similar to that of Example 3 was carried out.

[0168] The results are shown in Table 10. The lower detection limit of primer mix 2 with a tag addition rate of 100% was 500 fg, whereas primer mix 3 with a tag addition rate of 50% was able to detect 50 fg. This experiment confirmed the sensitization effect of using a primer mix having the configuration of one or more embodiments of the present invention.

[0169] [Table 10]

[0170] <Example 6> An investigation was carried out to determine whether the sensitization effect of a primer mix having the configuration of one or more embodiments of the present invention can be obtained in the RPA method.

[0171] (1) Primer design As primers used in the RPA reaction, a forward primer ftsY-1F (SEQ ID NO: 7) and a reverse primer ftsY-1R (SEQ ID NO: 8) that specifically bind to the ftsY gene of Staphylococcus aureus were designed.

[0172] To prepare ftsY-1F-Tag3, the 3'-end of a DNA tag sequence (Tag3) for membrane capture in nucleic acid chromatography was linked to the 5'-end of the forward primer ftsY-1F via a divalent group represented by formula I above that contains an azobenzene structure as a polymerase inhibitor. Tag3: 5'-TCGAGTGACAGCTAATGTGTGAT-3' (SEQ ID NO: 9)

[0173] To prepare ftsY-1R-Tag2, a DNA tag sequence (Tag2) (sequence number 4) for gold colloid labeling was linked to the 5' end of the reverse primer ftsY-1R via a divalent group represented by the above formula I containing an azobenzene structure as a polymerase inhibitor. The primers used in Example 6 are shown in Table 11.

[0174] [Table 11]

[0175] (2) Preparation of a membrane that holds the tag capture substance A solution containing an oligonucleotide probe represented by SEQ ID NO: 10 below as a tag capture substance was applied to a nitrocellulose membrane (Hi-Flow180) manufactured by Merck Millipore Co., Ltd. in a line shape of 1 mm width perpendicular to the direction of development using a dispenser. The membrane was then dried at 40°C for 30 minutes to obtain a membrane equipped with a tag capture substance holding portion. (Oligonucleotide probe): 5'-ATCACACATTAGCTGTCACTCGA-3' (SEQ ID NO: 10)

[0176] (3) Preparation of a lateral flow nucleic acid detection device A lateral flow type nucleic acid detection device was produced in the same manner as in Example 1, except that the membrane equipped with the tag capture substance retaining portion prepared in (2) above was used as the solid phase carrier 71.

[0177] (4) RPA reaction The RPA reaction was carried out using a TwistAmp Basic kit from TwistDx Inc. The primer mix used was a mixture of DNA-tagged primers and non-tagged primers in the ratio shown in Table 12. In addition, as Comparative Example 4, a primer mix consisting of only DNA-tagged primers was prepared.

[0178] A resolubilization solution was prepared by mixing 4.8 μL of primer mix, 29.5 μL of Primer Free Rehydration Buffer, 2 μL of serial dilutions (5 ng / μL to 50 fg / μL) of DNA extracted from Staphylococcus aureus (ATCC25923), and 11.2 μL of sterile distilled water. The resolubilization solution was added to the tube containing the freeze-dried RPA reaction pellet to redissolve the reagent. Then, 2.5 μL of 280 mM magnesium acetate was added, mixed, and the reaction was carried out at 37 °C for 20 minutes.

[0179] [Table 12]

[0180] After the reaction, 5 μL of the reaction solution was added to the sample pad of a lateral flow type nucleic acid detection device, 80 μL of developing solution was added, and the colored line was visually judged. If very strong coloring was observed, it was marked as "+++", if coloring was observed, it was marked as "++", if weak coloring was observed, it was marked as "+", and if no coloring was observed, it was marked as "-".

[0181] The results are shown in Table 13. The detection limit in Comparative Example 4, in which a primer mix consisting of only DNA-tagged primers was used, was 10 ng, whereas primer mixes 1 to 3 allowed detection down to 1 pg to 10 pg, confirming the improvement in detection limit performance due to the coexistence of primers without DNA tags.

[0182] [Table 13]

[0183] <Example 7> An investigation was carried out to determine whether the sensitization effect of a primer mix having the configuration of one or more embodiments of the present invention can be obtained in the LAMP method.

[0184] (1) Primer design The primers used in the LAMP reaction were a primer set that amplifies the Nuc gene of Staphylococcus aureus described in Non-Patent Document 1 (Frontiers in Microbiology. vol. 8, Article 192). The primers used are shown in Table 14.

[0185] [Table 14]

[0186] Sau-LF-Tag1 was prepared by linking the 3'-end of a DNA tag sequence (Tag1) (sequence number 3) for membrane capture in nucleic acid chromatography to the 5'-end of the loop primer Sau-LF (sequence number 15) via a divalent group represented by the above formula I containing an azobenzene structure as a polymerase inhibitor.

[0187] Sau-Tag2 was prepared by linking the 3'-end of a DNA tag sequence (Tag2) (sequence number 4) for gold colloid labeling to the 5'-end of the loop primer Sau-LB (sequence number 16) via a divalent group represented by the above formula I containing an azobenzene structure as a polymerase inhibitor.

[0188] (2) LAMP reaction A reaction solution containing 20 mM Tris buffer (pH 8.8), 10 mM potassium chloride, 10 mM ammonium sulfate, 0.1% Tween 20, 0.9 M betaine, 0.8 mM deoxynucleotide triphosphate, 6 mM magnesium sulfate, 8 units of Bst DNA polymerase, 1.6 μM Sau-FIP, 1.6 μM Sau-BIP, 0.8 μM Sau-F3, 0.8 μM Sau-B3, 0.4 μM Sau-LF, 0.4 μM Sau-LB, 0.4 μM Sau-LF-Tag1, and 0.4 μM Sau-LB-Tag2 was used. As a comparative example, a reaction solution was prepared using 0.8 μM Sau-LF and 0.8 μM Sau-LB as the loop primers of the above reaction solution. 2 μL of DNA sample was added to this reaction solution and reacted at 62 ° C. for 1 hour.

[0189] After the reaction, 5 μL of the reaction solution was added to the sample pad of a lateral flow type nucleic acid detection device similar to that in Example 2, and 80 μL of a developing solution was added. After drying, the color intensity of the line was measured using an immunochromatograph manufactured by Hamamatsu Photonics KK

[0190] In the comparative example where all of the loop primers were DNA-tagged primers, the line coloring intensity was 25 mAbs, whereas when half of the loop primers were replaced with primers without DNA tags, the intensity reached 50 mAbs, confirming an increase in the amount of tagged product.

[0191] <Example 8> An investigation was carried out to determine whether the sensitization effect of a primer mix having the configuration of one or more embodiments of the present invention can be obtained in a PCR method.

[0192] (1) PCR reaction For verification of the PCR reaction, the same primers as in Example 6 were used to prepare a primer mix shown in Table 15. As Comparative Example 5, a primer mix consisting only of primers having a DNA tag added thereto was prepared.

[0193] [Table 15]

[0194] The reaction solution was prepared according to the manual for 2xHSTaq perfect Mix UNG plus (Takara Bio). 10 μL of 2xHSTaq perfect Mix was mixed with 2 μL of 1 μM primer mix, 2 μL of DNA sample extracted from Staphylococcus aureus (100 pg / μL to 10 fg / μL), and 6 μL of sterile distilled water to prepare 20 μL of PCR reaction solution.

[0195] The PCR reaction solution was placed in a thermal cycler (Bior, LifeEco) and reacted at 94°C for 3 minutes, after which 35 cycles of 94°C-5 seconds / 58°C-5 seconds / 72°C-15 seconds were performed.

[0196] After the reaction, 5 μL of the reaction solution was added to the sample pad of a lateral flow type nucleic acid detection device, 80 μL of developing solution was added, and the colored line was visually judged. If very strong coloring was observed, it was marked as "+++", if coloring was observed, it was marked as "++", if weak coloring was observed, it was marked as "+", and if no coloring was observed, it was marked as "-".

[0197] The results are shown in Table 16. The detection limit of Comparative Example 5, in which a primer mix consisting of only DNA-tagged primers was used, was 20 pg, whereas primer mixes 1 to 3 all enabled detection down to 2 pg, demonstrating an improvement in detection limit performance due to the coexistence of primers without DNA tags.

[0198] [Table 16]

[0199] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A primer set for preparing an amplification product of a target nucleic acid detectable on a solid phase carrier by a nucleic acid amplification reaction, comprising: the primer set includes a first primer, a second primer, and a third primer, The first primer comprises a first polynucleotide having at its 3' end a polynucleotide A capable of hybridizing to a complementary strand of a partial polynucleotide A' at the 5' end of a target nucleic acid, and a first polynucleotide tag which is a polynucleotide independent of a nucleic acid amplification reaction and is linked to the 5' end of the first polynucleotide; the second primer comprises a second polynucleotide comprising at its 3' end a polynucleotide B capable of hybridizing to a partial polynucleotide B' at the 3' end of the target nucleic acid; the third primer comprises a third polynucleotide comprising, at its 3' end, a polynucleotide C capable of competitively hybridizing to the polynucleotide A of the first primer with respect to a complementary strand of the target nucleic acid, and does not comprise a polynucleotide independent of a nucleic acid amplification reaction; a ratio of the third primer to a total amount of the first primer and the third primer is 1 mol % or more and 90 mol % or less, the polynucleotide C of the third primer is capable of hybridizing with a polynucleotide having 50% or more of the number of bases contained in a complementary strand of the partial polynucleotide A' of the target nucleic acid; The primer set.

2. The primer set according to claim 1 , wherein a ratio of the third primer to a total amount of the first primer and the third primer is 2.5 mol % or more and 75 mol % or less.

3. The primer set according to claim 1 , wherein the second primer further comprises a labeling substance linked to the second polynucleotide.

4. The primer set further comprises a fourth primer, The second primer further comprises a second polynucleotide tag linked to the 5' end of the second polynucleotide, the second polynucleotide tag being a polynucleotide independent of a nucleic acid amplification reaction; the fourth primer comprises a fourth polynucleotide comprising, at its 3' end, a polynucleotide D capable of competitively hybridizing to the polynucleotide B of the second primer with respect to the target nucleic acid, and does not comprise a polynucleotide independent of a nucleic acid amplification reaction; a ratio of the fourth primer to a total amount of the second primer and the fourth primer is 1 mol % or more and 90 mol % or less, The polynucleotide D of the fourth primer is capable of hybridizing with a polynucleotide having 50% or more of the number of bases contained in the partial polynucleotide B' of the target nucleic acid. The primer set according to claim 1 or 2.

5. The primer set according to claim 4 , wherein a ratio of the fourth primer to a total amount of the second primer and the fourth primer is 2.5 mol % or more and 75 mol % or less.

6. A nucleic acid detection device comprising the primer set according to any one of claims 1 to 3 and a solid phase carrier, The solid phase carrier includes a capture substance holder including a capture substance capable of binding to the first polynucleotide tag. A kit for detecting a target nucleic acid.

7. A nucleic acid detection device comprising the primer set according to claim 4 or 5 and a solid phase carrier, The solid phase carrier includes a capture substance holder including a capture substance capable of binding to the first polynucleotide tag, and a labeling substance holder including a labeling substance capable of binding to the second polynucleotide tag. A kit for detecting a target nucleic acid.

8. A nucleic acid amplification step comprising subjecting a test substance that may contain a target nucleic acid to a nucleic acid amplification reaction using the primer set according to any one of claims 1 to 5; A detection step of detecting a target nucleic acid in the reaction product obtained in the nucleic acid amplification step. A method for detecting a target nucleic acid.

9. The method according to claim 8 , wherein in the detection step, the target nucleic acid is detected using a solid phase carrier.

10. The method according to claim 8 or 9, wherein the nucleic acid amplification reaction in the nucleic acid amplification step is carried out using a strand displacement polymerase.

11. The primer set according to any one of claims 1 to 5 is included, the target nucleic acids amplified by the nucleic acid amplification reaction using the two or more primer sets are different from each other; Multiplex primer set.

Images