JP4728663B2 - Target substance detection probe set and target substance detection method - Google Patents

Description

  The present invention relates to a probe set for detecting a target substance contained in a sample and a method for detecting a target substance using the probe set.

  Conventionally, as a method for detecting a target nucleic acid in a sample, Southern hybridization, Northern hybridization, in situ hybridization and the like are well known. These are methods for detecting the presence and / or position of a target nucleic acid by hybridizing a labeled probe to a membrane or a fixed cell blotted with the target nucleic acid and detecting the label. Since these methods do not amplify the target nucleic acid, especially when the amount of the target nucleic acid in the sample is very small, the signal is weak and the detection sensitivity is low.

  As one of the methods for detecting nucleic acid in a sample by performing nucleic acid amplification, the method described in Patent Document 1 can be used. The method includes the following steps. A sample containing the target nucleic acid, probe A, and probe B are mixed to produce a reaction mixture. Probe B is hybridized circularly to the target nucleic acid, and the 5 'end and 3' end of probe B are bound by ligase. The reaction mixture is heated to 90 ° C. or higher to thermally denature the hybridization between the target nucleic acid and the probe B. When heat-denatured, a structure in which a single-stranded target nucleic acid penetrates the circular probe B can be taken. The temperature of the reaction mixture is lowered, probe A is circularly hybridized to circular probe B, and the 5 'end and 3' end of probe A are bound by ligase. In this way, probe A and probe B are bound to one molecule of the target nucleic acid in a chain form. By labeling the probe A and the probe B bound to the target nucleic acid, even a nucleic acid contained in a trace amount in the sample can be detected with a strong signal.

  However, the above method targets only nucleic acids and cannot detect other substances such as proteins. Moreover, the step which heat-denatures the reaction liquid mixture at 90 degreeC or more is required, and the apparatus which keeps the reaction liquid mixture high temperature for a fixed time is needed.

Japanese Patent Laid-Open No. 9-23899

  An object of the present invention is to provide a probe set that can detect a target substance such as nucleic acid, protein, and hapten contained in a sample with a strong signal and a target substance detection method that does not require a heat denaturation step.

  As a result of intensive studies, the present inventors have found that a target binding substance capable of binding to a target substance, a primary probe having a base sequence X1 and a base sequence X2, and a base sequence X1c capable of hybridizing to the base sequence X1 at the end. A second probe having a base sequence X2c capable of hybridizing with the base sequence X2 at the other end, a base sequence Y1c and a base sequence Y2 at a portion not including the end, and the base sequence at the end A target substance can be detected with a strong signal by a probe set comprising a tertiary probe having a base sequence Y1 capable of hybridizing to Y1c and having a base sequence Y2 capable of hybridizing to the base sequence Y2c at the other end. The present invention relating to a method for detecting a target substance that does not require a heat denaturation step has been completed.

That is, this invention consists of the following.
1. A target binding substance capable of binding to the target substance, a primary probe having a base sequence X1 and a base sequence X2, and
It has a base sequence X1c that can hybridize with the base sequence X1 at the end, a base sequence X2c that can hybridize with the base sequence X2 at the other end, and a base sequence Y1c and a base at a portion not including the end a secondary probe having the sequence Y2 c,
A tertiary probe having a base sequence Y1 capable of hybridizing to the base sequence Y1c at the end and a base sequence Y2 capable of hybridizing to the base sequence Y2c at the other end;
Probe set containing.
2. 2. The probe set according to item 1, wherein the primary probe has a base sequence Z and a base sequence Zc capable of hybridizing with the base sequence Z.
3. 3. The probe set according to item 1 or 2, wherein the tertiary probe includes a base sequence X1 and a base sequence X2 in a portion not including a terminal.
4). 4. The probe according to item 3 above, wherein the base sequence X1 and base sequence X2 of the primary probe are adjacent, the base sequence Y1c and base sequence Y2c of the secondary probe are adjacent, and the base sequence X1 and base sequence X2 of the tertiary probe are adjacent. set.
5. 5. The probe set according to item 4, wherein the primary probe has a plurality of sequences in which the base sequence X1 and the base sequence X2 are adjacent.
6). 6. The probe set according to item 4 or 5, wherein the secondary probe has a plurality of sequences in which the base sequence Y1c and the base sequence Y2c are adjacent, and the tertiary probe has a plurality of sequences in which the base sequence X1 and the base sequence X2 are adjacent.
7). 7. The probe set according to any one of items 1 to 6, wherein the target substance is selected from the group consisting of nucleic acids, proteins, polysaccharides, and hormones.
8). 7. The probe set according to any one of items 1 to 6 , wherein the target binding substance is selected from the group consisting of a nucleic acid, an antibody, and a receptor.
9. A reagent for detecting a target substance comprising the probe set according to any one of items 1 to 8 .
10. Reagents including the probe set according to any one of 1 to 8 above , and each probe forming a detection complex in which a target substance, a primary probe, a secondary probe, and a tertiary probe included in the probe set are combined. A reagent kit for detecting a target substance, comprising a reagent containing ligase for linking adjacent ends of each other .
11. A target substance detection method including the following steps:
(A) a sample containing a target substance, a target binding substance capable of binding to the target substance, a primary probe having a base sequence X1 and a base sequence X2, and a base sequence X1c capable of hybridizing to the base sequence X1 at the end and the other at one end has the base sequence X2 capable of hybridizing nucleotide sequence X2c, a secondary probe having a partial free end a nucleotide sequence Y1c and base sequence Y2 c, the a terminal base sequence Y1c Mixing a tertiary probe having a base sequence Y1 capable of hybridizing with the base sequence Y2 at the other end and having the base sequence Y2 capable of hybridizing with the base sequence Y2c;
(B) a step of binding the primary probe and a target substance;
(C) a step of hybridizing the primary probe and the secondary probe;
(D) A step of hybridizing the secondary probe and the tertiary probe.
12 For detection, the target substance, the primary probe, the secondary probe, and the tertiary probe are bound to each other in the order of the step (a), the step (b), the step (c), and the step (d). 12. The target substance detection method according to 11 above, wherein a complex is formed.
13. For detection in which the target substance, the primary probe, the secondary probe, and the tertiary probe are bound to each other in the order of the step (a), the step (c), the step (d), and the step (b). 12. The target substance detection method according to 11 above, wherein a complex is formed.
14 The tertiary probe has a base sequence X1 and a base sequence X2 in a portion not including a terminal, and (e) a tertiary probe constituting the detection complex and a free secondary probe not constituting the detection complex 14. The method for detecting a target substance according to item 12 or 13, comprising a step of hybridizing, and a step (f) enlarging the detection complex by repeating the step (d) and the step (e).
15 . 15. The target substance detection method according to any one of the above items 11 to 14, further comprising a step of linking adjacent ends of each probe forming the detection complex using ligase.

  When the probe set of the present invention is used, a detection complex is formed by binding a primary probe to a target substance such as a nucleic acid, protein, or hapten contained in a sample, and further binding a secondary probe and a tertiary probe to the primary probe. And the target substance can be detected with a strong signal based on this detection complex. Further, according to the target substance detection method of the present invention, a detection complex can be formed without the need for a heat denaturation step, and the target substance can be detected with a strong signal based on this detection complex. it can.

  The term “nucleic acid” used herein refers not only to DNA and RNA, but also to artificially synthesized nucleic acids (hereinafter referred to as artificial nucleic acids) such as PNA (Peptide Nucleic Acid), BNA (Bridged Nucleic Acid), and analogs thereof. Including). Further, the “base sequence (hereinafter also simply referred to as sequence)” in the present specification includes not only DNA and RNA base sequences but also base sequences of artificial nucleic acids such as PNA, BNA, and analogs thereof.

<Target substance>
The target substance detected by the probe set of the present invention is not particularly limited. For example, nucleic acids, proteins, hormones, neurotransmitters, otachoids, haptens, etc. Cells such as bacteria and pollen can also be target substances.

<Probe set>
The probe set used in this embodiment includes three types of probes: a primary probe, a secondary probe, and a tertiary probe. When the target substance is detected, a detection complex in which the target substance and these probes are bound is formed.
Hereinafter, each probe will be described.

(1) Primary probe The primary probe has a substance capable of binding to a target substance (hereinafter referred to as “target binding substance”), a base sequence X1, and a base sequence X2. It is preferable that the arrangement | sequence X1 and arrangement | sequence X2 are adjacent. Thereby, when the below-mentioned secondary probe is hybridized to the primary probe, both ends of the secondary probe can be linked. By linking both ends of the secondary probe, durability against decomposition and washing operations can be improved.

  The “target binding substance” is not particularly limited as long as it is a substance that can bind to the target substance described above. For example, when the target substance is a nucleic acid, a nucleic acid having a base sequence complementary to the nucleic acid is used. When the target substance is a protein, an antibody or aptamer specific to this protein is used. When the target substance is an antibody, an antigen that specifically binds to the antibody may be used. When the target substance is a hormone, neurotransmitter, or otacoid, a receptor capable of binding to these is used. When the target substance is a hapten, an antibody specific for this hapten is used. When the target substance is a cell, an antibody specific for the substance exposed on the surface of the cell is used.

  The primary probe preferably has a base sequence S and a base sequence Sc capable of hybridizing to the sequence S. In this case, the sequence S hybridizes with the sequence Sc, and this portion of the primary probe becomes a double strand to form a stem loop structure. By forming the stem loop structure, the primary probe can be stabilized. The length of the sequence S is not particularly limited as long as it can be hybridized with the sequence Sc so as to form the above structure, but the Tm value of the sequence S and the sequence Sc is higher than the reaction temperature when forming the detection complex. It is preferable to be high. Specifically, the length of the sequence S is preferably 7 bases or more.

(2) Secondary probe The secondary probe has a sequence X1c capable of hybridizing to the sequence X1 at the end and a sequence X2c capable of hybridizing to the sequence X2 at the other end. Further, the secondary probe has a base sequence Y1c and a sequence Y2c in a portion not including the terminal.
The array Y1c and the array Y2c are preferably adjacent to each other. This makes it possible to connect both ends of the tertiary probe when a later-described tertiary probe is hybridized to the secondary probe. By linking both ends of the tertiary probe, durability against decomposition and washing operations can be improved.

(3) Tertiary probe The tertiary probe has a sequence Y1 capable of hybridizing to the sequence Y1c at the end and a sequence Y2 capable of hybridizing to the sequence Y2c at the other end. Moreover, it is preferable that the tertiary probe has the sequence X1 and the sequence X2 in a portion not including the terminal. As a result, a free secondary probe that does not form a detection complex can be hybridized to the tertiary probe. Thereafter, the secondary probe and the tertiary probe are sequentially hybridized to the detection complex. The body can be enlarged.
In addition, when the tertiary probe has the sequence X1 and the sequence X2, it is preferable that these sequences are adjacent to each other. Thereby, when the secondary probe is hybridized to the tertiary probe, both ends of the secondary probe can be linked. By linking both ends of the secondary probe, durability against decomposition and washing operations can be improved.

  The primary probe preferably has a plurality of sequences in which the sequences X1 and X2 are adjacent. The secondary probe preferably has a plurality of sequences in which the sequences Y1c and Y2c are adjacent. The tertiary probe preferably has a plurality of sequences in which the sequences X1 and X2 are adjacent. Since each probe has these characteristics, a plurality of probes are hybridized to each probe, and the detection complex is enlarged exponentially. Thereby, the target substance can be detected with a stronger signal.

  The lengths of the sequence X1, the sequence X1c, the sequence X2, the sequence X2c, the sequence Y1, the sequence Y1c, the sequence Y2 and the sequence Y2c are not particularly limited, but preferably 4 to 20 bases, more preferably 4 to 10 bases, most preferably Is 4 to 8 bases.

  In the secondary probe, between the sequence X1c and the sequence adjacent to the sequence Y1c and the sequence Y2c, and between the sequence adjacent to the sequence Y1c and the sequence Y2c and the sequence X2c, nucleic acid, polyether alcohol such as hexaethylene glycol, etc. It is preferable to arrange | position through a polymer | macromolecule. When the array and the array are connected via the polymer as described above, the intervening portion is referred to as an intervening portion in this specification. The length of the interposition part is not particularly limited, but when the interposition part is a nucleic acid, it is preferably 3 bases or more. If the intervening portion is long, the probe ring becomes large when a complex for detection is formed, and a plurality of probes are easily bonded to one probe structurally. Therefore, the longer the interposition part is, the better.

<Detection of target substance>
Various methods are conceivable as a method for detecting the target substance, and are not particularly limited. For example, there is a method using a plate. According to this method, first, a substance contained in a sample is fixed to a well of a plate, and the probe set is added thereto to cause a reaction. When the target substance is contained in the sample, the target substance fixed to the well and the probe bind to each other, so that the detection complex is fixed in the well even if the reaction solution is removed.

  There is also a method using magnetic beads as shown in FIG. According to this method, first, a magnetic bead is added to a target binding substance, and a substance having magnetism or a substance that adsorbs to magnetism such as iron (hereinafter referred to as a capture region) is provided inside or outside of the target binding substance. Flow in capillary. At the same time or after this, the probe set and sample are flowed into the capillary. As shown in FIG. 1, when the target substance is contained in the sample, the target binding substance having magnetic beads and the target binding substance of the complex for detection bind in a sandwich form via the target substance. This sandwich-like complex is fixed to a capture region in the capillary. A detection complex that cannot be bound to the labeling substance, a complex in which only the secondary probe and the tertiary probe are bound, and the like are not trapped in the capture region and flow as they are. In addition, it is preferable that the site | part of the target substance which the target binding substance which has a magnetic bead recognizes, and the site | part of the target substance which the target binding substance of a detection complex recognizes are different. Instead of magnetic beads, a substance that adsorbs to magnetism such as iron may be used, and a substance having magnetism may be provided on a part of the inside or outside of the capillary.

As described above, the detection complex immobilized on the well of the plate or the capture region in the capillary is labeled as shown below, and the target substance can be detected based on the signal.
For example, an intercalating dye such as ethidium bromide, oregon green, or SYBR GREEN is bound to the double-stranded portion of the detection complex. The fluorescence emitted by the intercalating dye becomes a signal.
Further, by previously binding a fluorescent substance such as FITC to at least one probe constituting the probe set, the fluorescence from the fluorescent substance can be used as a signal. Instead of FITC, radioisotopes such as ¹²⁵ I and ³² P can also be bound, and radiation emitted from these can be used as signals.
In addition, alkaline phosphatase is bound to at least one probe, and a luminescent / coloring substance such as digoxigenin or acridinium ester, a luminescent substance such as dioxetane, a fluorescent substance such as 4-methylumbelliferyl phosphate, etc. The signal can be luminescence, fluorescence, or coloration generated by reacting with.
In addition, avidin or biotin is bound to at least one probe, and further, biotin or avidin to which a fluorescent substance, a coloring substance, or the like is bound is added to the target substance, and these are reacted to thereby cause the fluorescence, coloring, etc. It can be a signal.
In addition, a donor fluorescent dye and an acceptor fluorescent dye are bound to at least one probe, and fluorescence emitted from these can be used as a signal by using fluorescence resonance energy transfer (FRET).

  In addition, when the intervening part of each probe is a nucleic acid, a signal can be obtained using a labeled nucleic acid probe. First, a single-stranded nucleic acid probe capable of binding to the intervening portion is prepared, and a labeled substance such as a radioisotope, a fluorescent / luminescent / chromogenic substance is added thereto to prepare a labeled nucleic acid probe. As shown in FIG. 2, since the intervening portion of each probe is in a single-stranded state even if each probe forms a detection complex, the labeled nucleic acid probe is interposed between each probe forming the detection complex. It can hybridize to the part. When this labeled nucleic acid probe is hybridized to a detection complex, radiation, fluorescence, luminescence, color development, etc. emitted from the labeled nucleic acid probe can be used as a signal.

  In addition, when the intervening part of each probe is a nucleic acid and a signal is obtained using an intercalating dye, the signal can be increased by using a nucleic acid probe that can bind to the intervening part. Even if each probe forms a circular detection complex, the intervening portion of each probe is in a single-stranded state. By binding a single-stranded nucleic acid probe having a sequence that can bind to this single-stranded intervening part to the intervening part, the intervening part to which the nucleic acid probe is bound constitutes a single strand, and the intercalating dye is detected. The region that binds to the complex for use increases, and a stronger signal can be obtained.

  In order to make the understanding of the present invention more reliable, examples will be shown and described below, but it goes without saying that the present invention is not limited to these examples.

(Example)
A probe having the following characteristics is produced.
(1) Primary Probe As shown in FIG. 3a, the primary probe has a target binding substance, a sequence A1c, a sequence A2c, a sequence S, and a sequence Sc that can hybridize to the sequence S.
The target binding substance is located at the end of the primary probe. The sequence A1c and the sequence A2c are adjacent. There are three sequences adjacent to the sequence A1c and the sequence A2c in the probe. The sequence S is located at the end to which the target binding substance is not bound.
Intervening portions exist between the sequence Sc and the sequence A1c, between the sequence A2c and the sequence A1c, and between the sequence A2c and the sequence S, respectively.
(2) Secondary probe The secondary probe has sequence A1, sequence A2, sequence B1, and sequence B2. Sequence A1 and sequence A2 are located at both ends of the secondary probe, respectively. Array B1 and array B2 are adjacent. There are three sequences adjacent to the sequence B1 and the sequence B2 in the probe.
Intervening portions exist between the array A1 and the array B1, between the array B2 and the array B1, and between the array B2 and the array A2.
(3) Tertiary probe The tertiary probe has sequence B1c, sequence B2c, sequence A1c, and sequence A2c. The sequence B1c and the sequence B2c are located at both ends of the tertiary probe, respectively. The sequence A1c and the sequence A2c are adjacent. There are three sequences adjacent to the sequence A1c and the sequence A2c in the probe.
Intervening portions exist between the sequence B1c and the sequence A1c, between the sequence A2c and the sequence A1c, and between the sequence A2c and the sequence B2c.

  It is preferable that either end of the secondary probe is phosphorylated. Moreover, it is preferable that either end of the tertiary probe is phosphorylated.

  When the intervening portion is a base sequence, the sequence of the intervening portion does not hybridize with any of the sequence A1, the sequence A1c, the sequence A2, the sequence A2c, the sequence B1, the sequence B1c, the sequence B2, the sequence B2c, the sequence S, and the sequence Sc. Designed as such.

  Of the above sequences, the sequence A1 is designed to hybridize with the sequence A1c, the sequence A2 with the sequence A2c, the sequence B1 with the sequence B1c, and the sequence B2 with the sequence B2c. Thereby, the secondary probe and the tertiary probe are hybridized in a circular pattern with the primary probe bound to the target substance, and a detection complex as shown in FIG. 3b is formed. The kind of base constituting each sequence is not particularly limited.

  It is preferable that the end of each probe constituting the detection complex is linked. As a result, the detection complex becomes more stable, and the durability against decomposition and washing operations can be increased. When the base sequence of each probe is a DNA or RNA base sequence, the ends of the probes can be linked using ligase. When ligating each probe using ligase, the 5 'end or 3' end of each probe needs to be phosphorylated in advance. Further, when the base sequence of each probe is an artificial nucleic acid such as PNA or BNA, the ends of the probes can be linked by photobonding or the like. For example, a method described in US Pat. No. 6,659,088B1 can be used as a method for linking artificial nucleic acids by photobinding.

  The probe set as described above can be provided as a reagent for detecting a target substance in a sample. Alternatively, it can be provided as a reagent kit containing each probe and / or ligase separately.

(Experimental example 1)
Hybridization and ligation are performed using a primary probe and a secondary probe that have a stem-loop structure and biotin is added to the 5 'end, and electrophoresis is performed to determine whether or not the primary probe and the secondary probe are bound. Confirmed by FIG. 4 schematically shows the reaction in this experiment.

<Material>
1) Primary probe
5'-BIOTIN-GGGCCGGGCCGGGCCTTTTTTTTTTGTGAGTAGATAGTTTTTTTTTTGGCCCGGCCCGGCCC-3 '(SEQ ID NO: 1)
2) Secondary probe
5'-ACTCACTTTTTGTACATACTCTTTTTGTACATACTCTTTTTGTACATACTCTTTCTATCT-3 '(SEQ ID NO: 2)
3) TE buffer (50 mM Tris-HCL (pH7.4), 1 mM EDTA)
4) T4 ligase (T4 Ligase; manufactured by TAKARA BIO) and attached T4 ligase buffer (× 10)
5) Low melting point agarose (NuSieve GTG Agarose; manufactured by TAKARA BIO)
6) Marker (20bp DNA Ladder; manufactured by TAKARA BIO)
The 5 ′ end of the secondary probe is phosphorylated.

<Method>
The following substances were accommodated in 0.5 ml sterilized microtubes (i) to (iii).
(i) Microtube marker (20bp DNA Ladder)
(ii) Microtube Primary probe (100pmol / μl): 0.5μl
Secondary probe (100pmol / μl): 0.5μl
T4 ligase: 0.5 μl
T4 ligase buffer (x10): 1μl
TE buffer: up to 10μl
Micro tube of (iii) Primary probe (100pmol / μl): 0.5μl
TE buffer: up to 10μl

  Microtubes (i) to (iii) containing the above substances were allowed to stand at room temperature for 30 minutes. Using these, electrophoresis was performed on a 4% agarose gel at 100 V for 15 minutes. The agarose gel after electrophoresis was stained with ethidium bromide to confirm whether the secondary probe bound to the primary probe.

<Result>
The results are shown in FIG. FIG. 5 is a photograph showing the results of electrophoresis in the above experiment. Microtube (i) marker (20bp DNA Ladder) is in lane (i), microtube (ii) reaction mixture is in lane (ii), and microtube (iii) mixture is in lane (iii). Washed away. In lane (ii), in addition to a band indicating only the primary probe, a band indicating a substance having a molecular weight larger than that of the primary probe was observed. This band shows the substance which the primary probe and the secondary probe couple | bonded. Therefore, the above experiment confirmed that the secondary probe was hybridized to the primary probe.

(Experimental example 2)
Using a primary probe having a stem-loop structure and biotin added to the 5 ′ end, a secondary probe, and a tertiary probe, the signal is amplified by binding the probe to the detection complex in a circular manner. It was confirmed by fluorescence measurement.

<Material>
1) Fluorescent probe
5'-FITC-CTATCTCACTCACAAACTATCTACTCAC-3 '(SEQ ID NO: 3)
2) Tertiary probe
5'-GTACAATTTGTGAGTAGATAGTTTGTGAGTAGATAGTTTGTGAGTAGATAGTTTGAGTAT-3 '(SEQ ID NO: 4)
3) Fluorescent dye (YOYO-1; manufactured by Molecular Probes)
4) Fluorescence measuring instrument (Genius; manufactured by TECAN)
5) Solid-phase plate (SA plate; manufactured by Labsystems) Black 8-well plate with streptavidin as a coating
The same primary probe, secondary probe, T4 ligase, T4 ligase buffer and TE buffer as those used in Experimental Example 1 were used. The 5 ′ end of the tertiary probe is phosphorylated.

<Method>
49 μl of TE buffer is contained in each of 4 0.5 ml sterilized macro tubes, and 1 μl of primary probe of 0.1 pmol / μl, 0.2 pmol / μl, 1 pmol / μl, 2 pmol / μl is added to each reaction mixture. Was prepared. These were respectively transferred to the wells of the solid-phased plate and allowed to stand at room temperature for 30 minutes to react the streptavidin and primary probe on the plate.
After the reaction, each well of the plate was washed three times with TE buffer. After washing, the following substances were added to each well and left at room temperature for 30 minutes for hybridization and ligation.
Secondary probe (100pmol / μl): 10μl
T4 ligase: 5μl
T4 ligase buffer: 10 μl
TE buffer: up to 100μl
Next, each well of the plate was washed three times with TE buffer. After washing, the following substances were added to each well and left at room temperature for 30 minutes for hybridization and ligation again.
Tertiary probe (100pmol / μl): 10μl
T4 ligase: 5μl
T4 ligase buffer: 10 μl
TE buffer: up to 100μl
Next, each well of the plate was washed three times with TE buffer. After washing, a staining solution prepared by mixing 1 μl of 100 pmol / μl fluorescent probe and 99 μl of TE buffer was added, and allowed to stand at room temperature for 15 minutes for fluorescent staining. Further, each well of the plate was washed three times with TE buffer, and fluorescence measurement was performed with a fluorescence measuring machine. The measurement results are shown in the graph of FIG.

  On the other hand, as a negative control, a reaction mixture without hybridization and ligation was prepared. 49 μl of TE buffer is contained in each of 4 0.5 ml sterilized macro tubes, and 1 μl of primary probe of 0.1 pmol / μl, 0.2 pmol / μl, 1 pmol / μl, 2 pmol / μl is added to each reaction mixture. Was prepared. These were transferred to each well of the solid-phased plate and allowed to stand at room temperature for 30 minutes to react the streptavidin on the plate with the primary probe. Next, each well of the plate was washed three times with TE buffer. After washing, a staining solution prepared by mixing 1 μl of 100 pmol / μl fluorescent probe and 99 μl of TE buffer was added, and allowed to stand at room temperature for 15 minutes for fluorescent staining. Further, each well of the plate was washed three times with TE buffer, and fluorescence measurement was performed with a fluorescence measuring machine. The measurement results are shown in the graph (N) in FIG.

<Result>
From FIG. 6, it was possible to detect stronger fluorescence when hybridization and ligation were performed to form a detection complex than when measurement was performed without hybridization and ligation. Therefore, it was found that the target substance can be detected with a stronger signal by performing hybridization and ligation using the probe set of the present embodiment.

When the probe set of the present invention is used, a detection complex is formed by binding a primary probe to a target substance such as a nucleic acid, protein, or hapten contained in a sample, and further binding a secondary probe and a tertiary probe to the primary probe. And the target substance can be detected with a strong signal based on this detection complex. Further, according to the target substance detection method of the present invention, a detection complex can be formed without the need for a heat denaturation step, and the target substance can be detected with a strong signal based on this detection complex. it can.
By the above method, a trace component contained in a sample can be detected with high sensitivity, and it can be applied to a measurement system that requires detection of various trace components such as clinical tests and hygiene tests. Moreover, the probe set of the present invention is useful as a reagent for detecting trace components contained in a sample.

It is a schematic diagram which shows the measurement principle at the time of using a magnetic bead. It is a schematic diagram which shows the composite_body | complex for detection formed using a probe set. (Example) It is a schematic diagram which shows a target substance and a probe set. (Example) It is a schematic diagram which shows the minimum unit of the composite_body | complex for detection formed using a probe set. (Example) It is a reaction schematic diagram using a primary probe to which biotin having a stem loop structure is added. (Experimental example) It is a photograph which shows the result of electrophoresis. (Experimental example 1) It is a figure which shows the detection result using the probe set of this invention. (Experimental example 2)

Explanation of symbols

Fig. 6 (A) Complex for detection using probe set
(N) Negative control

Claims (15)

A target binding substance capable of binding to the target substance, a primary probe having a base sequence X1 and a base sequence X2, and
It has a base sequence X1c that can hybridize with the base sequence X1 at the end, a base sequence X2c that can hybridize with the base sequence X2 at the other end, and a base sequence Y1c and a base at a portion not including the end a secondary probe having the sequence Y2 c,
A tertiary probe having a base sequence Y1 capable of hybridizing to the base sequence Y1c at the end and a base sequence Y2 capable of hybridizing to the base sequence Y2c at the other end;
Probe set containing. The probe set according to claim 1, wherein the primary probe has a base sequence Z and a base sequence Zc capable of hybridizing with the base sequence Z. The probe set according to claim 1 or 2, wherein the tertiary probe includes a base sequence X1 and a base sequence X2 in a portion not including a terminal. The base sequence X1 and the base sequence X2 of the primary probe are adjacent to each other, the base sequence Y1c and the base sequence Y2c of the secondary probe are adjacent to each other, and the base sequence X1 and the base sequence X2 of the tertiary probe are adjacent to each other. Probe set. The probe set according to claim 4, wherein the primary probe has a plurality of sequences in which the base sequence X1 and the base sequence X2 are adjacent. The probe set according to claim 4 or 5, wherein the secondary probe has a plurality of sequences in which the base sequence Y1c and the base sequence Y2c are adjacent, and the tertiary probe has a plurality of sequences in which the base sequence X1 and the base sequence X2 are adjacent. The probe set according to any one of claims 1 to 6, wherein the target substance is selected from the group consisting of a nucleic acid, a protein, a polysaccharide, and a hormone. Probe set according to any one of claims 1 to 6, wherein the target-binding substance is a nucleic acid, is selected from the group consisting of antibodies and receptors. A reagent for detecting a target substance comprising the probe set according to any one of claims 1 to 8 . Reagents comprising the probe set according to any one of claims 1 to 8 , and each forming a complex for detection in which a target substance, a primary probe, a secondary probe and a tertiary probe contained in the probe set are combined. A reagent kit for detecting a target substance, comprising a reagent containing ligase for linking adjacent ends of a probe . A target substance detection method including the following steps:
(A) a sample containing a target substance, a target binding substance capable of binding to the target substance, a primary probe having a base sequence X1 and a base sequence X2, and a base sequence X1c capable of hybridizing to the base sequence X1 at the end and the other at one end has the base sequence X2 capable of hybridizing nucleotide sequence X2c, a secondary probe having a partial free end a nucleotide sequence Y1c and base sequence Y2 c, the a terminal base sequence Y1c Mixing a tertiary probe having a base sequence Y1 capable of hybridizing with the base sequence Y2 at the other end and having the base sequence Y2 capable of hybridizing with the base sequence Y2c;
(B) a step of binding the primary probe and a target substance;
(C) a step of hybridizing the primary probe and the secondary probe;
(D) A step of hybridizing the secondary probe and the tertiary probe. For detection, the target substance, the primary probe, the secondary probe, and the tertiary probe are bound to each other in the order of the step (a), the step (b), the step (c), and the step (d). The target substance detection method according to claim 11, wherein a complex is formed. For detection in which the target substance, the primary probe, the secondary probe, and the tertiary probe are bound to each other in the order of the step (a), the step (c), the step (d), and the step (b). The target substance detection method according to claim 11, wherein a complex is formed. The tertiary probe has a base sequence X1 and a base sequence X2 in a portion not including the end,
(E) a step of hybridizing a tertiary probe constituting the detection complex and a free secondary probe not constituting the detection complex; and (f) repeating the steps (d) and (e). The method for detecting a target substance according to claim 12, further comprising a step of enlarging the detection complex. The target substance detection method according to claim 11, further comprising a step of linking adjacent ends of each probe forming the detection complex using ligase.