JP6202455B2 - Target nucleic acid detection method - Google Patents

Description

  The present specification relates to a method for detecting a target nucleic acid.

  In detecting the detection of the target nucleic acid, a nucleic acid sample that can contain the target nucleic acid is amplified by PCR or the like using specific primers, and the presence or absence of the amplified product is analyzed by a known method. As a method for detecting an amplification product, conventionally, the presence or absence of a target nucleic acid is detected by electrophoresis or the like depending on the length and size of the amplification product. In recent years, for example, a detection method using a probe that specifically hybridizes to a specific sequence derived from a target nucleic acid in an amplification product, or a specific sequence derived from a tag attached to the amplification product by a primer. A method of detecting using a hybridizing probe has come to be used.

  For example, as the former method, an oligonucleotide having a reporter at the 5 ′ end and a quencher at the 3 ′ end and specifically hybridizing to a target nucleic acid is used as a probe (non-patented). References 1, 2). In these methods, as a result of the PCR amplification reaction, the probe hybridized to the target nucleic acid is degraded by the 5′-3 ′ exonuclease activity of Taq DNA polymerase, so that the distance between the reporter and the quencher is increased. The amount of PCR product is measured by detecting the fluorescence signal generated by.

  Further, as a PCR amplification product, a partial double-stranded nucleic acid having a single-stranded portion having a single-stranded tag derived from a primer is obtained, and a solid phase on which a complementary tag that specifically hybridizes to this tag is fixed It has also been disclosed to efficiently detect using chromatography using a carrier (Patent Document 1).

International Publication No. 2013/038534

Higuchi, R., Fockler, C., Dollinger, G., and Watson, R. 1993. Kinetic PCR: Real time monitoring of DNA amplification reactions.Biotechnology 11: 1026-1030. Analytical Sciences, 2014, 30 (3), 427

  In the methods of Non-Patent Documents 1 and 2 above, the specificity to the target nucleic acid is high, but there is a limit to detecting many types of target nucleic acids at the same time, and the design and synthesis of fluorescent probes are problematic in terms of cost and labor There is.

  In the method of Patent Document 1, the amplification product is captured by a probe including a complementary tag that hybridizes with a single-stranded tag derived from a primer in the amplification product. For this reason, there is an advantage that the probe can be universalized. On the other hand, as shown in FIG. 16, the primer does not appropriately amplify the target nucleic acid, but a primer dimer obtained by hybridizing the primers and performing an amplification reaction may be by-produced as an amplification product. Primer dimers, as well as appropriate amplification products, can be equipped with tags and labels derived from primers and can cause false positives. Furthermore, in the method described in Patent Document 1, when an unreacted primer hybridizes with a complementary tag, there is a possibility of adverse effects such as false positives on detection of an appropriate amplification product.

  In view of these problems, the present specification provides a method for detecting a target nucleic acid simply and reliably by suppressing or avoiding the influence of an unintended product in a nucleic acid amplification reaction.

  As a result of various studies, the present inventors have identified a target nucleic acid having an identification region that specifically hybridizes with a nucleic acid extension region of a genuine amplification product amplified by a primer set in a nucleic acid amplification reaction using a DNA polymerase and a primer. The presence of the probe, and at least a part of the target nucleic acid identification probe or at least a part of the authentic amplification product is captured, and the presence or absence of the authentic amplification product is determined by using the at least part of the target nucleic acid identification probe. The knowledge that it can evaluate simply and reliably was acquired. That is, it has been found that such a method can avoid the influence of unintended false products such as primer dimers.

  In addition, in the amplification step in the presence of such a target nucleic acid identification probe, the present inventors have various aspects of the target nucleic acid identification probe depending on the presence or absence of the exonuclease activity in the 5′-3 ′ direction of the DNA polymerase used. It has been found that at least a portion or at least a portion of the authentic amplification product can be captured in various ways. Furthermore, it discovered that such various aspects can contribute to evaluation of the presence or absence of a genuine amplification product.

  In addition, when capturing at least a part of such a target nucleic acid identification probe or at least a part of a genuine amplification product, a target nucleic acid identification probe or primer is provided with a tag for capturing in advance, thereby easily identifying the target nucleic acid. It has been found that at least part of the probe or at least part of the authentic amplification product can be captured.

  The present specification provides the following means based on these findings.

(1) A method for detecting a target nucleic acid,
In the presence of a nucleic acid sample, a primer set for amplifying the target nucleic acid, and a target nucleic acid identification probe comprising an identification region that specifically hybridizes to the nucleic acid extension reaction region of the authentic amplification product amplified by the primer set An amplification step of performing a nucleic acid amplification reaction using a DNA polymerase;
Capture that captures on a solid phase carrier at least part of the target nucleic acid identification probe or at least part of the authentic amplification product that may be present in the amplification step according to the 5'-3 'exonuclease activity of the DNA polymerase Process,
With
A method for evaluating the presence or absence of the authentic amplification product using at least a part of the target nucleic acid identification probe.
(2) The amplification step is a step using the DNA polymerase that does not have exonuclease activity in the 5′-3 ′ direction.
The capture step is a step of capturing a hybridized product obtained by hybridizing the target nucleic acid identification probe and the nucleic acid extension region in the authentic amplification product via the target nucleic acid identification probe. Method.
(3) The method according to (2), wherein the target nucleic acid identification probe includes a region capable of suppressing or stopping nucleotide extension reaction by the DNA polymerase on the 5 ′ end side and / or 3 ′ end side of the identification region. .
(4) The amplification step is a step using the DNA polymerase having exonuclease activity in the 5′-3 ′ direction,
The method according to (1), wherein the capturing step is a step of capturing the target nucleic acid identification probe, a degradation product thereof, or the authentic amplification product.
(5) The method according to (4), wherein the capturing step is a step of capturing the target nucleic acid identification probe or a degradation product thereof.
(6) The target nucleic acid identification probe includes a first tag adjacent to one end of the identification region, and a first capture capable of capturing the first tag on the solid phase carrier. (5) The method of description.
(7) The method according to (6), wherein the first tag has one of a pair of proteins that specifically bind to each other.
(8) The method according to (6), wherein the first tag has one of a pair of polynucleotides each having a base sequence that specifically hybridizes to each other.
(9) The first capture is a solid phase carrier having a porous portion, and the first tag is a particle having a size and / or shape captured by the porous portion of the solid phase carrier. The method according to (6).
(10) The target nucleic acid identification probe further includes a second tag adjacent to the other end of the identification region,
The method according to (6), wherein the target nucleic acid identification probe or a degradation product thereof is captured using a second capture that captures the second tag.
(11) The method according to any one of (4) to (10), wherein the presence or absence of a nucleic acid amplification reaction of the target nucleic acid is evaluated after the amplification step and before the capture step or in the capture step.
(12) The method according to any one of (4) to (11), wherein the capturing step is a chromatography step.
(13) capturing at least a part of the target nucleic acid identification probe in the capturing step;
In the capture step or after the capture step,
A target nucleic acid comprising: contacting a target nucleic acid amplification product with a target nucleic acid detection probe under conditions that allow hybridization with a target nucleic acid detection probe that specifically hybridizes with the authentic amplification product; The method in any one of (4)-(12) provided with a detection process.
(14) The target nucleic acid identification probe includes a first tag adjacent to one end of the identification region, and includes a second tag and a third tag adjacent to the other end.
Using the first capture that captures the first tag and the second capture that captures the second tag to capture the target nucleic acid identification probe or a degradation product thereof;
The third tag has a polynucleotide having a base sequence that specifically hybridizes with the amplification product of the target nucleic acid,
The method according to (13), wherein the third tag captured via the second tag is used for the second capture as the target nucleic acid detection probe.
(15) The detection step uses a solid phase carrier provided with a target nucleic acid detection probe for detecting the target nucleic acid, and captures the target nucleic acid identification probe or a degradation product thereof on the solid phase carrier. ) Or the method according to (14).
(16) The method according to any one of (13) to (15), wherein the detection step is a chromatography step.
(17) The method according to (16), wherein the target nucleic acid identification probe or a degradation product thereof is captured upstream of the target nucleic acid detection probe in a development direction in chromatography.
(18) The method according to any one of (1) to (17), wherein the target nucleic acid identification probe comprises a labeling element.
(19) The method according to (18), wherein the labeling element is provided adjacent to the other end of the identification region or in the identification region.
(20) The method according to any one of (1) to (19), wherein the primer set is capable of amplifying a partial double-stranded nucleic acid having a single-stranded region only in one strand as an amplification product of the target nucleic acid. .
(21) A solid phase carrier for detecting a target nucleic acid,
A solid support;
A capture for capturing a target nucleic acid identification probe comprising an identification region that specifically hybridizes to a nucleic acid extension reaction region of a genuine amplification product amplified by a primer set for amplifying the target nucleic acid, and held on the solid phase carrier Captured capture,
A solid phase carrier comprising:
(22) The solid phase carrier according to (21), further comprising a target nucleic acid detection probe for detecting the target nucleic acid.
(23) A kit for detecting a target nucleic acid,
A primer set for amplifying the target nucleic acid;
A target nucleic acid identification probe comprising an identification region that specifically hybridizes to a nucleic acid extension reaction region of a genuine amplification product amplified by a primer set for amplifying the target nucleic acid;
A kit comprising:
(24) The kit according to (23), further comprising a solid phase carrier holding a capture probe that captures at least a part of the target nucleic acid identification probe.
(25) The kit according to (24), wherein the solid phase carrier further holds a target nucleic acid detection probe for detecting the target nucleic acid.

It is a figure which shows the outline | summary of 1 aspect of the detection method of the target nucleic acid disclosed by this specification. It is a figure which shows the outline | summary of other one aspect | mode of the detection method of the target nucleic acid disclosed by this specification. It is a figure which shows an example of the identification probe used for this detection method, and its utilization aspect. It is a figure which shows an example of the identification probe used for this detection method, and its utilization aspect. It is a figure which shows an example of the identification probe used for this detection method, and its utilization aspect. It is a figure which shows another example of the identification probe used for this detection method, and its utilization aspect. It is a figure which shows another example of the identification probe used for this detection method, and its utilization aspect. It is a figure which shows another example of the identification probe used for this detection method, and its utilization aspect. It is a figure which shows another example of the identification probe used for this detection method, and its utilization aspect. It is a figure which shows the chromatography strip used for a comparative example. It is a figure which shows the result in a comparative example. 1 is a diagram showing a chromatography strip used in Example 1. FIG. It is a figure which shows the result in Example 1. FIG. 6 is a diagram showing a chromatography strip used in Example 2. FIG. It is a figure which shows the result in Example 2. FIG. It is a figure which shows the result in Example 3. FIG. It is a figure which shows the problem in the conventional method.

  The present specification relates to a method for detecting a target nucleic acid and a material therefor. According to the method for detecting a target nucleic acid disclosed in this specification, in a nucleic acid amplification step using a DNA polymerase, a primer set and a nucleic acid extension reaction region of a genuine amplification product amplified by the primer set are specifically hybridized. By using a target nucleic acid identification probe having an identification region for soybean, the target nucleic acid identification probe may or may not be degraded according to the exonuclease activity in the 5′-3 ′ direction of DNA polymerase.

  For example, as schematically shown in FIG. 1A, when a nucleic acid amplification reaction is performed using a DNA polymerase that does not have exonuclease activity in the 5′-3 ′ direction, if a target nucleic acid is present, a genuine amplification product is generated. To do. After melting of the genuine amplification product duplex, a hybrid product between the single strand containing the nucleic acid extension region in the amplification product and the target nucleic acid identification probe can be generated. By capturing the hybridized product via a tag derived from the target nucleic acid identification probe, the presence of a genuine amplification product can be confirmed.

  On the other hand, when the target nucleic acid does not exist, even if a primer dimer is generated, a genuine amplification product is not generated. For this reason, the hybridized product of a target nucleic acid identification probe and an amplification product cannot be generated, and since it is not captured, the presence of a genuine amplification product can be denied.

  In addition, as schematically shown in FIG. 1B, when the nucleic acid amplification reaction is performed using a DNA polymerase having exonuclease activity in the 5′-3 ′ direction, if a target nucleic acid is present, a genuine amplification product is generated. Is done. At the same time, the target nucleic acid identification probe is degraded. By capturing the degradation product of the target nucleic acid identification probe, the presence of a genuine amplification product can be confirmed.

  On the other hand, when the target nucleic acid does not exist, even if a primer dimer is generated, a genuine amplification product is not generated. In addition, the target nucleic acid identification probe is not degraded. For this reason, the presence of an authentic amplification product can be denied by capturing the undegraded target nucleic acid identification probe.

  As described above, the target nucleic acid identification probe may or may not be degraded depending on the presence or absence of the exonuclease activity in the 5'-3 'direction of the DNA polymerase and the presence or absence of the authentic amplification product. That is, the degradability of the target nucleic acid identification probe is related to the 5'-3 'exonuclease activity of the DNA polymerase used and the presence or absence of the target nucleic acid. For this reason, the presence or absence of a genuine amplification product can be evaluated from the presence mode of the target nucleic acid identification probe (undegraded product) and its degraded product after the amplification step.

  Therefore, according to this detection method, by capturing at least a part of the target nucleic acid identification probe on the solid phase carrier, the presence or absence of a genuine amplification product can be determined using at least a part of the target nucleic acid identification probe. Can be evaluated. Further, the presence or absence of the genuine amplification product can be evaluated without necessarily capturing and detecting the genuine amplification product, and the target nucleic acid can be detected.

  In addition, according to this detection method, the detection of the signal corresponding to the target nucleic acid is true positive by the intended amplification product (genuine amplification product), or false positive by primer dimer or the like (incorrect amplification product). Can be determined.

  In addition, according to this detection method, even if an appropriate amplification reaction is accompanied by an inappropriate amplification reaction, the target nucleic acid is detected positively as long as the appropriate amplification reaction occurs. Can be determined.

  It is also possible to detect authentic amplification products. Thereby, the target nucleic acid can be detected with high accuracy.

  Hereinafter, some embodiments relating to the disclosure of the present specification will be described with reference to the drawings as appropriate. In the present specification, “nucleic acid” means a nucleotide polymer, and the number thereof is not particularly limited. Nucleic acids include oligonucleotides in which several tens of nucleotides are linked, and longer polynucleotides are also included. The nucleic acid includes DNA single-stranded or double-stranded, RNA single-stranded or double-stranded, DNA / RNA hybrid, DNA / RNA chimera, and the like. Further, the nucleic acid may include a natural base, a nucleotide, and a nucleoside, and may include a non-natural base, a nucleotide, and a nucleoside in part. Nucleic acids include all DNA and RNA including cDNA, genomic DNA, synthetic DNA, mRNA, total RNA, hnRNA and synthetic RNA, as well as artificial nucleic acids such as peptide nucleic acid, morpholino nucleic acid, methylphosphonate nucleic acid and S-oligonucleic acid. Contains synthetic nucleic acids. Moreover, it may be single-stranded or double-stranded.

  In the present specification, the “target nucleic acid” is not particularly limited, and is any nucleic acid whose presence and / or amount is to be detected. The target nucleic acid may be natural or artificially synthesized. Examples of natural target nucleic acids include genetic indicators in organisms such as humans and non-human animals, such as constitution, genetic diseases, onset of specific diseases such as cancer, disease diagnosis, treatment prognosis, selection of drugs and treatments, etc. Or a base sequence. Typically, polymorphisms such as SNP and congenital or acquired mutations can be mentioned. Further, nucleic acids derived from microorganisms such as pathogenic bacteria and viruses are also included in the target nucleic acid. Examples of synthetic target nucleic acids include nucleic acids that are artificially synthesized for some kind of identification. Moreover, the amplification product obtained by performing nucleic acid amplification reaction with respect to a certain kind of natural or artificial nucleic acid is mentioned.

  As used herein, “nucleic acid sample” refers to a sample that may contain a target nucleic acid. Although the sample collection source is not particularly limited, examples of samples that can contain the target nucleic acid include various biological samples (blood, urine, sputum, saliva, tissue, cells (cultivated animal cells and cultured plant cells derived from various animals). , Etc.), or a DNA extraction sample obtained by extracting DNA from such a biological sample. Furthermore, the DNA sample etc. which extracted RNA from the said biological sample and converted into DNA are also contained. Fractions containing nucleic acids from these various samples can be obtained by those skilled in the art with reference to conventional techniques as appropriate.

(Target nucleic acid detection method)
The method for detecting a target nucleic acid disclosed in the present specification includes an amplification step in which a nucleic acid amplification reaction is performed using a DNA polymerase, and the amplification step according to the 5'-3 'exonuclease activity of the DNA polymerase. A capture step of capturing at least a part of the target nucleic acid identification probe that may be present on a solid phase carrier. Furthermore, this detection method can also comprise a detection step for authentic amplification products. Hereinafter, the amplification process, the capture process, and the detection process will be described, and the evaluation of the presence / absence of the genuine amplification product will be sequentially described.

(Amplification process)
The amplification step in this detection method comprises a target comprising a nucleic acid sample, a primer set for amplifying the target nucleic acid, and an identification region that specifically hybridizes to the nucleic acid extension reaction region of the authentic amplification product amplified by the primer set. In this step, a nucleic acid amplification reaction is performed using a DNA polymerase in the presence of a nucleic acid identification probe. This step intends to generate a genuine amplification product. At the same time, this step intends to change the identification probe according to the presence or absence of the nucleic acid extension reaction region in the authentic amplification product by using a target nucleic acid identification probe (hereinafter simply referred to as an identification probe).

(Primer set)
A primer set for amplifying a target nucleic acid can be designed by a known method. The form of the authentic amplification product to be amplified by the primer set is not particularly limited, and is a complete double strand, a partial double strand having a single strand region only on one strand, and a portion in which both strands each have a single strand region. Can be double stranded. The partial double strand is preferable in that the melting of the double strand can be omitted in the hybridization with the detection probe. Particularly, the partial double strand having the single strand region only on one strand is efficiently used as the detection probe. It is more preferable in that hybridization with can be performed.

  The primer set is configured such that a region that specifically hybridizes to a detection probe associated in advance can be added to the authentic amplification product in a subsequent detection step. The authentic amplification product has a region that can specifically hybridize with the detection probe at any site. For example, such a hybridizing region in the authentic amplification product is provided in one of the double stranded portions in the complete double stranded form, and in the double stranded portion or single stranded region in the partial double stranded form. Preferably provided in the single-stranded region. The hybridizing region is preferably provided by a primer. As a sequence preferable as a hybridizing region and a detection probe, for example, the base sequence of SEQ ID NOs: 1 to 100 in which mutual mishybridization is suppressed and its complementary sequence can be appropriately used.

  In addition, the form of the primer set for obtaining a partial double-stranded nucleic acid is disclosed in International Publication No. WO2013 / 038534, and various forms can be adopted. In addition, the site (region) that suppresses or stops the DNA elongation reaction of the DNA polymerase incorporated in the primer to obtain such a partially double-stranded nucleic acid can be described later, and compounds described in the publications can be used. .

(Signal element)
The primer set may also contain a primer that retains the labeling element in the authentic amplification product or can impart the labeling element. Examples of the labeling element include a labeling substance and a labeling substance binding substance.

  In this specification, the “labeling substance” is a substance that makes it possible to distinguish a substance or molecule to be detected from others. The labeling substance is not particularly limited, but typically, a labeling substance using fluorescence, radioactivity, enzyme (for example, peroxidase, alkaline phosphatase, etc.), phosphorescence, chemiluminescence, coloring and the like can be mentioned.

  The labeling substance is preferably a luminescent substance or a chromogenic substance that presents luminescence or color development that can be detected visually (with the naked eye). That is, it is preferably a substance that itself can directly generate a signal that is visible to the naked eye without the need for other components. The detection process can be performed quickly and easily. Such materials typically include various colorants such as various pigments and dyes. In addition to this, in addition to noble metals such as gold and silver, various metals or alloys such as copper, or organic compounds containing the metal (may be complex compounds) may be used. In addition, inorganic compounds such as mica, which are similar to the colorant, can be used.

  This kind of labeling substance typically includes various dyes, various pigments, luminol, isoluminol, acridinium compounds, olefins, enol ethers, enamines, aryl vinyl ethers, dioxene, aryl imidazoles, lucigenin, luciferin and eclion. A chemiluminescent substance is mentioned. Moreover, particles such as latex particles labeled with such a labeling substance are also included. Furthermore, colloids or sols including gold colloids or sols or silver colloids or sols can be mentioned. Furthermore, a metal particle, an inorganic particle, etc. are mentioned.

  As described above, a part of the labeling substance may include particles. The average particle diameter of particles such as latex particles constituting a part of the labeling substance is not particularly limited, and is, for example, from 0.1 nm to 20 μm, and can be appropriately selected depending on the pore diameter of the solid phase carrier.

  Preferred particles are particles that can be suspended in an aqueous solution and consist of a water-insoluble polymeric material. Examples include polyethylene, polystyrene, styrene-styrene sulfonate copolymer, acrylic acid polymer, methacrylic acid polymer, acrylonitrile polymer, acrylonitrile-butadiene-styrene, polyvinyl acetate-acrylate, polyvinyl pyrrolidone, or vinyl chloride-acrylate. Mention may also be made of latex particles having active groups on their surface, for example carboxyl, amino or aldehyde groups.

  Examples of the labeling substance binding substance include substances that can finally bind the labeling substance using protein-protein interaction, low molecular compound-protein interaction, nucleic acid-nucleic acid interaction, and the like. For example, antibodies in antigen-antibody reactions, biotin in avidin (streptavidin) -biotin system, digoxigenin in anti-digoxigenin (DIG) -digoxigenin (DIG) system, haptens represented by FITC in anti-FITC-FITC system, and the like Examples include oligonucleotides that can be hybridized. In this case, the labeling substance finally used for detection is the other molecule or substance that interacts with the labeling substance binding substance (for example, antigen, ie, streptavidin, anti-FITC, oligonucleotide, etc.) It is comprised so that it may provide as a site | part for the coupling | bonding with a binding substance.

  Such labeling substances and labeling substance-binding substances are commercially available, and the production of labeling substances and labeling substance-binding substances and methods for labeling labeling substances etc. are also known. And can be obtained. Furthermore, binding between such a labeling substance or particles labeled with a labeling substance or a labeling substance binding substance and an oligonucleotide such as DNA can be appropriately performed via a functional group such as an amino group, and as such is itself in the field. It is well known.

  The labeling element only needs to be provided at any location of the genuine amplification product to be finally detected. For example, the nucleic acid amplification reaction may be performed using a nucleoside triphosphate composition containing a nucleoside derivative triphosphate having a labeling substance or a labeling substance binding substance, which may be imparted to a genuine amplification product in an amplification step by a primer set. When implemented, the labeling element may be incorporated into the authentic amplification product. Furthermore, the labeling element may be applied in the detection step. For example, when the authentic amplification product includes a labeling substance binding substance, in the detection step or prior to this step, the labeling substance binding substance of the genuine amplification product and a labeling substance having a site that binds to the labeling substance binding substance; The authentic amplification product is detected by the labeling substance.

(DNA polymerase)
In this amplification step, for the exonuclease activity in the 5′-3 ′ direction, either a DNA polymerase not having the activity or a DNA polymerase having the activity can be used. The exonuclease activity in the 5′-3 ′ direction is an activity that degrades DNA from the 5 ′ side to the 3 ′ side.

  In one embodiment of this amplification step, a DNA polymerase having no exonuclease activity in the 5'-3 'direction can be used. A DNA polymerase that does not have an exonuclease activity in the 5'-3 'direction is a DNA polymerase classified as Family B type (α type). Examples of the DNA polymerase include ultrahigh heat Archaea-derived DNA polymerases represented by KOD DNA polymerase, Pfu DNA polymerase and the like. When this type of DNA polymerase is used, the identification probe of the first aspect described later is used. This type of DNA polymerase cannot degrade an identification probe that hybridizes to a template strand of a primer in a nucleic acid sample.

  In another embodiment of this amplification step, a DNA polymerase having exonuclease activity in the 5'-3 'direction can be used. A DNA polymerase having an exonuclease activity in the 5'-3 'direction is a DNA polymerase classified as Family A type (Pol I type). This type of DNA polymerase can be appropriately selected from DNA polymerases derived from thermophilic aerobic eubacteria such as the known Thermos genus such as Thermus aquaticus and Thermus thermophilus. This type of DNA polymerase can degrade an identification probe that hybridizes to a template strand of a primer in a nucleic acid sample.

  Various embodiments of the DNA polymerase are commercially available, for example, as KOD DNA polymerase, Pfu DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, and the like.

(Identification probe)
The identification probe is a probe for evaluating the presence or absence of a genuine amplification product.

(Identification area)
The identification probe can include an identification region that specifically hybridizes to a nucleic acid extension reaction region to be extended by a primer set for amplifying the target nucleic acid. When a DNA polymerase that does not have exonuclease activity in the 5′-3 ′ direction is used as the DNA polymerase, the identification region is not degraded regardless of the presence or absence of a genuine amplification product. However, the identification probe can hybridize to the nucleic acid extension reaction region by the primer set via the identification region.

  In addition, when a DNA polymerase having exonuclease activity in the 5′-3 ′ direction is used as the DNA polymerase, the identification region of the identification probe is 5′-3 ′ when an appropriate amplification reaction of the target nucleic acid occurs. Degraded by directional exonuclease activity.

  The identification region is not particularly limited as long as the target nucleic acid primer does not hybridize, that is, an extension reaction region. Although it is illustration and it does not limit, it can be set as the area | region which hybridizes specifically to the target area | region about 5-100 base length of an extension reaction area | region. From the viewpoint of melting temperature (Tm), the base length of the identification region, that is, the base length of the target region is preferably 10 to 50 bases. Also, the identification can typically be a region that is completely complementary to the target region.

  In addition to the identification region, the identification probe can include a tag region for subsequent capture of the identification probe and the like. The identification probe can also comprise a label element. The labeling element can be provided inside the identification area or outside the identification area.

  The identification probe has various aspects. Hereinafter, first, an identification probe used when a DNA polymerase having no 5'-3 'exonuclease activity is used will be described.

(First aspect of identification probe)
As shown in FIG. 2, the identification probe can include a first tag adjacent to one end of the identification region. The first tag is associated in advance so as to be captured by a first capture provided on the solid phase carrier. The first tag and the first capture and their interaction will be described in detail later.

  Such an identification probe can form a hybridized product with a single strand having a nucleic acid extension reaction region in the DNA duplex of the authentic amplification product. This hybridized product is captured by the first capture via the first tag. In addition, this hybridized product is associated with the first capture when the single strand of the authentic amplification product has a target element (for example, immobilization of the probe as the first capture on the solid support). The signal derived from the labeling element can be presented at the site.

  On the other hand, this identification probe can no longer form a hybridized product when there is no authentic amplification product. In addition, the identification probe is captured by the first capture via the first tag, but it cannot present a signal based on the label element included in the authentic amplification product.

  The identification probe is provided with a region (hereinafter also simply referred to as a suppression region) capable of suppressing or stopping the DNA elongation reaction by DNA polymerase on either or both of the 5 ′ end side and the 3 ′ end side of the identification region. it can. For example, a suppression region can be provided between the first tag and the identification region and / or at the 3 ′ end of the identification region or both. By providing a suppression region between the first tag and the identification region, the primer strand does not extend beyond the suppression region even if the identification probe hybridizes with the primer by mistake. In addition, the 3 'terminal side of the identification probe is not extended by the DNA polymerase. Therefore, even if the identification probe and the primer are mishybridized, a complete dimer is not generated. Thereby, the bad influence by such a dimer can be avoided or suppressed.

  The region (inhibition region) capable of suppressing or stopping the DNA extension reaction by DNA polymerase can stop the DNA extension reaction by DNA polymerase in DNA, and when it is contained in the template strand, This is a region where the nucleotide extension reaction of the complementary strand of the template strand can be suppressed. Such a region is known to those skilled in the art, and typically includes a region not containing a natural base or a derivative of a natural base (such as a natural base) paired with the natural base. Therefore, the region may be only a skeleton chain having no natural base or the like. That is, it may be a sugar-phosphate skeleton or a skeleton applied to other known artificial oligonucleotides.

  In addition, the suppression region may be a chain-like linking group including a single-chain structure having 2 to 40 elements adjacent to the nucleotide via a phosphodiester bond. This is because if the number of elements is 1 or less, the DNA polymerase reaction is likely to be incompletely inhibited or stopped, and if the number of elements exceeds 40, the solubility of nucleotides may be reduced. Considering the effect of suppressing or stopping the DNA polymerase reaction, the chain linking group element is preferably 2 or more and 36 or less, more preferably 3 or more and 16 or less.

  The suppression region preferably includes an alkylene chain or a polyoxyalkylene chain that has 2 to 40 elements and may be substituted. Such a chain-like connection structure is structurally simple and can be easily introduced as a connection site.

As such a connection part, the connection part represented by the following formula | equation (1) is mentioned, for example.
5′-O—C _m H _2m —O-3 ′ Formula (1)
(In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents a phosphate atom of a phosphodiester bond on the 3 ′ side, and m represents an integer of 2 to 40. Represents.)

  In the formula (1), m is preferably 2 or more and 36 or less, and more preferably 3 or more and 16 or less. Typical examples of the substituent of H in formula (1) include an alkyl group, an alkoxy group, and a hydroxyl group. It is preferable that carbon number of an alkyl group and an alkoxy group is 1-8, More preferably, it is 1-4. Moreover, when it has two or more substituents, the substituents may be the same or different. Furthermore, it is also preferable that it does not have a substituent.

Moreover, as another suppression area | region, suppression represented by the following formula | equation (2) is mentioned.
5 ′-(OC _n H _2n ) _l —O-3 ′ Formula (2)
(In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents a phosphate atom of a phosphodiester bond on the 3 ′ side, and n represents an integer of 2 or more and 4 or less. And l is an integer of 2 or more, and (n + 1) × l represents an integer of 40 or less.)

  In the formula (2), (n + 1) × l is preferably 2 or more and 36 or less, and more preferably 3 or more and 16 or less. The same aspect as the substituent in Formula (1) is applied to the substituent of H in Formula (2).

  Examples of the suppression region include the following chain sites.

  Next, an identification probe used when a DNA polymerase having 5'-3 'exonuclease activity is used will be described. Such identification probes include second to sixth aspects.

(Second aspect of identification probe)
As shown in the left column of FIG. 3, the identification probe can include, for example, a labeling substance and a quencher that can quench the labeling substance by the proximity effect. The identification probe holds the labeling substance and the quencher in an undecomposed state so that the labeling substance can be maintained in the quenched state. By way of example and not limitation, a labeling substance can be provided adjacent to one end of the identification region and a quencher can be provided adjacent to the other end. Although not shown, a labeling substance and a quencher can be provided in the identification region. Furthermore, either one of a labeling substance and a quencher can be provided adjacent to one end of the identification region, and the other can be provided in the identification region. Typically, as shown in FIG. 3, a labeling substance is provided adjacent to one end of the identification region, and a quencher is provided adjacent to the other end.

  In such an arrangement, an identification probe comprising a labeling substance and a quencher, when undegraded, the labeling substance is quenched by the quencher, but there is a genuine amplification product and exonuclease activity in the 5′-3 ′ direction. If the identification region is decomposed by the above, the labeling substance is separated from the quencher, so that the original emission signal can be exhibited. Therefore, according to this aspect, the genuine amplification product can be reliably detected.

  Examples of such a labeling substance include fluorescent labels. Those skilled in the art can appropriately select such labeling substances and quenchers. For example, as an example of such an identification probe, there is a commercially available so-called Taqman probe (trade name).

(Other aspects of identification probe)
The identification probe may further include other elements in addition to the identification region. The other element is an element for capturing at least a part of the identification probe. Such an element serves as an element for separating the identification probe or a degradation product thereof from the environment for hybridizing the authentic amplification product and the target nucleic acid detection probe (hereinafter, this environment is referred to as a target nucleic acid detection system or simply a detection system). be able to. As a result, detection of the target nucleic acid can be carried out without hindering their hybridization by the identification probe and / or a degradation product thereof, and the accuracy of the amplification reaction can be increased.

  Hereinafter, an identification probe including a tag as such an element will be described.

(Third Aspect of Identification Probe)
For example, as shown in the left column of FIG. 4, the identification probe can include a labeling substance and a quencher and a first tag, as in the second mode. For example, the first tag can be provided on the labeling substance side. The first tag is configured to be captured in advance by the first capture. The first tag and the first capture and their interaction will be described in detail later.

  As shown in FIG. 4, the identification probe comprising the labeling substance, the first tag, and the quencher in such an arrangement has the labeling substance quenched by the quencher in an undegraded state, but the authentic amplification product is If the identification region is decomposed by the exonuclease activity in the 5′-3 ′ direction, the labeling substance is separated from the quencher and thus exhibits an original luminescence signal, and further, via the first tag. Captured by the first capture. Therefore, according to this aspect, the genuine amplification product can be reliably detected.

(Fourth aspect of identification probe)
For example, as shown in FIG. 5, the identification probe can include a first tag adjacent to one end of the identification region. The first tag is configured to be captured in advance by the first capture. Hereinafter, the first tag and the first capture will be described.

  For example, the first tag and the first capture can constitute a relationship in which the first capture captures the first tag by interaction. In general, such interactions are non-covalent interactions and include, for example, hydrogen bonds, ionic bonds, electrostatic bonds, hydrophobic bonds, and the like. It may also be a physical interaction.

  Such interactions typically include various organic compound-organic compound interactions. Preferably, such interactions in the organism can be utilized. Examples of such interaction include protein-protein interaction, protein-nucleotide interaction, nucleotide-nucleotide interaction, organic compound-protein interaction, and the like. Preferably, such interactions are specific.

  Such an interaction preferably has a stability equal to or higher than that of hybridization between a target nucleic acid amplification product and a target nucleic acid detection probe. Furthermore, the interaction is preferably irreversible. Such an interaction is known and can be appropriately selected by those skilled in the art.

  For example, the following aspect is mentioned as a 1st tag and 1st capture for capturing an identification probe or its decomposition product.

  The first tag and the first capture are based on protein-protein interactions, and the first tag is one of a pair of proteins that specifically bind to each other, and the first capture is the other of the pair. can do. Examples of such pairs include hapten-antibodies such as antigen protein (peptide) and antibody, streptavidin (avidin) and biotin, FITC and anti-FITC, anti-digoxigenin (DIG) -digoxigenin (DIG), and the like.

  In addition, for example, the first tag and the first capture can be one of the pair of polynucleotides such as DNA each having a base sequence that specifically hybridizes to each other and the other, respectively.

  In this case, the first tag is preferably associated in advance with the probe used in the detection step, and is configured to specifically hybridize to the probe.

  The length contributing to the hybridization of the first tag is not particularly limited, and can be set as appropriate by those skilled in the art. In addition, it is preferable to employ a sequence in which mishybridization is suppressed as the first tag region. As the first tag region, for example, the base sequence of SEQ ID NOS: 1 to 100 in which mutual mishybridization is suppressed and its complementary sequence can be appropriately used.

  Furthermore, for example, each of the first tag and the first capture can be a particle having a predetermined size and / or form and a porous body capable of suppressing the movement of the particle. For example, the particles as the first tag cannot pass through the porous body based on the size (for example, average particle diameter, etc.) and / or morphology, or the movement inside the porous body is suppressed or trapped. Can be equalized.

  The material, form, and size of the particles as the first tag are not particularly limited. The particles may be beads such as plastics and ceramics, and may be particles that can serve as the labeling elements described above.

  The material and form of the porous body are not particularly limited. The material for constituting the porous body may be any material as long as it has an appropriate pore size or void and can suppress the movement of particles as the first tag. Moreover, the form includes a disk, a film, a sheet | seat etc. other than the particle | grains of various shapes. For example, the porous body may take various forms, and may be in the form of a column filled with a particulate porous body, or may be various shaped articles obtained by further shaping these porous particles. . Furthermore, in addition to a film or sheet such as a non-woven fabric, a molded body such as a disk formed by assembling fibrous bodies may be used.

  When using the first tag and the first capture of such an embodiment, for example, the result of the nucleic acid amplification reaction that may include an identification probe including the first tag and / or a degradation product thereof is the first capture. By making contact with the material, the identification probe or the like can be captured inside the porous material. Further, by loading the resultant product so as to move within the porous body, the identification probe and the like can be separated via the porous body. That is, the identification probe or the like can be separated from the passing material of the porous body by, for example, preventing the identification probe from entering the porous body or blocking its passage inside the porous body. Identification probes and their degradation products can be captured.

  Further, for example, the porous body may be provided as a part of a chromatography carrier when the detection step intends hybridization by chromatography. That is, for example, the porous portion as the first tag can be configured to have an average pore size distribution such that particles cannot pass or are captured. The first capture may be provided at the end of the chromatography carrier that contacts the chromatography medium.

  The identification probes of these embodiments are captured by the first capture and associated with the first capture when the identification probe is undegraded (for example, the probe as the first capture on the solid support). The signal derived from the labeling element can be presented at the fixing site. On the other hand, since the identification probe is no longer separated from the first tag and the label element when the identification area is decomposed, even if the first tag area is captured by the first capture, The signal associated with one tag cannot be presented.

(Fifth aspect of identification probe)
As shown in the left column of FIG. 6, the identification probe can include a first tag, a second tag, and a label element in addition to the identification region. The 1st tag can be made into various modes like the 4th mode of the above-mentioned identification probe. The second tag is associated in advance with the second capture used in the detection step, and is configured to be captured by the second capture by interacting with the second capture.

  The identification probe includes a first tag adjacent to one end of the identification region, a second tag adjacent to the other end, and a label element adjacent to the second tag. Can be provided.

  The first tag and the second tag only need to be separated from the detection system by interaction with the first capture and the second capture, respectively. Therefore, the first tag and the second tag may be based on the same type of interaction, but may be based on different interactions. In FIG. 5, the first tag and the second tag are illustrated as if they are based on different interactions.

  For example, if the first tag and the second tag are polynucleotides such as DNA and are captured by the first capture and the second capture, respectively, by hybridization based on base pairing, the first tag The length of the second tag is not particularly limited and can be appropriately set by those skilled in the art. Moreover, it is preferable to employ | adopt the arrangement | sequence in which mishybridization was suppressed for the 1st tag and the 2nd tag. As the base for the first tag and the second tag, for example, the base sequence of SEQ ID NOs: 1 to 100 in which mutual mishybridization is suppressed and its complementary sequence can be appropriately used.

  Since the identification probe comprises a second tag in addition to the first tag, when the identification probe is undegraded, it is captured by the first capture and associated with the first capture (e.g., The signal derived from the labeling element can be presented at the first capture immobilization site on the solid support). At the same time, even when captured by the second capture, it is possible to present a signal derived from the label in association with the second capture.

  On the other hand, in the identification probe, when the identification region is decomposed, the first tag region and the label element are separated. For this reason, even if the first capture captures the first tag region, no signal is presented, but the second capture captures the second tag region and a signal is presented associated with the second capture. Is done.

(Sixth aspect of identification probe)
As shown in FIG. 7, the identification probe can include a first tag, a second tag, and a third tag. Similar to the fifth aspect of the identification probe described above, the first tag is associated with the first capture in advance and is configured to specifically interact with the capture. Similar to the fifth aspect of the identification probe described above, the second tag is associated in advance with the second capture used in the detection step, and is configured to specifically interact with the capture. The first tag and the second tag only need to be separated from the detection system by interaction with the first capture and the second capture, respectively.

  The identification probe can comprise a third tag. The third tag is a region that specifically hybridizes with at least a part of the amplification product of the target nucleic acid. Therefore, the third tag region can function as a detection probe for detecting the amplification product of the target nucleic acid. Therefore, the third tag is a polynucleotide such as DNA and has a base sequence that specifically hybridizes with a part of the amplification product.

  The identification probe includes a first tag adjacent to one end of the identification region, and includes a second tag or a third tag adjacent to the other end, and the second or third tag A second or third tag can be provided adjacent to.

  In addition, when the first tag, the second tag, and the third tag region are polynucleotides such as DNA and hybridize specifically with the first to third captures, the hybridization is performed. The length of the base sequence that contributes to is not particularly limited, and can be appropriately set by those skilled in the art. Moreover, it is preferable to employ a sequence in which mishybridization is suppressed for the first tag, the second tag, and the third tag. As the first to third tags, for example, the base sequence of SEQ ID NOs: 1 to 100 in which mutual mishybridization is suppressed and its complementary sequence can be appropriately used. FIG. 6 illustrates the case where each of the first to third tags is DNA.

  The identification probe is captured by the first capture when the identification probe is in an undegraded state, and is associated with the first capture (for example, a fixing site of the first capture on the solid phase carrier). The amplification product of the target nucleic acid can be captured via the third tag. At the same time, when contacted with the second capture, it is captured by the second capture, and the amplification product of the target nucleic acid can be captured via the third tag region in association with the second capture. is there.

  On the other hand, when this identification probe is decomposed, only the portion including the first tag region in the first capture is captured. In addition, the second capture captures a portion including the second tag region and the third tag region, and captures the amplification product of the target nucleic acid via the third tag region.

  When this identification probe is used, the amplification product of the target nucleic acid captured in association with the first capture and / or the second capture via the third tag is preliminarily provided with a labeling element on the amplification product itself. It can be presented as a signal based on the labeling element as appropriate.

  Although not shown, labeling elements such as a labeling substance and a label binding substance may be provided in the identification region.

(Seventh aspect of identification probe)
As illustrated in FIG. 8, the identification probe can include a first tag, a second tag, and a third tag, as in the identification probe of the sixth aspect illustrated in FIG. 7. Similar to the fourth aspect of the identification probe described above, the first tag is associated in advance with the first capture used in the detection step, and is configured to specifically interact with the capture. Similar to the fifth aspect of the identification probe described above, the second tag is associated in advance with the second capture used in the detection step, and is configured to specifically interact with the capture.

  The identification probe can comprise a third tag. The third tag in the seventh aspect is an element for labeling the identification probe itself. That is, in this aspect, a label probe including a third capture that specifically interacts with the third tag and a label element can be further provided. The third tag is sufficient if it allows the identification probe itself to specifically interact with the labeled probe to retain the label. Therefore, the third tag and the third capture can be appropriately selected from the aspects already described.

  The identification probe of this aspect also includes the first tag adjacent to one end of the identification region and the second tag or second adjacent to the other end, as in the identification probe of the sixth aspect. 3 tags, and the third or second tag can be provided adjacent to the second or third tag.

  In addition, when the first tag, the second tag, and the third tag region are polynucleotides such as DNA and hybridize specifically with the first to third captures, the hybridization is performed. The length of the base sequence that contributes to is not particularly limited, and can be appropriately set by those skilled in the art. Moreover, it is preferable to employ a sequence in which mishybridization is suppressed for the first tag, the second tag, and the third tag. As the first to third tags, for example, the base sequence of SEQ ID NOs: 1 to 100 in which mutual mishybridization is suppressed and its complementary sequence can be appropriately used. FIG. 7 illustrates the case where each of the first to third tags is DNA.

  This identification probe can be captured by the first capture when the identification probe is undecomposed. At the same time, when touched by the second capture, it can be captured by the second capture. In each case, the undegraded identification probe is labeled with a labeled probe via a third tag.

  On the other hand, when this identification probe is decomposed, only the portion including the first tag region in the first capture is captured. In the second capture, a portion including the second tag region and the third tag is captured and labeled with a labeling probe via the third tag.

  Although various aspects of the identification probe have been described above, the present invention is not limited to these aspects, and the identification region, various tags (and captures), and label elements can be configured in various combinations depending on the purpose. .

  In this amplification step, a nucleic acid amplification reaction is performed on the nucleic acid sample using a primer set, an identification probe, and a DNA polymerase. Various known methods can be applied to the nucleic acid amplification method, and typically, various PCRs such as PCR and multiplex PCR are used. A person skilled in the art can appropriately set the solution composition, temperature control, time, etc. in carrying out the nucleic acid amplification step.

  This detection method is particularly significant when obtaining amplification products of a plurality of target nucleic acids. Usually, a plurality of primer sets are used simultaneously to obtain a plurality of target nucleic acids. In such a case, it is often difficult to suppress the formation of primer dimers, and there is a high possibility of exhibiting false positives.

(Authentication product detection process)
This method may include a detection step of detecting a genuine amplification product in addition to the capture step described later. This detection step includes contacting the result of the nucleic acid amplification reaction with the target nucleic acid detection probe under conditions that allow hybridization of the target nucleic acid detection probe that specifically hybridizes with the authentic amplification product. Can do. By this detection step, an authentic amplification product can be detected. As described above, the authentic amplification product can be provided with labeling elements in various forms. In this detection step, the target nucleic acid is detected by detecting signals based on the labeling elements by various methods known to those skilled in the art. Can be detected. In addition, the detection means according to the label | marker element used at a detection process is known to those skilled in the art, and the skilled person can use the detection means according to the label | marker element to be used suitably.

  In addition, this detection process may be accompanied by the capture process in this method, which will be described later, or may be performed as a process independent of the capture process. As long as the presence / absence of the authentic amplification product described below is evaluated, this detection step is not necessarily required, but the accuracy of detection of the genuine amplification product can be improved by performing this detection step.

  Contacting the result of the amplification reaction with a probe or the like under predetermined hybridization conditions is generally referred to as nucleic acid hybridization (step). The hybridization step is usually performed on a probe bound to a solid phase carrier. The probe and solid phase carrier will be described later. The form of hybridization is not limited to the form of hybridization (immersion-type hybridization) performed by supplying a hybridization solution to the surface of a solid phase carrier on which a probe is immobilized. For example, it is a form of chromatography (development type hybridization) in which a hybridization solution that is also a mobile phase is supplied to a part of a solid phase carrier, and the hybridization solution is developed in a predetermined direction with respect to the solid phase carrier. May be. In this detection method, it is preferable to employ a chromatographic form. This is because the significance of improvement in detection accuracy is higher in a hybridization process by chromatography that is simple and excellent in rapidity. In addition, according to chromatography, since the order of contact with the probe can be controlled, it is possible to more easily determine the accuracy and the like.

  A person skilled in the art can appropriately set hybridization conditions in the detection step. That is, those skilled in the art can appropriately set the hybridization time, such as the hybridization temperature, the salt concentration of the hybridization solution, and the type of solvent. In this detection method, particularly in the case of chromatographic hybridization, for example, about 10 ° C. to 50 ° C., preferably about 15 ° C. to 35 ° C., about several minutes to several tens of minutes, typically within 30 minutes. Preferably, the hybridization step can be performed within 20 minutes.

  When the hybridization step is performed in a chromatographic form, the development medium used for the hybridization step is preferably an aqueous medium. The aqueous medium can be prepared by appropriately blending known components used for such chromatography.

  The probe is immobilized on a carrier. As such a carrier, a solid phase carrier can be used. For example, the carrier may be plastic or glass, and the material is not particularly limited. In particular, examples of the solid phase carrier used for hybridization by chromatography include so-called porous materials mainly composed of polymers such as polyethersulfone, nitrocellulose, nylon, and polyvinylidene fluoride. Cellulose materials such as filter paper can also be preferably used.

  The shape of the carrier may be a flat plate shape, but may be a bead shape, and the shape is not particularly limited. The solid phase carrier is preferably an array (particularly a microarray) in which the carrier is in the form of a solid phase plate and a plurality of detection probes are fixed in a fixed arrangement. The array can fix a large number of probes, and is convenient for comprehensively detecting various target nucleic acids at the same time. In addition, the chromatographic form is preferable because the order of contact between the resulting product and the probe can be controlled by the position at which the probe is immobilized.

  Examples of the solid phase carrier used for the hybridization by chromatography include so-called porous materials mainly composed of polymers such as polyethersulfone, nitrocellulose, nylon, and polyvinylidene fluoride. Cellulose materials such as filter paper can also be preferably used. The probe-immobilized body for chromatography does not necessarily need to be composed of a single solid phase carrier. As long as the development medium can be moved by capillary action as a whole, it may be connected by a plurality of solid phase carriers. Moreover, the whole form of the probe immobilized body for chromatography is not particularly limited. Any form such as a sheet form or a thin bar form capable of developing and diffusing the chromatographic liquid by the capillary phenomenon may be used. Preferably, it is an elongate body, and one edge part along the longitudinal direction contacts the developing medium of chromatography.

(Capture process)
The method can comprise a capture step of capturing at least a portion of the identification probe that may be present in the amplification step on a solid phase carrier in accordance with the 5′-3 ′ exonuclease activity of the DNA polymerase. According to this step, capturing at least a part of the target nucleic acid identification probe can contribute to the evaluation of the presence or absence of a genuine amplification product using at least a part of the target nucleic acid identification probe.

  At least a part of the identification probe can include an undegraded separate probe and a degradation product of the identification probe. This is because, as described above, the identification region of the identification probe can be degraded by the exonuclease activity in the 5'-3 'direction of DNA polymerase.

  In order to capture at least a part of the identification probe, a tag included in at least a part of the identification probe is captured by a capture probe on a solid phase carrier. The combination of tag and capture has already been described. In addition, in order to capture at least a part of the identification probe on the solid phase carrier, the tag included in the identification probe and the probe on the solid phase carrier are allowed to interact with each other. In addition, as described in the detection step, the capture by such interaction can use the contact form between the probe and the capture in the immersion form or the chromatographic form applied to the nucleic acid hybridization.

  This detection method is intended for the detection of target nucleic acids, ie the detection of authentic amplification products. However, the primers may hybridize unintentionally, and the extension reaction may proceed to produce an unintentional unsynthetic product in the form of a primer dimer and its amplification product (incorrect amplification product). Such a primer dimer is no different from a genuine amplification product except that it does not contain a site corresponding to the target nucleic acid. In particular, in the case where a single-stranded tag region is provided in the region hybridized with the detection probe, the detection step using nucleic acid hybridization behaves in the same manner as a genuine amplification product. Therefore, even when the target nucleic acid is not present, this false amplification product is detected in the same manner as the genuine amplification product, and as a result, a state that is apparently positive is caused.

  Therefore, it is useful for this detection method to evaluate the presence or absence of a genuine amplification product using at least a part of the identification probe. Further, since the presence of the genuine amplification product is nothing but detection of the target nucleic acid, it is useful in that the target nucleic acid can be detected without detecting the target nucleic acid itself.

  The evaluation of the presence or absence of a genuine amplification product is an evaluation in which a change in signal is detected by using at least a part of the identification probe captured on the solid phase carrier in the capture step, that is, the undegraded identification probe and / or its degradation product. This is done by carrying out the process. In some cases, a change in signal can be detected in the amplification process. Moreover, you may implement the evaluation process which detects a signal etc. in a capture process or separately from a capture process.

  Therefore, when this detection method implements the detection process of a genuine amplification product, it can separate from the said detection process and can implement a capture process before or after a detection process. On the other hand, the detection step can also be performed as an evaluation step using the identification probe and / or a decomposition product thereof.

  In addition, when a DNA polymerase having exonuclease activity in the 5′-3 ′ direction is used in the amplification step, the intended target nucleic acid can be obtained by capturing or detecting the undegraded product of the identification probe at any of the stages described above. It is possible to affirm the possibility that the amplification reaction is not generated (the possibility that no amplification reaction has occurred and the possibility of primer dimer formation).

  Furthermore, in the case of using a DNA polymerase having exonuclease activity in the 5′-3 ′ direction in the amplification step, the target nucleic acid can be detected by capturing or detecting both the degradation product of the identification probe and the undegraded product of the identification probe. It is possible to evaluate the presence / absence of both an authentic amplification reaction and an unauthentic amplification reaction. That is, even if it can be affirmed that there is an inaccurate amplification reaction, the possibility of a false positive can be eliminated if the existence of a genuine amplification reaction can be affirmed.

  As described above, according to the present detection method, the presence or absence of a genuine nucleic acid amplification reaction can be evaluated, so that the target nucleic acid can be detected by itself. Moreover, according to this detection method, false positives can be determined. Furthermore, even if there is an authentic amplification reaction, the authentic amplification reaction can be affirmed. This makes it easy to avoid false positive discrimination and detection accuracy, detection sensitivity, reproducibility, etc. due to false amplification reactions, regardless of the presence or absence of false amplification reactions such as primer dimers. . Therefore, it is possible to eliminate the cost and labor of eliminating the false amplification product by suppressing the false amplification reaction.

  The following various aspects are mentioned as an embodiment which evaluates the presence or absence of the amplification reaction of a target nucleic acid using an identification probe. In the following description, a mode in which the evaluation process is simultaneously performed in the detection process will be described. However, the evaluation process can be performed separately from the detection process.

(Evaluation by the identification probe of the first aspect)
(First aspect of identification probe)
A detection method using the identification probe of this embodiment is shown in FIG. The discrimination probe of this embodiment involves an amplification step using a DNA polymerase that does not have 5′-3 ′ exonuclease activity. In the following first embodiment, the case where a primer set for obtaining a complete double-stranded DNA as an amplification product is used will be described.

  As shown in FIG. 2, when the identification probe of this embodiment is used, a genuine amplification product of the target nucleic acid is formed if a genuine amplification reaction has occurred.

  In such an amplification step, or upon denaturation by heating of the amplification reaction solution after the amplification step and subsequent reannealing, a hybridization product of the identification probe and one strand of the nucleic acid amplification product duplex can be formed. Like that. That is, when a genuine amplification product is generated, in the cooling (cooling) step after the denaturation (melting) of the double-stranded DNA of the genuine amplification product, together with the hybridization product of the genuine amplification product, the identification probe and the genuine amplification product A hybridized product with a single-stranded DNA containing a nucleic acid extension reaction region can also be formed. The denaturation of double-stranded DNA includes thermal denaturation and denaturation with chemical substances such as alkali denaturation.

  As shown in FIG. 2 as an example, such a hybridized product can be detected by a tag derived from an identification probe and a labeling element derived from a primer set. That is, the presence of the authentic amplification product is detected not only by the signal from the hybridization product of the identification probe and one strand of the authentic amplification product, but also affirmed by the signal based on the authentic amplification product by the detection probe.

  In order to detect the signal level and evaluate it with higher accuracy, it is preferable to carry out the amplification step and the capture step of this detection method under the same conditions as the control except that the nucleic acid sample is free and / or primer free. When this identification probe is used, the capturing step is the same regardless of whether it is immersion type hybridization or chromatography. The same applies to the following embodiments.

(Evaluation by the identification probe of the second aspect)
A detection method using the identification probe of this embodiment is shown in FIG. The signal column indicates the presence / absence of a signal by the identification probe / the presence / absence of a signal by the detection probe (hereinafter the same in FIGS. 4 to 7). In the following second to sixth embodiments, a case where a primer set for obtaining a single-stranded partial double-stranded DNA of only one strand as an amplification product is used as an example, and the seventh embodiment shown in FIG. In the embodiment, a case where a primer set for obtaining a complete double-stranded DNA as an amplification product is used is exemplified.

  As shown in the upper part of FIG. 3, when the identification probe of this embodiment is used, the identification region is decomposed by the exonuclease activity in the 5′-3 ′ direction of the DNA polymerase. Since it is not quenched, a signal such as fluorescence is presented. These signals can affirm a genuine amplification reaction. When a genuine amplification reaction occurs, the genuine amplification product is captured by the detection probe, and a signal based on the label element included in the genuine amplification product is presented. The presence of the genuine amplification product, that is, the presence of the target nucleic acid is detected not only by the signal of the labeling substance based on the degradation product of the identification probe but also affirmed by the signal from the detection probe.

  In addition, as shown in the middle part of FIG. 3, when a false amplification reaction has occurred and a primer dimer is generated, the identification probe is not decomposed, so that the labeling substance remains quenched, but the primer dimer Can be captured by the detection probe to present a signal based on the labeling element. The absence of the authentic nucleic acid amplification reaction, that is, the absence of the target nucleic acid is affirmed only by the fact that the signal of the labeling substance based on the undegraded product of the identification probe is not detected.

  Furthermore, as shown in the lower part of FIG. 3, when the amplification reaction has not occurred, the identification probe is not decomposed, so that the labeling substance remains quenched and no signal is captured by the detection probe. Not. The absence of a genuine amplification reaction, that is, the absence of the target nucleic acid, is affirmed only by the fact that the signal of the labeling substance based on the undegraded product of the identification probe is not detected.

(Evaluation by the identification probe of the third aspect)
A detection method using identification of this aspect is shown in FIG. As shown in the upper part of FIG. 4, when a genuine amplification product is present, the identification region is decomposed, so that the fluorescent labeling substance, the first tag region, and the quencher are separated. Therefore, a signal based on the fluorescent labeling substance is presented in the first capture probe. Further, the genuine amplification product is captured by the detection probe, and a signal based on the labeling element included in the genuine amplification product is presented. The presence of the genuine amplification product, that is, the presence of the target nucleic acid is detected not only by the generation or increase of the signal of the labeling substance based on the degradation product of the identification probe, but also by the signal at the detection probe.

  On the other hand, as shown in the middle part of FIG. 4, when a genuine amplification product is not present, or when a primer dimer is generated, the identification region is not decomposed. Therefore, in the first capture probe, the identification probe has a quencher. Accompanying. For this reason, in the detection step, a signal based on the fluorescent labeling substance is not presented in the first capture probe. In addition, the primer dimer can be captured by the detection probe and a signal based on the labeling element can be presented. By presenting these signals, the presence of a false amplification product such as a primer can be affirmed. The absence of the true amplification product, that is, the absence of the target nucleic acid is also affirmed only by the signal of the labeling substance based on the undegraded product of the identification probe.

  Furthermore, as shown in the lower part of FIG. 4, when the amplification reaction has not occurred, the signal from the labeling substance is not presented in the first capture probe, but since no signal is captured by the detection probe, No signal is presented. The absence of a genuine amplification reaction, that is, the absence of a genuine amplification product is affirmed only by the fact that the signal of the labeling substance based on the undegraded product of the identification probe is not detected.

(Evaluation by the identification probe of the fourth aspect)
FIG. 5 shows a detection method using the identification probe of this embodiment. In the upper column of the column of capture form, the capture form in immersion hybridization is shown, and the lower part shows the capture form in chromatography.

  As shown in the upper part of FIG. 5, when this identification probe is used, if a genuine amplification reaction occurs, the identification region is decomposed, and as a result, the first tag region and the label element are separated. . Therefore, the signal based on the label element is not presented in the first capture probe. In addition, the genuine amplification product is captured by the detection probe, and a signal based on the labeling element included in the genuine amplification product is presented. The presence of a genuine nucleic acid amplification reaction, that is, the presence of the target nucleic acid is detected not only by the decrease or disappearance of the signal of the labeling substance based on the degradation product of the identification probe, but also affirmed by the signal in the detection probe.

  On the other hand, as shown in the middle part of FIG. 5, when a genuine amplification reaction has not occurred and a primer dimer has occurred, the identification region is not decomposed, so the first tag region in the first capture probe is With a sign element. For this reason, in the detection step, a signal based on the label element is presented in the first capture probe. In addition, the primer dimer can be captured by the detection probe and a signal based on the labeling element can be presented. By showing these signals, it is possible to affirm the generation of primer dimers in an incorrect amplification reaction. The absence of the authentic nucleic acid amplification reaction, that is, the absence of the target nucleic acid is affirmed only by the signal of the labeling substance based on the undegraded product of the identification probe.

  Furthermore, as shown in the lower part of FIG. 5, when no amplification reaction has occurred, a labeling element is presented in the first capture probe, but no signal is captured by the detection probe. Not presented. The absence of a genuine amplification reaction, that is, the absence of the target nucleic acid, is affirmed only by the fact that the signal of the labeling substance based on the undegraded product of the identification probe is not detected.

(Evaluation by the identification probe of the fifth aspect)
A detection method using the identification probe of this embodiment is shown in FIG. In the upper column of the capture form column, the capture form in immersion type hybridization is shown, and the lower stage shows the capture form in chromatography (the same in FIG. 7).

  As shown in the upper part of FIG. 6, when this identification probe is used, if a genuine amplification reaction has occurred, the identification region is decomposed so that the first tag region and the second tag region are separated from each other. , And the label element is attached only to the second tag region. Therefore, the signal based on the label element is not presented in the first capture probe, and the signal based on the label element is presented only in the second capture probe. Further, the genuine amplification product is captured by the detection probe, and a signal based on the label element is presented. The presence of a genuine nucleic acid amplification reaction, that is, the presence of the target nucleic acid is detected only by “no / yes” of the labeling substance signal (first capture probe / second capture probe) based on the degradation product of the identification probe. In addition, the signal at the detection probe is also affirmed. Further, both of these can clearly affirm a genuine amplification reaction. In this embodiment, the detection step is the same for both immersion hybridization and chromatography.

  This identification probe can selectively detect the degradation product by the second tag region and the labeling element. For this reason, the presence of a genuine amplification reaction can be detected with high accuracy.

On the other hand, as shown in the middle part of FIG. 6, since the identification area is not decomposed, the first tag area also includes a label element. For this reason, a signal based on the label element is presented in the first capture probe, and a signal can be presented in the second capture probe as well. In addition, when a genuine amplification reaction has not occurred and a primer dimer is generated, the primer dimer is captured by the detection probe and a signal based on the labeling element is presented. The absence of the authentic nucleic acid amplification reaction, that is, the absence of the target nucleic acid is detected not only by the presence / absence of the signal of the labeling substance based on the undegraded product of the identification probe, but also by the signal in the detection probe. Even affirmed. Furthermore, both can clearly affirm an incorrect amplification reaction.
In this embodiment, in the case of immersion type hybridization, the capture mode is as described above. However, according to the preceding hybridization (chromatography) described later, an undegraded identification probe in advance by the first capture probe. Therefore, the capture of the primer dimer with the second capture probe is suppressed or avoided, and more explicit distinction is possible.

  Furthermore, as shown in the lower part of FIG. 6, when no amplification reaction occurs, a labeling element is presented in the first capture probe and the second capture probe, but detection is not performed by the detection probe. No signal is displayed in the probe for use. The absence of the authentic nucleic acid amplification reaction, that is, the absence of the target nucleic acid is detected not only by the presence / absence of the signal of the labeling substance based on the undegraded product of the identification probe, but also by the signal of the detection probe. It is affirmed even by the absence. Furthermore, both of these clearly affirm the absence of the target nucleic acid. In this embodiment, in the case of immersion type hybridization, the capture mode is as described above. However, according to the preceding hybridization (chromatography) described later, an undegraded identification probe in advance by the first capture probe. Therefore, the capture of the primer dimer with the second capture probe is suppressed or avoided, and more explicit distinction is possible.

  In addition, according to the above embodiment, the presence or absence of a target nucleic acid can be determined without detecting a signal using a detection probe. That is, the presence (positive) of the target nucleic acid can be distinguished from the absence (false positive and negative) of the target nucleic acid only by signal detection with the degradation product and undegraded product of the identification probe as a result of the amplification reaction. .

(Preceding hybridization)
Preferably, prior to the detection step after the amplification step or in the detection step, the first capture probe and the resultant product of the amplification reaction can be hybridized with the identification probe and / or its degradation product and the first capture probe. It is preferable to contact under conditions (hereinafter, such hybridization is also referred to as pre-hybridization). In this way, in particular, the presence or absence of the genuine amplification reaction and the false amplification reaction can be more clearly evaluated by eliminating the undegraded identification probe in advance.

  The form of prior hybridization is not particularly limited. In the case of immersion type hybridization, capture may be performed via the first capture probe prior to immersion type hybridization. Alternatively, an interaction element such as biotin may be added in advance to the first tag region and captured by the corresponding action element. Pre-hybridization can be easily realized by employing chromatography in the detection step. In other words, the first capture probe is provided on the upstream side (start side or base point side) in the development direction of the hybridization solution, and the second capture probe and the detection probe are provided on the further downstream side. Hybridization is realized. In order to reliably recover the undegraded identification probe, it is preferable to immobilize a sufficient amount of the first capture probe on the solid phase carrier.

  Hybridization prior to such a detection step is easily realized by performing the hybridization step in a chromatographic form. That is, the first capture probe may be provided on the upstream side (development start side) in the development direction of the hybridization solution.

(Evaluation by the identification probe of the sixth aspect)
A detection method using the identification probe of this embodiment is shown in FIG. When using an identification probe, as shown in the upper part of FIG. 7, when a genuine amplification reaction occurs, the identification region is decomposed, resulting in the first tag region, the second tag region, and the third tag. It is separated from the area. Therefore, in the detection step, only the first tag region is captured by the first capture probe, the amplification product is captured only by the second capture probe, and a signal based on the labeling element included in the amplification product is presented. . In this embodiment, the detection step is the same for both immersion hybridization and chromatography.

  This identification probe can selectively detect the decomposition product by the second tag region and the third tag region. For this reason, the presence of a genuine amplification reaction can be detected with high accuracy.

  On the other hand, as shown in the middle part of FIG. 7, when a primer dimer is generated by an incorrect amplification reaction without generating an authentic amplification reaction, undegraded in the first capture probe and the second capture probe. As the identification probe is captured, in any case, the primer dimer is captured and a signal based on the labeling element is presented. In this embodiment, in the case of immersion type hybridization, the capture mode is as described above. However, according to the preceding hybridization (chromatography), the undegraded identification probe is previously removed by the first capture probe. Therefore, the capture of the primer dimer with the second capture probe is suppressed or avoided, and a more explicit distinction becomes possible.

  Furthermore, as shown in the lower part of FIG. 7, when an authentic amplification reaction has not occurred and an amplification reaction itself has not occurred, an undecomposed identification probe is present in the first capture probe and the second capture probe. Although it is captured, no signal is presented in either case because there is no amplification product. Further, since the identification region is not decomposed, the first tag region is also accompanied by a label element. For this reason, in the detection step, a signal based on the labeling element is presented in the first capture probe, and a signal can be presented in the same manner in the second capture probe. In this embodiment, the detection step is the same for both immersion hybridization and chromatography.

(Evaluation by the identification probe of the seventh aspect)
FIG. 8 shows a detection method using the identification probe of this embodiment. In the upper column of the column of capture form, the capture form in immersion hybridization is shown, and the lower part shows the capture form in chromatography. This embodiment is a modification of the fifth aspect, and includes a third tag region and a label probe in place of the label element of the identification probe.

  In the description of this embodiment, a primer set for amplifying a target nucleic acid is different from the above-described embodiments, and a primer set for obtaining an amplification product of a complete double-stranded nucleic acid is used.

  As shown in the upper part of FIG. 8, when this identification probe is used, if a genuine amplification product is present, the identification region is decomposed along with the amplification reaction. As a result, the first tag region and the second tag The region and the third tag region are separated from each other, and only the second tag region and the third tag region are accompanied by a label element via the label probe. Accordingly, in the detection step, the signal based on the label element is not presented in the first capture probe, and the signal based on the label element is presented only in the second capture probe. In addition, the genuine amplification product is separately captured by the detection probe, and a signal based on the labeling element is presented. The presence of the authentic nucleic acid amplification reaction, that is, the presence of the target nucleic acid, is also detected only by “none / yes” of the labeling substance signal (first capture probe / second capture probe) based on the degradation product of the identification probe. In addition, the signal at the detection probe is positive. Furthermore, both of them can clearly affirm the presence of the genuine amplification product. In this embodiment, the detection step is the same for both immersion hybridization and chromatography.

  This identification probe can selectively detect the degradation product by the second tag region and the third tag region. For this reason, the presence of the genuine amplification product can be detected with high accuracy.

  On the other hand, as shown in the middle part of FIG. 8, when there is no authentic amplification product and a primer dimer is generated, the identification region of the identification probe is not decomposed. Is accompanied. For this reason, in the detection step, a signal based on the labeling element is presented in the first capture probe, and a signal can be presented in the same manner in the second capture probe. In addition, since a dimer based on a primer set for obtaining a complete double-stranded DNA cannot hybridize to a probe that should be originally hybridized, the primer dimer cannot be captured by a detection probe. The absence of the true amplification product, that is, the absence of the target nucleic acid is detected not only by the presence / absence of the signal of the labeling substance based on the undegraded product of the identification probe, but also by the signal in the detection probe. Is done. Furthermore, both can clearly affirm the false amplification product. In this embodiment, in the case of immersion type hybridization, the capture mode is as described above. However, according to the prior hybridization (chromatography) described later, an undegraded identification probe is previously obtained by the first capture probe. Therefore, the capture of the primer dimer with the second capture probe is suppressed or avoided, and more explicit distinction is possible.

  Furthermore, as shown in the lower part of FIG. 8, when there is no authentic amplification product, a labeling element is displayed in the first capture probe and the second capture probe, but detection is not performed by the detection probe. No signal is displayed in the probe for use. The absence of the genuine amplification product, that is, the absence of the target nucleic acid is detected not only by the presence / absence of the signal of the labeling substance based on the undegraded product of the identification probe, but also by the signal in the detection probe. Affirmed. Furthermore, both of these can clearly affirm the presence of the false amplification product. In this embodiment, in the case of immersion type hybridization, the capture mode is as described above. However, according to the preceding hybridization (chromatography) described later, an undegraded identification probe in advance by the first capture probe. Therefore, the capture of the primer dimer with the second capture probe is suppressed or avoided, and more explicit distinction is possible.

  In this embodiment, as in the fifth embodiment, the presence or absence of the target nucleic acid can be determined without accompanying signal detection using a detection probe. That is, the presence (positive) of the target nucleic acid can be distinguished from the absence (false positive and negative) of the target nucleic acid only by signal detection with the degradation product and undegraded product of the identification probe as a result of the amplification reaction. .

  For evaluation with higher accuracy, it is preferable to carry out the amplification step of the present detection method under the same conditions as the control except that the nucleic acid sample is free and / or primer free. More preferably, it is preferable to carry out the preceding hybridization described above.

(Solid phase carrier for detecting target nucleic acid)
A solid phase carrier for detecting a target nucleic acid disclosed in the present specification can include a capture probe that captures at least a part of an identification probe.

  According to this solid phase carrier, even if a false amplification product is generated in detecting the target nucleic acid, it is possible to efficiently perform discrimination with high accuracy by eliminating adverse effects such as false positives caused by it. it can. The solid phase carrier may further include a target nucleic acid detection probe for detecting a genuine amplification product.

  The various aspects already described can be applied to the identification probe. Alternatively, as the capture for capturing the identification probe, the various types of capture described above that interact with a tag that the identification probe may be provided may be applied. The various aspects already described can be applied to the solid phase carrier. Preferably, the solid phase carrier is a chromatographic solid phase carrier. In this case, a capture probe for capturing the identification probe can be provided on the upstream side, and a target nucleic acid detection probe can be provided on the downstream side.

(Kit for detecting target nucleic acid)
The kit disclosed in the present specification can include a primer set for amplifying a target nucleic acid, and an identification probe including an identification region that specifically hybridizes to a nucleic acid extension reaction region by the primer set. According to this kit, even if a false amplification reaction product is generated when a target nucleic acid is amplified and detected via a nucleic acid amplification reaction, it is possible to discriminate with high accuracy by eliminating adverse effects such as false positives caused by it. Can be performed efficiently.

  Further, the kit can include a capture probe that captures at least a portion of the identification probe. This kit may include a target nucleic acid detection probe. These probes may be provided on the solid phase carrier described above. Various aspects already described can be applied to identification, solid phase carrier, capture, and the like provided in this kit.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, a following example does not limit this invention. In the following examples,% means mass%.
(Comparative example) Membrane type DNA microarray (example of false positives)
In the comparative example, the target nucleic acid was detected by the following method.

(1) Preparation of chromatographic strip A capture DNA probe solution having a base sequence shown in the following table on a Hi-Flow Plus membrane sheet (60 mm x 600 mm) manufactured by Merck Millipore is described in JP-A-2003-75305. Spotting was performed using GENISHOT (registered trademark) spotter using NGK Corporation using a discharge unit (inkjet method). The synthetic oligo DNA sequence used was polyT (20) at the 3 ′ end side of any 8 sequences out of 100 sequences D1_1 to D1_100 described in Supplementary Table 1 of the literature (Analytical Biochemistry 364 (2007) 78-85). Using the sequence to which is added as a probe, strips for development hybridization (for chromatography) were prepared in the arrangement shown in FIG. In addition, polyT (20) -biotin is spot-fixed in the flow control of FIG.

As a spot treatment, immobilization was performed by irradiating UV light of about 300 mJ / cm ² at a wavelength including a component of 280 nm using a UV irradiation apparatus (XL-1500 UV Crosslinker) manufactured by Spectroline.

(2) Amplification of sample gene As a sample used for amplification, human-derived mRNA was reverse-transcribed into cDNA using Life Technologies SuperScript® VILO cDNA Synthesis Kit and Master Mix, and the sequences shown in Table 2 were used. Amplification was performed using primers (SEQ ID NO: 101, 102) consisting of The amplification product by this primer set is prepared so as to hybridize with the probe D1-006.

  The ligation site X was synthesized according to a general oligonucleotide synthesis method using GlenResearch Spacer Phosphoramidite C3 (SpacerC3: X).

  Next, cDNA was amplified using these primers under the following conditions. In addition, AmpliTaq Gold Fast PCR Master Mix of Life Technologies was used as a sample amplification reagent. As a thermal cycler, Applied Biosystems GeneAmp PCR System 9700 was used.

(Reagent preparation)
DNA-free water 5.5μl
AmpliTaq Gold Fast PCR Master Mix (x2) 10.0μl
Primer mixture (10μM, each) 2.0μl
Sample: cDNA (10ng / μl) or DNA-free water 2.5μl
Total 20.0μl

  Next, the amplification reagent is transferred to a thermal cycle plate, and the thermal cycle reaction (after 95 minutes at 95 ° C .; 40 cycles of 95 ° C. for 15 seconds, 60 ° C. for 1 minute, then 72 ° C. for 10 seconds, then 4 ° C.) ). The amplified sample was purified with QIAGEN's MinElute PCR Purification Kit, and then it was confirmed that the amplified sample was amplified with the intended length by agarose electrophoresis (only those subjected to PCR with cDNA).

(3) Detection using development type chromatography The reaction and procedure for development type hybridization using the sample amplified in (2) were as follows.

Sample composition developing solution 20.0 μl
Latex solution 2.0 μl
Sample 20.0 μl
Total 42.0 μl

  In addition, the latex solution used was prepared by using a chromatographic developing solution to prepare an arbitrary concentration of polystyrene latex beads containing a blue colorant coated with avidin (streptavidin). . Further, Phosphate buffered saline was used as the chromatographic developing solution.

(Chromatography and color reaction)
42 μl of each sample was added to a 0.2 ml tube, and the reaction was started by inserting the prepared slip. The sample solution was all sucked up in about 20 minutes, and the reaction was completed. After completion of the reaction, the developed chromatography was air-dried, and an image was taken.

(Detection judgment)
The presence or absence of color development by a chromatographic reaction was confirmed. The results are shown in FIG. The right side of FIG. 10 shows the chromatographic result of the product obtained by performing PCR on human-derived cDNA, and the left side was similarly subjected to PCR using DNA-free water instead of the human DNA solution. The chromatographic result about a result is shown.

  As shown on the right side of FIG. 9, according to the PCR product using human-derived cDNA, hybridization could be confirmed at a position corresponding to the probe D1-006. On the other hand, hybridization was confirmed at the position corresponding to the probe D1-006 in the PCR result using DNA-free water. This means that a dimer is generated as a by-product of the PCR primer, and since it has a tag chain and biotin to be hybridized to the probe D1-006 included in the primer dimer, the probe D1-006 has an appropriate amplification product as well as an appropriate amplification product. It was thought that hybridization had occurred. That is, it was considered that a false positive occurred.

(Detection method 1 using a DNA polymerase having exonuclease activity in the 5′-3 ′ direction)
In this example, a nucleic acid amplification reaction was performed using a DNA polymerase having exonuclease activity in the 5′-3 ′ direction, and the target nucleic acid was detected by the following method.
(1) Preparation of chromatographic strip A capture DNA probe solution comprising a base sequence shown in the following table on a Hi-Flow Plus membrane sheet (60 mm x 600 mm) manufactured by Merck Millipore is described in JP-A-2003-75305. Spotting was performed using GENISHOT (registered trademark) spotter using NGK Corporation using a discharge unit (inkjet method). The synthetic oligo DNA sequence used was polyT (20) on the 3 ′ end side of the following 5 sequences out of 100 types from D1_1 to D1_100 described in Supplementary Table 1 of the literature (Analytical Biochemistry 364 (2007) 78-85). Using the sequence to which is added as a probe, a strip for chromatography was prepared in the arrangement shown in FIG.

As a spot treatment, immobilization was performed by irradiating UV light of about 300 mJ / cm ² at a wavelength including a component of 280 nm using a UV irradiation apparatus (XL-1500 UV Crosslinker) manufactured by Spectroline.

(2) Amplification of target gene The DNA samples used for amplification were the same as those used in the comparative example, primers (SEQ ID NOs: 103 and 104) having the sequences shown in Table 4 and probes shown in Table 5 (SEQ ID NO: 105) was used to amplify the target gene. The identification probe shown in Table 5 has biotin at the 5 ′ end, a specific hybridizing region for human albumin, a region complementary to probe D1-001 and a region complementary to probe D1-006. Yes.

  Next, cDNA was amplified using these primers as follows. In addition, AmpliTaq Gold Fast PCR Master Mix of Life Technologies was used as a sample amplification reagent. As a thermal cycler, Applied Biosystems GeneAmp PCR System 9700 was used.

(Reagent preparation)
DNA-free water 3.5μl
AmpliTaq Gold Fast PCR Master Mix (x2) 10.0μl
Primer mixture (10μM, each) 2.0μl
Probe aqueous solution (0.1μM) 2.0μl
Sample: cDNA (10ng / μl) or DNA-free water 2.5μl
Total 20.0μl

Next, the amplification reagent is transferred to the thermal cycle plate, and the thermal cycle reaction (after 95 minutes at 95 ° C .; 30 seconds at 95 ° C., 1 second at 80 ° C., 40 minutes for 6 minutes at 64 ° C., then lowered to 10 ° C.) ) Thereafter, unreacted biotin-modified probe was removed using Streptavidin Mag Sepharose ^™ of GE Healthcare.

(3) Detection Using Chromatography The reaction to the chromatography strip using the sample amplified in (2) and the procedure were as follows.

(Sample composition)
Developing solution 20.0 μl
Latex solution 2.0 μl
Sample 20.0 μl
total 42.0 μl

  In addition, the latex-modified DNA aqueous solution has the amino acid-modified 5 ′ end of the sequence shown in Table 6 (SEQ ID NO: 106) and the surface of polystyrene latex beads containing a blue colorant carboxyl-modified. Were covalently bonded in advance and prepared with a chromatographic developing solution so as to have an arbitrary concentration. This DNA has a sequence complementary to the 3 'terminal sequence of the probe. Further, Phosphate buffered saline was used as the chromatographic developing solution.

(Chromatography and color reaction)
42 μl of each of the above prepared samples was added to a 0.2 ml tube and developed chromatography was inserted to start the reaction. The sample solution was all sucked up in about 20 minutes, and the reaction was completed. After completion of the reaction, the developed chromatography was air-dried, and the reaction site was visually confirmed and an image was taken.

(Detection judgment)
The presence or absence of color development by a chromatographic reaction was confirmed. The results are shown in FIG. The left side of FIG. 12 shows the chromatographic result of the product obtained by performing PCR on human-derived cDNA, and the right side was similarly subjected to PCR using DNA-free water instead of the human DNA solution. The chromatographic result about a result is shown.

  As shown on the left side of FIG. 12, coloration by latex beads could be confirmed at a position corresponding to the probe D1-001. This is because, as a result of obtaining an appropriate amplification product by the primer, the sequence specific to human albumin of the probe is degraded, and a region complementary to the probe D1-006 on the 3 ′ end side of the probe as the degradation product of the probe is present. The region hybridized to the modified DNA and complementary to the probe D1-001 was considered to be the result of hybridization to the probe D1-001 on the chromatography.

  On the other hand, depending on the PCR product using DNA-free water shown on the right side of FIG. 12, color development could be detected only on the flow control on the chromatography. This was thought to be due to the reaction of the latex that had not been captured by the capture probe via the amplification product with the flow control located downstream.

  In addition, according to the present example, since it is configured to detect a degradation product of the identification probe by-produced only when a genuine amplification product is generated, the genuine amplification product is detected regardless of the presence or absence of the primer dimer. It can be detected accurately. That is, it is not necessary to prepare a probe for detecting the genuine amplification product itself or the primer dimer itself.

(Detection method 2 using a DNA polymerase having exonuclease activity in the 5′-3 ′ direction)
In this example, a nucleic acid amplification reaction was performed using a DNA polymerase having exonuclease activity in the 5′-3 ′ direction, and the target nucleic acid was detected by the following method.
(1) Production of chromatographic strip A chromatographic strip was produced in the same manner as in Example 1 except that a probe having the following base sequence was used and the arrangement was as shown in FIG.

(2) Amplification of target gene The DNA sample used for amplification was the same as that used in the comparative example, and the primers consisting of the sequences shown in Table 4 and the identification probe (SEQ ID NO: 107) shown in Table 8 were used. Was used to amplify the target gene. The identification probe shown in Table 8 has a region complementary to probe D1-009 from the 5 ′ end, a specific hybridizing region for human albumin, and a region complementary to probe D1-001 and probe D1-006. It has a complementary region.

  Next, cDNA was amplified in the same manner as in Example 1 using these primers.

(3) Detection using chromatography The reaction to chromatography using the sample amplified in (2) and the procedure thereof were the same as in Example 1.

(Hybridization and color reaction)
After carrying out in the same manner as in Example 1, after the reaction was completed, the developed chromatography was air-dried, and the reaction site was visually confirmed and an image was taken.

(Detection judgment)
The presence or absence of color development by a chromatographic reaction was confirmed. The results are shown in FIG. The left side of FIG. 14 shows the chromatographic result of the product obtained by performing PCR on human-derived cDNA, and the right side thereof is similarly subjected to PCR using DNA-free water instead of the human-derived cDNA solution. The chromatographic result about the obtained result is shown.

  As shown on the left side of FIG. 14, coloration by latex beads could be confirmed at a position corresponding to the probe D1-001 on the strip. This is because, as a result of obtaining an appropriate amplification product by the primer, the sequence specific to human albumin of the identification probe is degraded, and complementary to the probe D1-006 on the 3 ′ end side of the probe as the degradation product of the identification probe. The region hybridized to the modified DNA, and the region complementary to probe D1-001 was thought to be the result of hybridization to probe D1-001 on chromatography. In addition, it was thought that the DNA of the region complementary to the probe D1-009, which was another degradation product, was captured by the trap.

  On the other hand, depending on the PCR product using DNA-free water shown on the right side of FIG. 14, color development could be detected only on the flow control on the chromatography. Moreover, this is considered that the false positive in the chromatography could be suppressed even if the primer dimer was generated by PCR.

  In addition, when trapping of unreacted probe by the D1-009 region of the chromatography strip is insufficient, there is a concern that the unreacted probe is developed in the downstream region from D1-009). However, from this result, since unreacted probes are not seen to develop downstream from D1-009, unreacted probes are efficiently trapped in the D1-009 region (by visual inspection). It was proved that light color was confirmed. From this result, it was found that the method is useful compared to Example 1 in that the probe cost and the labor of removing the unreacted probe after PCR can be omitted.

  From the above results, according to the method disclosed in the present specification, even when primer dimer is present in the PCR product, it is possible to perform the reaction by chromatography only when the target fragment is amplified. It was confirmed that this method can suppress the occurrence of false positives, which has been a problem until now, and contribute to the improvement of detection accuracy.

(Detection method 3 using a DNA polymerase having exonuclease activity in the 5′-3 ′ direction)
In this example, a nucleic acid amplification reaction was performed using a DNA polymerase having exonuclease activity in the 5′-3 ′ direction, and the target nucleic acid was detected by the following method.

(1) Preparation of chromatographic strip As shown in the left column of FIG. 15, a chromatographic strip including probes having base sequences of D1-001, 002, 003, and 005 was prepared in the same manner as in Example 1. .

(2) Amplification of target gene The gene xthA (exonuclease III) and NCBI-GeneID: 946254 of Escherichia coli K-12 MG1655 strain were used as target genes. The extracted genome of the above strain was used as a sample. Primer sequences (SEQ ID NOs: 108 and 109) for amplifying the target gene (amplified chain length 150 bp) are shown below.

  In addition, the base sequence (SEQ ID NO: 110) of the identification probe used simultaneously during amplification is shown below.

  A tag (DNA) having a base sequence complementary to the probe D1-001 of the chromatography strip prepared in (1) is added to the 5 'end side of the identification probe. In addition, a spacer (blocker: X = SpacerC3) was added to the 3 'end to reliably prevent the progress of the polymerase reaction.

  The gene amplification enzyme used was Takara Bio's Premix ExTaq, and the thermal cycler used Thermogen's Quick Bath.

  An amplification reaction solution was prepared as follows.

(Amplification reaction solution)
Sterile water 1.0 μL
Premix ExTaq (x2) 5.0 μL
Primer mixture (2.5 μM each) 1.0 μL
Probe solution (0.2 μM) 2.0 μL
Genomic DNA (10 ng / μL) or DNA-free water 1.0 μL
Total 10.0 μL

As a primer, a mixture of primer F and primer R is used. As an identification probe, tag DNA is added to an oligo DNA having the sequence shown in Table 10 whose Tm is about 20 ° C. lower than the PCR reaction temperature. Probe was used.

  After amplification reaction of the gene under the following conditions using a thermal cycler, development to chromatography was performed. That is, after heating at 94 ° C. for 2 minutes, 94 ° C. for 20 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds 30 times, 72 ° C. for 2 minutes, 97 ° C. for 10 seconds and 30 ° C. for 10 seconds, Stored at 4 ° C.

(3) Target gene detection by chromatography
The amplification reaction solution and various reagents prepared in (2) in a 1.5 mL tube were prepared as follows to obtain a developed sample solution.
(Development sample solution)
Amplification reaction solution 10.0 μL
Developing solution 10.0 μL
Latex solution 1.0 μL
Total 21.0 μL

  The chromatography strip prepared in (1) was inserted into a 1.5 mL tube containing the developed sample solution to start the reaction. The developed sample solution was completely sucked up to the top of the strip in about 15 minutes, and the reaction was completed. After completion of the reaction, coloration of the strip on the target gene detection line was visually confirmed. The results are shown in the middle column of FIG.

  As shown in the middle column of FIG. 15, when genomic DNA was used in target gene amplification, color development could be confirmed on the probe D1-001 on the strip. On the other hand, when DNA-free water was used, color development at the probe site on the strip was not confirmed. This means that the method can be detected only when the target gene is amplified (it does not become a false positive even if a primer dimer is formed).

  As an example corresponding to the conventional method, the effect of the primer dimer when a double-stranded DNA having a single strand only on one side was obtained as an amplification product was confirmed.

  That is, using the chromatography strip used in this example, the target gene of this example was subjected to normal PCR amplification reaction using the following primer set (SEQ ID NOs: 111 and 112) without using an identification probe. went.

  In the above PrimerF-T, oligo DNA complementary to the probe D1-002 of the chromatographic strip is added to the 5 'end of Primer F via a spacer (X = SpacerC3).

The composition of the amplification reaction solution is as follows, and the temperature condition is 94 ° C. for 2 minutes, then 94 ° C. for 20 seconds, 60 ° C. for 30 seconds and 72 ° C. for 30 seconds 30 times. The amplification reaction was carried out in the same manner as described above except that the mixture was stored in 4 minutes at 4 ° C, followed by chromatography. The results are also shown in the right column of FIG.
(Amplification reaction solution)
Sterile water 3.0 μL
Premix ExTaq (x2) 5.0 μL
Primer mixture (2.5 μM each) 1.0 μL
Genomic DNA (10 ng / μL) or DNA-free water 1.0 μL
Total 10.0 μL

  As shown in the right column of FIG. 15, in the target gene amplification, color development could be confirmed in the probe D1-002 on the strip both in the case of using genomic DNA and in the case of using DNA-free water. It is assumed that when DNA-free water was used, genomic DNA was not present, so the target gene was not amplified, but was detected by dimer formation between primers (false positive). This means that the amplification product can be detected, but it is detected on the strip even when a dimer (false positive) is formed.

(Detection method using DNA polymerase having no 5'-3 'exonuclease activity)
In this example, an amplification product of a nucleic acid amplification reaction using a DNA polymerase that does not have exonuclease activity in the 5′-3 ′ direction but has exonuclease activity in the 3′-5 ′ direction is used. The target nucleic acid was detected by the method.

(1) Production of chromatographic strip The strip produced in the comparative example was used.
(2) Amplification of target gene The gene xthA (exonuclease III) and NCBI-GeneID: 946254 of Escherichia coli K-12 MG1655 strain were used as target genes. In addition, the sample used the extracted genome of the said strain. Primer sequences for amplifying the target gene (amplified chain length 150 bp) are shown in the following table. The base sequence of the identification probe that specifically hybridizes to the amplification product is shown in another table. The Tm of the identification probe is set to be about 20 ° C. lower than the PCR reaction temperature, and the base sequence that specifically hybridizes to the amplification product and the chromatography prepared in (1) on the 5 ′ end side thereof. A complementary oligo DNA was added to the probe D1-001 on the strip.

  The gene amplification enzyme used was Pho DNA polymerase, which does not have exonuclease activity in the 5'-3 'direction of Nippon Gene Co., but has exonuclease activity in the 3'-5' direction. As the thermal cycler, Thermogen Quick Bath was used.

  An amplification reaction solution was prepared as follows. That is, the primer mixed solution used was a mixture of primer F and primer R, and the probe solution was an identification probe solution. Using a thermal cycler, a cycle of 94 ° C. for 2 minutes, 94 ° C. for 20 seconds, 60 ° C. for 30 seconds and 72 ° C. for 30 seconds was repeated 30 times, then 72 ° C. for 2 minutes and 97 ° C. for 10 minutes. After 10 seconds at 30 seconds and 30 ° C., the amplification reaction was carried out, and then stored at 4 ° C., and returned to room temperature in a timely manner for development into chromatography.

(3) Detection using chromatography
The amplification reaction solution and various reagents prepared in (2) in a 1.5 mL tube were prepared as follows to obtain a developed sample solution. The same developing solution and latex solution as in Example 1 were used.

(Development sample solution composition)
Amplification reaction solution 10.0 μl
Developing solution 10.0μl
Latex solution 1.0μl

  The chromatography strip prepared in (1) was inserted into a 1.5 mL tube containing the developed sample solution to start the reaction. The developed sample solution was completely sucked up to the top of the strip in about 15 minutes, and the reaction was completed. When the coloration of the target gene detection line of the strip was visually confirmed after completion of the reaction, the same result as in Example 3 (column in FIG. 15) was obtained.

  That is, when DNA polymerase that does not have exonuclease activity in the 5′-3 ′ direction is used, when genomic DNA is used in target gene amplification, color development may be confirmed on the probe D1-001 on the strip. However, when DNA-free water was used, color development at the probe site on the strip was not confirmed. This means that the method can be detected only when the target gene is amplified (it does not become a false positive even if a primer dimer is formed).

Sequence number 1-100, 105, 106, 107, 110: Probe sequence number 101-104, 108, 109, 111-112: Primer

Claims (15)

A method for detecting a target nucleic acid, comprising:
In the presence of a nucleic acid sample, a primer set for amplifying the target nucleic acid, and a target nucleic acid identification probe comprising an identification region that specifically hybridizes to the nucleic acid extension reaction region of the authentic amplification product amplified by the primer set An amplification step of performing a nucleic acid amplification reaction using a DNA polymerase having an exonuclease activity in the 5′-3 ′ direction;
A capture step of capturing on a solid support at least a portion of said target nucleic acid identification probes which may be present in the amplification step,
Evaluating the presence or absence of a false amplification product using at least a portion of the target nucleic acid identification probe;
A method comprising:   The target nucleic acid identification probe includes a first tag adjacent to one end of the identification region, and a first capture capable of capturing the first tag on the solid phase carrier. The method according to 1.   The method of claim 2, wherein the first tag has one of a pair of proteins that specifically bind to each other.   The method according to claim 2, wherein the first tag has one of a pair of polynucleotides each having a base sequence that specifically hybridizes to each other.   The first capture is a solid phase carrier having a porous portion, and the first tag is a particle having a size and / or shape captured by the porous portion of the solid phase carrier. The method in any one of 2-4. The target nucleic acid identification probe further comprises a second tag adjacent to the other end of the identification region,
The method according to claim 4 or 5, wherein the target nucleic acid identification probe or a degradation product thereof is captured or detected using a second capture that captures the second tag.   The method according to claim 1, wherein the capturing step is a chromatography step. Or in the rear capturing step before Symbol acquisition step,
A target nucleic acid comprising: contacting a target nucleic acid amplification product with a target nucleic acid detection probe under conditions that allow hybridization with a target nucleic acid detection probe that specifically hybridizes with the authentic amplification product; A detection step,
The target nucleic acid identification probe includes a first tag adjacent to one end of the identification region, and includes a second tag and a third tag adjacent to the other end.
Using the first capture that captures the first tag and the second capture that captures the second tag to capture the target nucleic acid identification probe or a degradation product thereof;
The third tag has a polynucleotide having a base sequence that specifically hybridizes with the amplification product of the target nucleic acid,
The method according to any one of claims 1 to 7 , wherein the third tag captured by the second capture via the second tag is used as the target nucleic acid detection probe. The detection step, the target nucleic acid using a solid phase support comprising a target nucleic acid detection probe for detecting the, capturing the target nucleic acid identification probe or a degradation product thereof in the solid phase carrier, according to claim 8 the method of. The method according to claim 8 or 9 , wherein the detection step is a chromatography step. The method according to claim 10 , wherein the target nucleic acid identification probe or a decomposition product thereof is captured upstream of the target nucleic acid detection probe in a development direction in chromatography. The target nucleic acid identification probe comprises a labeling element, the method according to any one of claims 1 to 11. 13. A method according to claim 12 , wherein the marking element is provided adjacent to or at the other end of the identification area. The method according to any one of claims 1 to 13 , wherein the primer set is capable of amplifying a partial double-stranded nucleic acid having a single-stranded region only in one strand as an amplification product of the target nucleic acid. A kit for use in the method for detecting a target nucleic acid according to any one of claims 1 to 14 ,
A primer set for amplifying the target nucleic acid;
An identification region that specifically hybridizes to a nucleic acid extension reaction region of a genuine amplification product amplified by a primer set for amplifying the target nucleic acid, and a first tag adjacent to one end of the identification region A target nucleic acid identification probe;
A probe for capturing the first tag on a solid support;
A kit comprising: