WO2017006859A1

WO2017006859A1 - Method for detecting target nucleic acid

Info

Publication number: WO2017006859A1
Application number: PCT/JP2016/069638
Authority: WO
Inventors: 創太郎佐野; 重彦宮本; 高橋　孝治
Original assignee: 株式会社カネカ
Priority date: 2015-07-03
Filing date: 2016-07-01
Publication date: 2017-01-12
Also published as: JP2018133998A

Abstract

The purpose of the present invention is to provide a detection method having a high determination accuracy, said detection method being for detecting a target nucleic acid and comprising conducting a nucleic acid amplification reaction and detecting a nucleic acid amplification product on a solid support, whereby nonspecific detection of a primer dimer can be avoided. A method for detecting a target nucleic acid that comprises conducting a nucleic acid amplification reaction by a Nested-PCR method and detecting a nucleic acid amplification product on a solid support.

Description

Target nucleic acid detection method

The present invention relates to a target nucleic acid detection method including amplification of a target nucleic acid using a two-step nucleic acid amplification reaction and detection of a nucleic acid amplification product on a solid phase carrier.

Today, a method for specifically detecting a target nucleic acid is a very important technique in the field of molecular biology research and clinical application such as genetic testing and pathogen testing. Among them, a method for detecting a target nucleic acid using a nucleic acid amplification reaction and nucleic acid chromatography has attracted attention because of its rapidity and simplicity. This detection method is generally performed by attaching a tag to a primer specific to the target nucleic acid and performing a nucleic acid amplification reaction such as polymerase chain reaction (hereinafter referred to as “PCR”) using the DNA containing the target nucleic acid as a template. The obtained amplification product is diffused and moved in the porous solid phase carrier by a capillary phenomenon, and is captured and bound to the label and the solid phase carrier via the tags attached to both ends of the amplification product. Is detected visually (Patent Document 1).

However, since this detection method relies on a tag derived from a primer attached to both ends of the amplification product to perform capture and binding to a label and a solid phase carrier, it is specific to the target nucleic acid. In some cases, the amplification product and the primer dimer formed by the non-specific reaction cannot be distinguished, and there is a possibility of performing false positive determination based on the detection of the primer dimer.

For this reason, in this detection method, it has been desired to improve such that non-specific detection of the primer dimer is avoided and high determination accuracy is obtained.

WO2012 / 070618

The present invention avoids non-specific detection of a primer dimer in a method for detecting a target nucleic acid including a nucleic acid amplification reaction and detection of a nucleic acid amplification product on a solid phase carrier (for example, a nucleic acid chromatography method). An object of the present invention is to provide a new detection method with high determination accuracy.

In order to prevent the formation of primer dimers in the nucleic acid amplification reaction, it is necessary to reduce the number of cycles of the nucleic acid amplification reaction. However, if the number of cycles is reduced, the copy number of the target nucleic acid can be determined by nucleic acid chromatography, etc. In some cases, the detection sensitivity cannot be increased to the extent that it can be visually detected, and the detection sensitivity can be lowered. Accordingly, there is a demand for a technique that can prevent the formation of primer dimers while increasing the copy number of the target nucleic acid to such an extent that it can be visually detected by a nucleic acid chromatography method or the like.

The present inventors carry out a two-step nucleic acid amplification reaction (Nested-PCR method) for amplification of a target nucleic acid, that is, a first-step nucleic acid amplification reaction and a tag that are performed using an untagged primer set. By performing the second-stage nucleic acid amplification reaction performed using the primer set to which the tag is attached, the number of copies of the target nucleic acid can be increased by the first-stage nucleic acid amplification reaction, and a primer with a tag The target nucleic acid can be detected specifically and with high sensitivity by a nucleic acid chromatography method, etc. by carrying out the nucleic acid amplification reaction in the second stage using the set with the number of cycles in which dimer formation does not occur. It came to complete.

That is, the present invention includes the following inventions.
[1] A method for detecting a target nucleic acid, comprising:
(A) consisting of a primer containing a sequence homologous to the first base sequence of the target nucleic acid and a primer containing a sequence homologous to the complementary strand sequence of the second base sequence located downstream of the first base sequence Using the first primer set to obtain a first nucleic acid amplification product by performing a nucleic acid amplification reaction using the test DNA as a template,
(B) a primer including a sequence homologous to a third base sequence located downstream of the first base sequence and upstream of the second base sequence; a downstream of the third base sequence; and A second primer set comprising primers containing a sequence homologous to the complementary strand sequence of the fourth base sequence located upstream of the second base sequence, wherein any one of the primers can bind to the labeling substance Using the second primer set, which is bound to a first tag and the other primer is bound to a second tag capable of binding to a solid phase carrier, the first nucleic acid amplification product is Performing a nucleic acid amplification reaction as a template to obtain a second nucleic acid amplification product to which each of the first tag and the second tag is added at each end;
(C) obtaining a labeled nucleic acid amplification product by bringing the second nucleic acid amplification product into contact with a labeling substance that can bind to the first tag;
(D) capturing the labeled nucleic acid amplification product on the solid phase carrier by contacting the labeled nucleic acid amplification product with a solid phase carrier; and (e) capturing the labeled nucleic acid amplification product on the solid phase carrier. Detecting the labeled nucleic acid amplification product;
Including the above method.
[2] The method according to [1], wherein the step (a) and the step (b) are continuously performed in the same reaction vessel.
[3] The method according to [1] or [2], wherein the annealing temperature of the nucleic acid amplification reaction in the step (a) is higher than the annealing temperature of the nucleic acid amplification reaction in the step (b).
[4] The number of amplification cycles of the nucleic acid amplification reaction in the step (b) is less than the number of amplification cycles of the nucleic acid amplification reaction in the step (a). Method.
[5] (a) a primer containing a sequence homologous to the first base sequence of the target nucleic acid, and a sequence homologous to the complementary strand sequence of the second base sequence located downstream of the first base sequence A first primer set comprising primers,
(B) a primer including a sequence homologous to a third base sequence located downstream of the first base sequence and upstream of the second base sequence; a downstream of the third base sequence; and A second primer set comprising primers containing a sequence homologous to the complementary strand sequence of the fourth base sequence located upstream of the second base sequence, wherein any one of the primers can bind to the labeling substance The second primer set, which is bound to a first tag and the other primer is bound to a second tag capable of binding to a solid phase carrier;
(C) a labeling substance capable of binding to the first tag, and
(D) a solid phase carrier capable of binding to the second tag,
A kit for detecting a target nucleic acid, comprising:
[6] The kit according to [5], wherein in the second primer set, at least one of the first tag and the second tag is an oligonucleotide tag.
[7] The kit according to [6], wherein the oligonucleotide tag is bound to a primer via a spacer capable of suppressing or stopping the DNA polymerase reaction.
[8] The kit according to [7], wherein the spacer is azobenzene, a fatty chain, or an inverted base.
[9] The kit according to any one of [6] to [8], wherein the first tag is a tag composed of an oligonucleotide, and the labeling substance includes an oligonucleotide having a sequence complementary to the oligonucleotide.
[10] The first tag is a tag made of any low molecular weight compound selected from biotin, digoxigenin, and FITC, and the labeling substance contains a protein that can bind to the tag. [6] to [9 ] One of the kits.
[11] The kit according to any one of [6] to [10], wherein the second tag is a tag comprising an oligonucleotide, and the solid phase carrier comprises an oligonucleotide having a sequence complementary to the oligonucleotide.
[12] The Tm value of one or both primers included in the first primer set is higher than the Tm value of one or both primers included in the second primer set. [5 ] To [11].

This specification includes the disclosure of Japanese Patent Application No. 2015-134659, which is the basis of the priority of the present application.

According to the present invention, non-specific detection of primer dimers is avoided in a method for detecting a target nucleic acid including a nucleic acid amplification reaction and detection of a nucleic acid amplification product on a solid phase carrier (eg, nucleic acid chromatography). Therefore, it is possible to provide a detection method with high determination accuracy.

The schematic diagram of a lateral flow type nucleic acid detection device is shown. 1: solid phase carrier, 2: conjugate pad, 3: sample pad, 4: absorption pad, 5: substrate, 6: capture means. The region corresponding to the recognition sequence of each primer used in the examples is shown. (A) It is a photograph figure which shows the result of having detected the amplification product obtained by Nested-PCR method using the outer primer set and the inner primer set in Example 1 using the nucleic acid detection device. (B) It is a photograph figure which shows the result of having detected the amplification product obtained by PCR method using only the inner primer in the comparative example 1 using the nucleic acid detection device. Each figure shows the amount of template DNA added to the reaction system.

I. Target Nucleic Acid Detection Method The target nucleic acid detection method according to the present invention generally includes the following steps:
Amplifying the target nucleic acid;
A step of labeling the amplified target nucleic acid, and a step of capturing and detecting the labeled target nucleic acid on a solid phase carrier. Hereinafter, each step will be described.

1. Step of Amplifying Target Nucleic Acid In the method of the present invention, the amplification of the target nucleic acid can be performed using a nested-PCR method. That is, using the first primer set, the first nucleic acid amplification reaction is performed using the test DNA as a template to obtain a first nucleic acid amplification product, and then each second primer set to which a tag is bound is used. A step of performing a second nucleic acid amplification reaction using the first nucleic acid amplification product as a template to obtain a second nucleic acid amplification product. According to this method, the first nucleic acid amplification reaction can be performed using the first primer set, and the copy number of the first nucleic acid amplification product containing the target nucleic acid (if present) can be increased. To reduce the number of cycles of the second nucleic acid amplification reaction using the second primer set to which the tag is bound, to suppress the formation of the primer dimer of the second primer set to which the tag is bound, and to detect A desired second nucleic acid amplification product containing a sufficient number of copies of the target nucleic acid (if present) can be obtained. Furthermore, through a two-step nucleic acid amplification reaction using the first primer set and the second primer set, respectively, the directivity for the target nucleic acid can be enhanced, and non-specific nucleic acid amplification different from the target nucleic acid. Can be reduced or avoided.

In the present invention, the “target nucleic acid” means any nucleic acid sequence or nucleic acid molecule to be detected, which may be natural or artificially synthesized, and is not particularly limited. Examples of the target nucleic acid include a specific trait, a genetic marker indicating the onset or possibility of onset of a specific disease, a gene derived from a pathogen such as a bacterium or virus, a gene derived from an allergen, a single nucleotide polymorphism, Examples include, but are not limited to, base sequences that exhibit congenital or acquired mutations.

In the method of the present invention, each primer (so-called forward primer and reverse primer) included in the “first primer set” and the “second primer set” is a common in the nested-PCR method. It can be designed according to a common technique (published by Hiroki Nakayama, “Bio-Experiment Illustrated, 3 Really PCR”, Shujunsha, 1997).

The “first primer set” includes a primer having a sequence homologous to a specific base sequence (first base sequence) on one strand of a double-stranded DNA that is a target nucleic acid, or consisting of the sequence, A sequence homologous to the complementary strand sequence of the specific base sequence (second base sequence) located downstream (3 ′ side) from the base sequence of 1 (ie, homologous to the base sequence of the other strand of the double-stranded DNA) Or a primer composed of the sequence. Here, the primer having a “homologous sequence” not only has the same sequence as the first base sequence and the same sequence as the complementary strand sequence of the second base sequence, but also binds to the target nucleic acid and amplifies the nucleic acid. As long as the reaction can be promoted, the base sequence having a deletion, substitution, addition or insertion of 1 to several bases in the complementary strand sequence of the first base sequence and the second base sequence, (BASIC (Basic Local Alignment Search at the National Center for Biological Information) (basic local alignment search tool of the National Center for Biological Information), etc.) Default parameter) When calculated had 85% or more, preferably 90% or more, more preferably 95% or more, more preferably also include a primer having a nucleotide sequence with a sequence identity of 99% or more. Here, “1 to several” is not particularly limited, but is, for example, 1 to 10, preferably 1 to 5 (hereinafter also used in the same meaning).

The size of the primer in the first primer set can be 8 bases or more, 12 bases or more, or 15 bases or more in length, and the upper limit is not particularly limited, but 50 bases or less, 40 bases or less, or 30 bases The length can be as follows. The size of each primer may be the same or different.

The first primer set has a higher annealing temperature than the annealing temperature in the second nucleic acid amplification reaction using the second primer set by 5 ° C or higher, preferably 10 ° C or higher, more preferably 15 ° C or higher. The target nucleic acid can be amplified under the conditions of one nucleic acid amplification reaction. That is, the Tm value of any one, more preferably both primers included in the first primer set is more than the Tm value of any one, more preferably both primers included in the second primer set. It can be designed to be 5 ° C. or higher, preferably 10 ° C. or higher, more preferably 15 ° C. or higher. Thereby, even when the second primer set is included in the first nucleic acid amplification reaction system using the first primer set, the first nucleic acid amplification is performed at a high annealing temperature according to the Tm value of the first primer set. By carrying out the reaction, binding of the second primer set to the target nucleic acid and formation of primer dimers can be suppressed or avoided. The “Tm value” of each primer in the second primer set means a value calculated based on a region not including the tag region or spacer region described below.

The “second primer set” is a specific base sequence located on the downstream side (3 ′ side) of the first base sequence and on the upstream side (5 ′ side) of the second base sequence ( (3rd base sequence) or a primer comprising this sequence, or a primer comprising the sequence, located downstream (3 ′ side) from the third base sequence, and upstream of the second base sequence ( Including a sequence homologous to a complementary strand sequence (that is, a sequence homologous to the base sequence of the other strand of double-stranded DNA) of a specific base sequence (fourth base sequence) located on the 5 ′ side), It can be combined with a primer comprising the sequence. That is, the second primer set can be designed in a region sandwiched between the first primer sets (that is, the first nucleic acid amplification product). Here, the primer having a “homologous sequence” not only has the same sequence as the third base sequence and the same sequence as the complementary strand sequence of the fourth base sequence, but also binds to the target nucleic acid and amplifies the nucleic acid. As long as the reaction can be promoted, a base sequence having a deletion, substitution, addition or insertion of one to several bases in the complementary strand sequence of the third base sequence and the fourth base sequence, 85% or more, preferably 90% or more, more preferably 95%, when calculated using the base sequence of the above and the complementary strand sequence of the fourth base sequence and BLAST or the like (for example, default or default parameters) In addition, a primer having a base sequence having a sequence identity of 99% or more is more preferable. Alternatively, in another embodiment, any one of the primers of the second primer set has the same sequence as the entire sequence of any one of the primers of the first primer set or a partial sequence thereof. Also good.

The primer size of the second primer set can be 8 bases or more, 12 bases or more, or 15 bases or more in length, and the upper limit is not particularly limited, but 50 bases or less, 40 bases or less, or 30 bases The length can be as follows. The size of each primer may be the same or different. Here, the “primer size” means a size that does not include the tag region and spacer region described below.

The design of each primer of the first primer set and the second primer set can be performed using a known primer design software / design site or the like. As such primer design software / design site, for example, OligoEvaluator (Sigma-Aldrich), Primer 3 (National Human Genome Research Institute), Primer-BLAST (NCBI), etc. can be used.

The production of each primer of the first primer set and the second primer set can be performed by a known method, and may be performed using a DNA synthesizer or a contracted synthesis service. Can do.

In the present specification, the primer included in the first primer set may be referred to as “outer primer” and the primer included in the second primer set may be referred to as “inner primer”.

A tag is bound to the 5 'end of each primer in the second primer set. The tag is selected from a first tag that can bind to the labeling substance and a second tag that can bind to the solid phase carrier, and each primer in the second primer set (forward primer and reverse primer) One of the tags is bound to each primer. The “first tag” only needs to be capable of binding to a labeling substance described in detail below, and is a nucleic acid (DNA, RNA, oligonucleotide, etc.), protein, peptide, or low molecular weight compound (eg, biotin, digoxigenin) , FITC, etc.) or a combination thereof can be used, and is not particularly limited. Preferably, it contains an oligonucleotide or consists of an oligonucleotide. The “second tag” only needs to be capable of binding to the solid phase carrier described in detail below, and is composed of a nucleic acid (DNA, RNA, oligonucleotide, etc.), protein, peptide, compound, or a combination thereof. A thing can be utilized and is not particularly limited. Preferably, it contains an oligonucleotide or consists of an oligonucleotide.

The primer and the tag can be bound by any means, and can be bound directly or indirectly. However, when the tag bound to the primer is composed of a nucleic acid, a spacer capable of suppressing or stopping the progress of the DNA polymerase reaction is used so that the tag region is not double-stranded together with the primer region by the nucleic acid amplification reaction. Thus, the primer and the tag are combined. As such a “spacer”, any spacer can be used as long as it can suppress or stop the progress of the DNA polymerase reaction when contained in the template strand and prevent the tag region from becoming double-stranded. For extension by DNA polymerase, a nucleic acid (or base) serving as a template is required, and the DNA strand does not extend without the template. That is, the spacer has a structure that cannot serve as a template for a DNA extension reaction by DNA polymerase. Examples of such a spacer include artificial nucleic acid bases such as L-type nucleic acids, peptide nucleic acids (PNA), cross-linked nucleic acids (Bridged Nucleic Acid (BNA) or Locked Nucleic Acid (LNA)), and are represented by the following formula I: Spacer with azobenzene structure, fatty chain such as alkylene chain or polyoxyalkylene chain, inverted base (natural nucleobase linked by 5'-5 'or 3'-3' bond), strong hairpin structure, Examples thereof include, but are not limited to, nucleic acid sequences having a pseudoknot structure.

When a fatty chain is used as the spacer, the number of chain length elements is preferably 2 or more and 40 or less. This is because when the number of elements is 1 or less, the inhibition of the elongation reaction of DNA polymerase is incomplete, and double stranding cannot be suppressed, and when the number of elements exceeds 40, the solubility in water decreases. Considering the inhibition efficiency of the DNA polymerase reaction, the chain length of the fatty chain is preferably 2 or more and 36 or less, more preferably 3 or more and 18 or less. Examples of the fatty chain spacer include spacers represented by the following formula II.
5′-O—C _m H _2m —O-3 ′ (Formula II)
(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. To express.)
In the formula II, m is preferably 2 or more and 36 or less, more preferably 3 or more and 18 or less, and further preferably 3 or 6.

The first nucleic acid amplification reaction and the second nucleic acid amplification reaction can be performed according to a general PCR method. That is, when PCR is performed on the template DNA containing the target nucleic acid using each of the first primer set and the second primer set, the PCR can be performed under PCR conditions that a desired region is amplified. .

In the first nucleic acid amplification reaction, test DNA is used as template DNA. “Test DNA” means a DNA sample that may contain a target nucleic acid, and is derived from a sample such as an animal, plant, or a part thereof (organ, tissue, cell, etc.), microorganism, virus, food or drink, etc. Examples include (but are not limited to) DNA and cDNA prepared from these samples. The test DNA may be in a form extracted and purified from a sample, or may be in a crudely purified form. Alternatively, a part of cells or tissues can be included in the reaction system as it is as test DNA.

In the second nucleic acid amplification reaction, the first nucleic acid amplification product produced by the first nucleic acid amplification reaction is used as the template DNA. The number of PCR cycles in the second nucleic acid amplification reaction is preferably smaller than the number of PCR cycles in the first nucleic acid amplification reaction. By reducing the number of PCR cycles in the second nucleic acid amplification reaction, formation of primer dimers by the second primer set can be suppressed or avoided. Presence or absence of primer dimer formation can be confirmed by electrophoresis of the second nucleic acid amplification product on an appropriate gel such as an agarose gel according to a conventional method. Second nucleic acid amplification that does not cause primer dimer formation The number of PCR cycles in the reaction can be set as appropriate.

The DNA polymerase used for PCR is not particularly limited as long as it is a heat-resistant DNA polymerase. In the present invention, commercially available DNA polymerase can be used, and for example, TaKaRa Ex Taq (registered trademark), KOD DNA polymerase, and the like can be suitably used. The temperature, time, buffer composition, etc. can be appropriately selected depending on the DNA polymerase used, the primer sequence, the size of the target nucleic acid amplification region, and the like.

The first nucleic acid amplification reaction and the second nucleic acid amplification reaction may be sequentially performed in separate reaction containers, or the first nucleic acid amplification reaction and the second nucleic acid amplification reaction may be performed in the same reaction container. You may carry out continuously. When the first nucleic acid amplification reaction and the second nucleic acid amplification reaction are sequentially performed in separate reaction vessels, the obtained first nucleic acid amplification product is taken out after the first nucleic acid amplification reaction (if necessary, This means that after the dilution, a second nucleic acid amplification reaction is performed in addition to a separately prepared second nucleic acid amplification reaction system comprising a DNA polymerase, a second primer set, and the like. On the other hand, performing the first nucleic acid amplification reaction and the second nucleic acid amplification reaction continuously in the same reaction vessel means that the first nucleic acid amplification product is not taken out after the first nucleic acid amplification reaction. It means that two nucleic acid amplification reactions are performed. In the case where the first nucleic acid amplification reaction and the second nucleic acid amplification reaction are continuously performed in the same reaction vessel, the second primer set is added to the reaction system in advance before the start of the first nucleic acid amplification reaction. Alternatively, it may be added to the reaction system after the first nucleic acid amplification reaction and before the start of the second nucleic acid amplification reaction. If necessary, DNA polymerase may be supplemented after the first nucleic acid amplification reaction and before the start of the second nucleic acid amplification reaction. Preferably, the first nucleic acid amplification reaction and the second nucleic acid amplification reaction are continuously performed in the same reaction vessel for ease of operation. Furthermore, preferably, the first nucleic acid amplification reaction and the second nucleic acid amplification reaction are continuously performed in the same reaction vessel, and the second primer set is preliminarily set in the reaction system before the start of the first nucleic acid amplification reaction. To be added. In this case, the Tm value of any one, more preferably both primers included in the first primer set, is more preferably the Tm value of any one, more preferably both primers included in the second primer set. Is preferably high. Thereby, the annealing temperature in the first nucleic acid amplification reaction using the first primer set can be performed under a temperature condition higher than the annealing temperature in the second nucleic acid amplification reaction using the second primer set. The binding of the second primer set to the target nucleic acid contained in the test DNA and the formation of primer dimers can be suppressed or avoided under the first nucleic acid amplification reaction conditions.

2. The step of labeling the amplified target nucleic acid The second nucleic acid amplification product containing the target nucleic acid (if present) obtained by the nucleic acid amplification includes the first tag and the second tag resulting from the second primer set At each end. The labeling of the second nucleic acid amplification product can be performed by contacting and binding the labeling substance with the first tag that can bind to the labeling substance. The labeling substance is not particularly limited as long as it can detect the second nucleic acid amplification product, but preferably allows visual detection of the second nucleic acid amplification product. Examples of such a labeling substance include colored particles, pigments, enzymes (peroxidase, alkaline phosphatase, luciferase, etc.), and preferably colored particles. Examples of the “colored particles” include, but are not limited to, metal colloid (eg, gold, silver, copper, platinum, etc.) particles, colored latex particles, silica nanoparticles including a pigment, and the like. The size of the labeling substance does not hinder the capture of the second nucleic acid amplification product on the solid phase carrier, and may be any material that develops color at the time of detection. It can select suitably so that it may become a size smaller than the hole diameter of a porous member. For example, the size of the labeling substance can be about 500 nm or less, preferably about 0.1 nm to 250 nm, more preferably about 1 nm to 100 nm.

The contact and binding between the labeling substance and the first tag may be direct binding or indirect binding, and the binding means is suitably suitable depending on the labeling substance to be used and the first tag. And nucleic acid-nucleic acid interaction, protein-protein interaction, low molecular weight compound-protein interaction, and the like can be used. For example, when the first tag includes an oligonucleotide, the labeling substance is hybridized by binding the labeling substance to an oligonucleotide containing a sequence complementary to the base sequence of the oligonucleotide, And the first tag can be indirectly coupled. The binding between the labeling substance and the oligonucleotide may be performed via a peptide, protein, nucleic acid, or the like, or may be performed via an appropriate functional group. The hybridization conditions are not particularly limited as long as hybridization occurs. For example, in a buffer solution (pH 6.5 to 7.5) containing 10 mM to 50 mM phosphate at 20 ° C. to 40 ° C. It can be performed by reacting with. In order to increase the hybridization efficiency, the buffer may further contain a salt such as sodium chloride. Alternatively, when the first tag contains biotin, which is a low molecular weight compound, both can be bound by binding the labeling substance to avidin (streptavidin). When the first tag contains digoxigenin or FITC, which are low molecular compounds, both can be bound by binding the labeling substance to the anti-DIG antibody or anti-FITC antibody.

3. Step of capturing and detecting the labeled target nucleic acid on a solid phase carrier The labeled second nucleic acid amplification product can be captured on a solid phase carrier and detected. Capture (ie, binding) of the second nucleic acid amplification product to the solid phase carrier is performed by bringing the solid phase carrier into contact with a second tag that can bind to the solid phase carrier in the second nucleic acid amplification product. be able to. The solid phase carrier is not particularly limited, but may be made of a resin, metal, polysaccharide, mineral or the like, and may be in the form of a membrane, film, nonwoven fabric, plate, gel or the like. Preferably, the solid phase carrier has a porous structure so that the second nucleic acid amplification product and the labeling substance in the solution can be developed. Examples of the solid phase carrier usable in the present invention include filter paper, nitrocellulose membrane, polyether sulfone membrane, nylon membrane, various dried gels (silica gel, agarose gel, dextran gel, gelatin gel) and the like. As the size and form of the solid phase carrier, those suitable for various operations and detection can be appropriately selected.

Contact and capture (that is, binding) between the solid phase carrier and the second tag may be direct binding or indirect binding, and the binding means uses the solid phase carrier and the second tag to be used. A suitable one can be selected depending on the case. For example, when the second tag contains an oligonucleotide, an oligonucleotide containing a sequence complementary to the base sequence of the oligonucleotide is immobilized on a solid phase carrier to serve as a tag capturing means, and both oligonucleotides are hybridized. Thus, the solid phase carrier and the second tag can be indirectly bound. Immobilization of the oligonucleotide to the solid phase carrier may be performed via a peptide, protein, nucleic acid, or the like, or may be performed via an appropriate functional group. When immobilizing an oligonucleotide on a solid phase carrier, the captured second nucleic acid amplification product is detected only in a predetermined region by immobilizing it in a specific region. Negative discrimination can be facilitated. Hybridization conditions can be performed according to the conditions described above.

The detection of the second nucleic acid amplification product can be performed by detecting, preferably visually detecting the labeling substance bound to the second nucleic acid amplification product captured on the solid phase carrier. When the target nucleic acid is present, the labeling substance of the nucleic acid amplification reaction product captured and bound to the solid phase carrier is detected. The presence or absence of the target nucleic acid in the second nucleic acid amplification reaction product can be determined using the presence or absence of the detection as an index.

4). Nucleic acid detection device The above-mentioned “step of labeling the amplified target nucleic acid” and “step of capturing and detecting the labeled target nucleic acid on a solid phase carrier” can be performed using a nucleic acid detection device utilizing nucleic acid chromatography. By using the nucleic acid detection device, the presence or absence of the target nucleic acid in the second nucleic acid amplification product can be detected and discriminated without requiring a special device, and the result can be obtained easily and quickly. it can.

As the nucleic acid detection device, a known nucleic acid detection device (WO2012 / 070618) used for detecting a nucleic acid amplification product labeled by a nucleic acid chromatography method can be used.

FIG. 1 shows a schematic diagram of an embodiment of a nucleic acid detection device that can be used in the present invention, but the nucleic acid detection device is not limited to this embodiment. In the following description, the reference numerals given to the respective members correspond to the reference numerals shown in FIG.

The nucleic acid detection device of FIG. 1 includes a sample pad (3) for adding a second nucleic acid amplification product, a conjugate pad (2) having a labeling substance disposed thereon, The solid phase carrier (1) and the absorption pad (4) for capturing the nucleic acid amplification product of 2 are sequentially stacked. On the solid phase carrier (1), means (capturing means) (6) (for example, the above-mentioned oligonucleotide etc.) for capturing the labeled second nucleic acid amplification product is locally arranged and immobilized. . The sample pad (3), the conjugate pad (2), the solid phase carrier (1) and the absorption pad (4) are composed of members having a porous structure that can be used as the solid phase carrier. These may be comprised from the same member, and may be comprised from a different member. The base material (5) can support various members disposed thereon and can facilitate the operation of the nucleic acid detection device. For example, a material made of resin, metal, mineral, or the like is used. Can do. When the labeling substance is mixed in the developing solution, the conjugate pad (2) can be omitted.

After the second nucleic acid amplification reaction, the obtained second nucleic acid amplification reaction product is added to the sample pad (3). The reaction solution after the nucleic acid amplification reaction may be dropped as it is, or may be dropped together with an appropriate developing solution (for example, phosphate buffer, Tris buffer, Good buffer, SSC buffer). The developing solution can further contain a surfactant, a salt, a protein, a nucleic acid and the like, if necessary. The second nucleic acid amplification reaction product added to the sample pad (3) develops by a capillary phenomenon from upstream to downstream in the direction indicated by the arrow in FIG.

When the second nucleic acid amplification reaction product passes through the conjugate pad (2) on which the labeling substance is disposed, the second nucleic acid amplification reaction product comes into contact with the labeling substance and is labeled with the labeling substance via the first tag.

Next, when the labeled second nucleic acid amplification reaction product passes through the solid phase carrier (1), it contacts the capture means (6) and is captured by the solid phase carrier (1) through the second tag. -Combined.

When the target nucleic acid is present, the labeling substance of the nucleic acid amplification reaction product captured and bound to the solid phase carrier (1) by the capture means (6) is detected in the region of the capture means (6). If the labeling substance can be visually confirmed, the region of the capturing means (6) is colored due to the labeling substance. The presence or absence of the target nucleic acid in the second nucleic acid amplification reaction product can be determined using the presence or absence of detection (coloration) of the labeling substance as an index.

II. Nucleic acid detection kit The nucleic acid detection kit according to the present invention includes the first primer set, the second primer set, a labeling substance, and a solid phase carrier, and can be used in the nucleic acid detection method of the present invention. The labeling substance and the solid phase carrier can be in the form of the nucleic acid detection device.

The nucleic acid detection kit can further contain a PCR buffer solution, dNTPs, DNA polymerase, a nucleic acid chromatography developing solution, and the like.

Hereinafter, the present invention will be specifically described by way of examples. However, the present invention is not limited to these examples.

(Example 1)
(I) Preparation of gold colloid-binding oligonucleotide Gold Colloid (40 nm, 9.0 × 10 ¹⁰ (number of particles / ml), manufactured by British Biocell International) and thiol group-containing oligonucleotide represented by SEQ ID NO: 1 are mixed And incubated at 50 ° C. for 16 hours. Centrifugation was performed at 6000 rpm for 15 minutes, the supernatant was removed, 0.05 M sodium chloride, 5 mM phosphate buffer (pH 7) was added and mixed, and then incubated again at 50 ° C. for 40 hours.

After incubation, centrifugation (6000 rpm, 15 minutes) was performed, the supernatant was removed, and 5 mM phosphate buffer (pH 7) was added. This buffer replacement was performed again.

The prepared gold colloid solution was uniformly added to a glass fiber pad, and then dried with a vacuum dryer to obtain a conjugate pad.
(Thiol group-containing oligonucleotide): 5′-CTAATAACCCAGTGAAAAATGTGCCA-SH-3 ′ (SEQ ID NO: 1)

(II) Preparation of membrane with immobilized tag capturing means As a tag capturing means, a solution containing the oligonucleotide probe represented by SEQ ID NO: 2 below was applied to a nitrocellulose membrane (Hi-Flow 180) manufactured by Merck Millipore, and a dispenser. And applied in a line. Thereafter, the membrane was dried at 40 ° C. for 30 minutes to obtain a membrane equipped with tag capturing means.
(Oligonucleotide probe): 5′-ATCACACATTAGCTGTTCACTCGATGCA-3 ′ (SEQ ID NO: 2)

(III) Production of Lateral Flow Type Nucleic Acid Detection Device The produced lateral flow type nucleic acid detection device was produced according to the detection device shown in the schematic diagram of FIG.

That is, a backing sheet made of polypropylene (Lohmann) as the base material (5), the conjugate pad prepared in the above (I) as the conjugate pad (2), the solid phase carrier (1) and the above as the capture means (6) ( The membrane provided with the tag capturing means prepared in II), the sample pad made of glass fiber as the sample pad (3), and the absorbent pad made of cellulose as the absorption pad (4) overlap each other as shown in FIG. The lateral flow type nucleic acid detection device was produced by bonding.

(IV) Design of target detection primer In this example, when PCR amplification is performed using the synthetic nucleic acid represented by SEQ ID NO: 3 as a template DNA, about 230 base pairs in a specific region of the template sequence are amplified. Inner primer F (SEQ ID NO: 4) and inner primer R (SEQ ID NO: 5) were designed.

Further, an outer primer F (SEQ ID NO: 6) and an outer primer R (SEQ ID NO: 7) were designed so that the recognition sequence of the inner primer was sandwiched between the upstream side (5 ′ end side) of each inner primer.

In designing, the Tm value of the outer primer was designed to be 10 ° C. higher than the Tm value of the inner primer.

Table 1 shows the sequence and Tm value of each primer. Moreover, the area | region corresponding to the recognition sequence of each primer is shown in FIG.

The following tag sequence 1 (SEQ ID NO: 8) and tag sequence 2 (sequence) are attached to the 5 ′ end of each inner primer via a spacer (expressed by the above formula (I)) having azobenzene which is a polymerase reaction inhibition region. Each of No. 9) was ligated to prepare inner primer-Tag1-F (SEQ ID NO: 10) and inner primer-Tag2-R (SEQ ID NO: 11).
(Tag sequence 1): 5'-TCGAGTGACAGCTCATAATGTGTATT-3 '(SEQ ID NO: 8)
(Tag sequence 2): 5′-ATTTTTCACTGGGTTTATAGT-3 ′ (SEQ ID NO: 9)
(Inner primer-Tag1-F): 5′-TCGAGTGACAGCTCATAGTGTGTATT-X-CGGAGGTTCCGCAAAAGATG-3 ′ (SEQ ID NO: 10)
(Inner primer-Tag2-R): 5'-ATTTTTCACTGGGTTTATAGT-X-CCTCCCAGAGTGATCGATGTTT-3 '(SEQ ID NO: 11)
(In the above sequences, “X” represents a spacer)

(V) Amplification reaction of target nucleic acid TaKaRa Ex Taq (registered trademark) Hot Start Version (Takara Bio) was used as a PCR reagent, 1 × EX Taq buffer, 0.2 mM dNTPs for each, 0.5 μM inner primer-Tag1-F, 0.5 μM inner primer-Tag2-R, 0.11 μM outer primer F, 0.11 μM outer primer R, 0.9375 Unit TaKaRa EX Taq (registered trademark), template synthetic DNA (SEQ ID NO: 3) (0 pg / test, 0. Nested-PCR reaction was performed in a reaction solution containing 5 pg / test, 5 pg / test, or 50 pg / test).

Reaction conditions were 95 minutes at 95 ° C. for 2 minutes, 55 cycles of 95 ° C. for 10 seconds and 72 ° C. for 20 seconds, then 90 ° C. for 5 seconds, 60 ° C. for 10 seconds, 72 ° C. for 10 seconds. As a set, 15 cycles were performed to obtain an amplification product. The obtained amplification product has a tag sequence consisting of single-stranded DNA resulting from each inner primer.

(VI) Detection of amplification product using lateral flow nucleic acid detection device The amplification product obtained in (V) above was detected by the lateral flow nucleic acid detection device prepared in (III) above.

Add the reaction solution (5 μL) after Nested-PCR to the sample pad on the device, and then add 80 μL of the developing solution (1 × SSC, 1% Tween 20, 0.1% SDS) to develop the reaction solution. did. After 10 minutes at room temperature, the tag capturing means fixed in a line on the membrane was checked for coloration.

The result is shown in FIG. Also in the sample to which 0.5 pg / test template synthetic DNA was added, a red line derived from the gold colloid was observed appropriately, and the amplification product derived from the template synthetic DNA could be detected. On the other hand, in the sample (0 pg / test) to which no template synthetic DNA was added, no coloration was observed, and it was confirmed that no nonspecific coloration not derived from the amplification product was exhibited.

(Comparative Example 1)
Using only the inner primer to which the tag sequence prepared in Example 1 was linked, PCR amplification was performed using the synthetic nucleic acid represented by SEQ ID NO: 3 as the template DNA. As PCR reagents, TaKaRa Ex Taq (registered trademark) Hot Start Version (Takara Bio) was used, and 1 × EX Taq buffer, 0.2 mM dNTPs for each, 0.4 μM inner primer-Tag1-F, 0.4 μM inner primer-Tag2- PCR reaction in a reaction solution containing R, 0.9375 Unit TaKaRa EX Taq (registered trademark), template synthetic DNA (SEQ ID NO: 3) (0 pg / test, 0.5 pg / test, 5 pg / test, or 50 pg / test) Went.

Reaction conditions were in accordance with a conventional method, and after 2 minutes at 95 ° C., 40 cycles were performed with 95 ° C. for 20 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds to obtain an amplification product. The obtained amplification product was detected by a lateral flow type nucleic acid detection device in the same manner as in Example 1.

The result is shown in FIG. Non-specific coloring was also observed in the sample (0 pg / test) to which no template synthetic DNA was added. When the amplification product was analyzed by agarose gel electrophoresis for the cause of this coloring, formation of a primer dimer was observed and it was found that this was detected non-specifically.

(Example 2)
Among the PCR conditions described in Example 1, the second amplification step is fixed to 15 cycles that do not cause non-specific amplification by the primer dimer, and the number of cycles of the first amplification step is in the range of 10 to 60 cycles. The sensitivity and specificity when increasing or decreasing were examined.

The results are shown in Table 2 below. When the number of cycles in the first amplification step was 25 or more, specific coloring of the template DNA was confirmed. In the case of 50 cycles or more, specific coloring was confirmed even when the template DNA was 0.5 pg / test. Under the condition that the second amplification step was 15 cycles, no false positive coloring was observed in the sample (0 pg / test) to which no template synthetic DNA was added.

(Comparative Example 2)
Among the PCR conditions described in Comparative Example 1, the number of cycles in PCR was changed in the range of 10 to 60 cycles, and the detectability under each PCR condition was evaluated. An amplification product detection test was carried out using a lateral flow type nucleic acid detection device under the same conditions as in Example 1.

The results are shown in Table 2 below. Coloring was confirmed when the number of cycles in PCR was 25 cycles or more. However, false positive coloring was also confirmed in the sample (0 pg / test) to which no template synthetic DNA was added. For this reason, a positive / negative determination was impossible under any conditions.

From the above results, according to the method of the present invention, detection of non-specific amplification products by primer dimers can be avoided, and amplification products derived from target nucleic acids can be detected simply, efficiently and with high sensitivity. can do.

The present invention can be used for various tests and research including a step of detecting a DNA fragment obtained by a nucleic acid amplification method. More specifically, according to the present invention, molecular biology research fields, pathogen detection, allergen detection in foods and drinks, livestock management, detection of nucleotide polymorphisms, detection of diseases (for example, cancer, etc.) are simplified. And can be done quickly. The present invention is expected to contribute greatly in such fields.

All publications, patents and patent applications cited in this specification are incorporated herein by reference in their entirety.

Claims

A method for detecting a target nucleic acid, comprising:
(A) consisting of a primer containing a sequence homologous to the first base sequence of the target nucleic acid and a primer containing a sequence homologous to the complementary strand sequence of the second base sequence located downstream of the first base sequence Using the first primer set to obtain a first nucleic acid amplification product by performing a nucleic acid amplification reaction using the test DNA as a template,
(B) a primer including a sequence homologous to a third base sequence located downstream of the first base sequence and upstream of the second base sequence; a downstream of the third base sequence; and A second primer set comprising primers containing a sequence homologous to the complementary strand sequence of the fourth base sequence located upstream of the second base sequence, wherein any one of the primers can bind to the labeling substance Using the second primer set, which is bound to a first tag and the other primer is bound to a second tag capable of binding to a solid phase carrier, the first nucleic acid amplification product is Performing a nucleic acid amplification reaction as a template to obtain a second nucleic acid amplification product to which each of the first tag and the second tag is added at each end;
(C) obtaining a labeled nucleic acid amplification product by bringing the second nucleic acid amplification product into contact with a labeling substance that can bind to the first tag;
(D) capturing the labeled nucleic acid amplification product on the solid phase carrier by contacting the labeled nucleic acid amplification product with a solid phase carrier; and (e) capturing the labeled nucleic acid amplification product on the solid phase carrier. Detecting the labeled nucleic acid amplification product;
Including the above method.
The method according to claim 1, wherein the step (a) and the step (b) are continuously performed in the same reaction vessel.
The method according to claim 1 or 2, wherein the annealing temperature of the nucleic acid amplification reaction in the step (a) is higher than the annealing temperature of the nucleic acid amplification reaction in the step (b).
The method according to any one of claims 1 to 3, wherein the number of amplification cycles of the nucleic acid amplification reaction in the step (b) is less than the number of amplification cycles of the nucleic acid amplification reaction in the step (a). .
(A) consisting of a primer containing a sequence homologous to the first base sequence of the target nucleic acid and a primer containing a sequence homologous to the complementary strand sequence of the second base sequence located downstream of the first base sequence First primer set,
(B) a primer including a sequence homologous to a third base sequence located downstream of the first base sequence and upstream of the second base sequence; a downstream of the third base sequence; and A second primer set comprising primers containing a sequence homologous to the complementary strand sequence of the fourth base sequence located upstream of the second base sequence, wherein any one of the primers can bind to the labeling substance The second primer set, which is bound to a first tag and the other primer is bound to a second tag capable of binding to a solid phase carrier;
(C) a labeling substance capable of binding to the first tag, and
(D) a solid phase carrier capable of binding to the second tag,
A kit for detecting a target nucleic acid, comprising:
The kit according to claim 5, wherein at least one of the first tag and the second tag in the second primer set is an oligonucleotide tag.
The kit according to claim 6, wherein the tag comprising the oligonucleotide is bound to a primer through a spacer capable of suppressing or stopping the DNA polymerase reaction.
The kit according to claim 7, wherein the spacer is azobenzene, a fatty chain, or an inverted base.
The kit according to any one of claims 6 to 8, wherein the first tag is a tag composed of an oligonucleotide, and the labeling substance includes an oligonucleotide having a sequence complementary to the oligonucleotide.
10. The tag according to claim 6, wherein the first tag is a tag composed of any low molecular compound selected from biotin, digoxigenin, and FITC, and the labeling substance includes a protein that can bind to the tag. Kit according to item.
The kit according to any one of claims 6 to 10, wherein the second tag is a tag composed of an oligonucleotide, and the solid phase carrier includes an oligonucleotide having a sequence complementary to the oligonucleotide.
The Tm value of one or both primers included in the first primer set is higher than the Tm value of one or both primers included in the second primer set. The kit as described in any one of.