JP6705190B2 - Measurement method using amplification reaction - Google Patents

Description

The present invention relates to a measuring method using an amplification reaction.

The measurement method using the principle of enzyme-linked immunosorbent assay (ELISA) is widely used in the fields of clinical diagnosis and basic research. However, the measurement by ELISA requires a washing step (B/F separation) for removing non-specific adsorption of labeled compounds, which requires a long operation time and weak interaction that cannot withstand the washing operation. There was a problem that the host molecule could not be used. Therefore, a homogeneous assay method that does not require B/F separation is used for high throughput screening in the field of biomedical research (Non-Patent Document 1). As a detection method in the conventional homogeneous assay, a method such as fluorescence resonance energy transfer (FRET), time-resolved fluorescence resonance energy transfer (TR-FRET), or fluorescence polarization (FP) is used for detection. Unlike the detection system using an enzyme such as ELISA, these detection systems do not amplify the signal in the reaction system, so that the detection sensitivity may be a problem.

For example, in Non-Patent Document 2, by utilizing the fact that a fluorescent molecule modified near the antigen-binding region of a single-chain antibody (ScFv) is quenched by tryptophan in a protein, a probe that recovers fluorescence by antigen-binding is developed. There is. Further, in Non-Patent Document 3, two DNA chains modified with a host molecule and a pyrene of a fluorescent dye form a double chain by association with a guest molecule, and as a result, the pyrene comes close to the pyrene. We are developing a probe that causes the switching of the emission of light. Both of these measurement methods function in a homogeneous system that does not require B/F separation, but they are not measurement systems having a signal amplification system.

As an example of the measuring method having a signal amplification function and working in a homogeneous system, there is a method utilizing the half reaction of luciferase described in Patent Document 1. However, this measurement method has a problem that the background signal is high. In addition, it is considered that the problem that the luciferase having a large molecular weight has to be modified in the host molecule will be a problem during probe preparation.

JP, 2010-268793, A

Chemistry and Biology Vol. 38 No. 8, 2000.555 J. Am. Chem. Soc. , 2011, 133(43), 17386 Bioconjugate Chem. , 2008, 19, 1132 Nucleic Acids Res, 2009, Vol. 37, No. 3 e20

An object of the present invention is to provide a measuring method capable of amplifying a signal and facilitating preparation of a probe.

The present invention made in view of the above problems includes the following aspects.
(1) Probe A having an association site and single-stranded DNA, and probe B having an association site and single-stranded DNA,
A method of measuring an object to be measured using
(I) the double-stranded sequence is contained in the single-stranded DNAs of probe A and probe B,
The promoter sequence and the amplified sequence are contained in the single-stranded DNA of probe A or probe B, respectively,
(Ii) associate the association site of probe A and the association site of probe B with the measurement target,
(Iii) hybridizing the double-stranded sequence of probe A and the double-stranded sequence of probe B to form a double strand of DNA,
(Iv) A double strand of the promoter sequence is formed by extending the DNA with DNA polymerase using it as a starting point,
(V) RNA is amplified by RNA polymerase using the antisense strand of the amplified sequence downstream of the promoter sequence as a template,
(Vi) detecting amplified RNA,
A measuring method characterized by the above.
(2) In probe A and probe B, one probe is bound to the association site and the 5'end side of the single-stranded DNA, and the other probe is attached to the association site and the 3'end side of the single-stranded DNA. The method according to (1), wherein
(3) The method according to (1), wherein in each of the probes A and B, the association site is bound to the 5'end side of the single-stranded DNA.
(4) The method according to (1), wherein in both probe A and probe B, the association site is bound to the 3'end side of the single-stranded DNA in both probes.
(5) The method according to any one of (1) to (4), which is carried out in a homogeneous system.
(6) The method according to any one of (1) to (5), wherein amplification of RNA is performed under constant temperature conditions.
(7) The method according to any one of (1) to (6), wherein the association site of probe A and/or probe B and the single-stranded DNA are bound via a spacer.
(8) The method according to (7), wherein the spacer is a DNA chain.

Hereinafter, the present invention will be described in detail.

(1) Measuring Method The measuring method of the present invention may be a qualitative measurement for measuring the presence or absence of a measurement target, or a quantitative measurement for measuring the amount of the measurement target. The measuring method of the present invention is preferably performed in a so-called homogeneous system that does not require B/F separation.

(2) Probe The probe A and the probe B used in the present invention have an association site and single-stranded DNA. The binding between the association site and the DNA chain may be performed at either the 3'-terminal side or the 5'-terminal side, and may be appropriately selected depending on the reaction principle. For example, in probe A and probe B, one probe binds the association site to the 5'end side of the single-stranded DNA, and the other probe binds the association site to the 3'end side of the single-stranded DNA. What is being carried out can be mentioned, and this can be used for the measurement principle 1 mentioned later, for example. In addition, in both probe A and probe B, the association site of both probes may be bound to the 5'end side of single-stranded DNA, and similarly, both probes may have one association site and an association site. An example is one in which the 3'end side of the strand DNA is bound. These can be used, for example, in the measurement principles 2, 3 and 4 described later.

The association site and the single-stranded DNA may be directly bound or may be indirectly bound via a spacer or the like. The binding method is not particularly limited. For example, as described in Non-Patent Document 3, the binding site may be bound to a single-stranded DNA by phosphoramidizing the association site and incorporating it into DNA solid phase synthesis. A reactive functional group such as an amino group or a thiol group may be introduced into the single-stranded DNA chain by solid-phase synthesis to react with the association site. The type of spacer is not particularly limited, but for example, a polyoxyethylene (PEG) chain may be used as the spacer, or a DNA chain may be used as the spacer. The DNA strand used as this spacer may be single-stranded or double-stranded. In the case of a double strand, a single strand DNA having a double-strand forming sequence may be bound to the DNA strand on the side bound to the association site, or conversely, a DNA strand bound to the association site Single-stranded DNA having a double-strand forming sequence may be bound to the complementary strand side.

(3) Association site The association site is included in the probe A and the probe B, and is not particularly limited as long as it can associate with the measurement target. For example, as an association site, a small molecule (cyclodextrin, crown ether, boronic acid, etc.) used as a host molecule, a protein (lectin, antibody, etc.) that has an affinity for the measurement target, or a nucleic acid (aptamer, etc.) that has an affinity ), avidin or biotin may be used. The association sites of the probe A and the probe B may be the same or different as long as both the probe A and the probe B can be in a state of being associated with the measurement target. The association sites of the probe A and the probe B may be associated with the measurement target first, or may be associated at the same time.

(4) Double Strand Forming Sequence The double strand forming sequences in the present invention are present in the single-stranded DNAs of probe A and probe B respectively, but the positions in the respective single-stranded DNAs are not particularly limited. It is not something that will be done. They have mutually complementary sequences, and the DNA chain length thereof is not particularly limited, but is preferably 3 to 10 bases, more preferably 4 to 6 bases.

(5) Promoter Sequence In the present invention, the promoter sequence is contained in the single-stranded DNA of probe A or probe B, but its position is not particularly limited. The sequence is not particularly limited as long as it is a promoter capable of performing transcription into RNA in the presence of RNA polymerase. Specific examples include T7 promoter, SP6 promoter, T3 promoter and the like. In particular, it is preferable to use the T7 promoter in the present invention.

(6) Amplified Sequence The amplified sequence in the present invention is contained in the single-stranded DNA of probe A or probe B, but its position is not particularly limited. The sequence is not particularly limited as long as it is an antisense strand or a sense strand serving as a template for RNA synthesis.

(7) Detection Detection of a signal in the present invention may be performed by detecting an RNA chain produced and amplified by RNA polymerase, and is not particularly limited. For example, for detection of RNA, a detection system such as a molecular beacon or an INAF probe that binds sequence-specifically and changes fluorescence may be used, or an intercalating fluorescent dye such as SYBRgreen may be used. Alternatively, a signal may be detected by designing a sequence so that the synthesized RNA sequence has RNAzyme activity and measuring the enzyme activity. Alternatively, visual detection may be performed using a nucleic acid chromatography method.

When a detection device is required, a real-time PCR device may be used. Further, the TRCR real-time monitor (TRCR Rapid-160 manufactured by Tosoh Corporation) used in this example may be used.

(8) Enzyme In the measuring method of the present invention, an enzyme having a DNA polymerase activity and an enzyme having an RNA polymerase activity are used. At that time, an enzyme having both DNA polymerase activity and RNA polymerase activity may be used, or different enzymes having respective polymerase activities may be used. For example, phi29, Bst, Csa, 96-7 or the like may be used as the DNA polymerase, and T7, SP6, T3 or the like may be used as the RNA polymerase. Particularly in the present invention, it is preferable to use 96-7 as the DNA polymerase and T7 as the RNA polymerase.

(9) Measurement principle 1
An example of the measurement principle of the present invention is shown in a schematic diagram (FIG. 1). The probe A has an association site bound to the 5'end of the single-stranded DNA and contains a double-strand forming sequence on the 3'side of the single-stranded DNA, and the probe B associates with the 3'end of the single-stranded DNA. The case where the sites are bound and includes a double-strand forming sequence, a promoter sequence, and an amplification sequence toward the 5'side of the single-stranded DNA will be described as an example, but the configuration of each probe and the sequence order are not limited to this. Not something.
(9-1) The association site of the probe A and the probe B is associated with the substance to be measured.
(9-2) As a result, the double-strand forming sequences in the probe A and the probe B are arranged at close positions, and a DNA double strand is formed.
(9-3) With this as a starting point, the DNA is extended by the DNA polymerase, and as a result, a double strand of the promoter sequence and a double strand of the amplification sequence are formed.
(9-4) Since the double strand of the promoter sequence is formed, RNA polymerase is repeatedly synthesized and amplified by RNA polymerase using the antisense strand of the amplified sequence downstream of the promoter sequence as a template.
(9-5) The amplified RNA is detected with a molecular beacon or the like.

(10) Measurement principle 2
An example of the measurement principle of the present invention is shown in a schematic diagram (FIG. 2). The probe A has an association site bound to the 5'end of the single-stranded DNA and contains a promoter sequence and a double-stranded sequence toward the 3'side of the single-stranded DNA. The probe B uses a DNA chain having a single-stranded portion and a double-stranded portion as a spacer, has an association site bound to its 5′ end, and has a double-stranded portion on its 3′ side and a single-stranded DNA linked thereto ( A double-strand forming sequence and an amplified sequence). Hereinafter, such a case will be described as an example, but the configuration of each probe and the order of arrangement are not limited to this.
(10-1) The association site of probe A and probe B is associated with the substance to be measured.
(10-2) As a result, the double-strand forming sequences in the probe A and the probe B are arranged at close positions, and a DNA double strand is formed.
(10-3) With this as a starting point, the DNA is extended by the DNA polymerase, and as a result, a double strand of the promoter sequence and a double strand of the amplification sequence are formed.
(10-4) Since the double strand of the promoter sequence is formed, RNA polymerase is repeatedly synthesized and amplified by RNA polymerase using the antisense strand of the amplification sequence downstream of the promoter sequence as a template.
(10-5) The amplified RNA is detected with a molecular beacon or the like.

(11) Measurement principle 3
An example of the measurement principle of the present invention is shown in a schematic diagram (FIG. 5). The probe A has an association site bound to the 5'end of the single-stranded DNA and contains a promoter sequence and a double-stranded sequence toward the 3'side of the single-stranded DNA, and the probe B is 5'of the single-stranded DNA. An example will be described in which the association site is bound to the end of the side and the amplified sequence and the double-strand forming sequence are included toward the 3'side of the single-stranded DNA. It is not limited to.
(11-1) The association site of probe A and probe B is associated with the substance to be measured.
(11-2) As a result, the double-strand forming sequences in the probe A and the probe B are arranged at close positions, and a DNA double strand is formed.
(11-3) With this as a starting point, DNA elongation is performed by a DNA polymerase, and as a result, a double strand of a promoter sequence and a double strand of an amplification sequence are formed.
(11-4) Since the double strand of the promoter sequence is formed, RNA polymerase is repeatedly synthesized and amplified by RNA polymerase using the antisense strand of the amplified sequence downstream of the promoter sequence as a template.
(11-5) The amplified RNA is detected with a molecular beacon or the like.

(12) Measurement principle 4
An example of the measurement principle of the present invention is shown in a schematic diagram (FIG. 8). Probe A having an association site at the 3'end of DNA and probe B having an association site at the 3'end of DNA are used. The DNA in probe A is composed of a DNA sequence No. 1 (having an association site at the 3'end) and a DNA sequence No. 2 having a partially complementary sequence, and the 5'end of DNA sequence No. 1 has the DNA sequence No. It is a sequence complementary to the 5'end of 2, and the DNA sequence 2 further contains a promoter sequence and a duplex forming sequence toward the 3'end. On the other hand, the DNA in the probe B is composed of a DNA sequence No. 3 (having an association site at the 3'end) and a DNA sequence No. 4 having a partially complementary sequence, and the 5'end of the DNA sequence No. 3 is DNA. Sequence 4 is a sequence complementary to the 5'end of the sequence 4, and the DNA sequence 4 further includes an amplification sequence and a duplex forming sequence toward the 3'end.

Hereinafter, such a case will be described as an example, but the configuration of each probe and the order of arrangement are not limited to this.
(12-1) The association site between probe A and probe B is associated with the substance to be measured.
(12-2) As a result, the double-strand forming sequences in the probe A and the probe B are arranged at close positions, and a DNA double strand is formed.
(12-3) With this as a starting point, DNA polymerase performs elongation of DNA, for example, strand displacement reaction, and as a result, a double strand of a promoter sequence and a double strand of an amplification sequence are formed.
(12-4) Since the double strand of the promoter sequence is formed, RNA polymerase is repeatedly synthesized and amplified by RNA polymerase using the antisense strand of the amplified sequence downstream of the promoter sequence as a template.
(12-5) The amplified RNA is detected with an INAF probe or the like.

(13) Measurement principle 5
An example of the measurement principle of the present invention is shown in a schematic diagram (FIG. 12). The probe A has an association site bound to the 5'end of the single-stranded DNA and contains a promoter sequence and a double-stranded sequence toward the 3'side of the single-stranded DNA, and the probe B is 5'of the single-stranded DNA. The association site binds to the'side end, and includes an amplification sequence and a duplex formation sequence toward the 3'side of the single-stranded DNA. Also, in the reaction system, a first primer (Promoter Primer) having a promoter sequence and a sequence homologous to a part of the amplified RNA, and a second primer having a sequence complementary to a part of the amplified RNA. In the following, the case of including the primer (RT Primer), the reverse transcriptase, and the enzyme having RNase H activity will be described as an example, but the configuration of each probe and the order of the sequences are not limited thereto.

(13-1) The association site of probe A and probe B is associated with the measurement target substance.
(13-2) As a result, the double-strand forming sequences in the probe A and the probe B are arranged at the adjacent positions, and a DNA double strand is formed.
(13-3) With this as a starting point, the DNA is extended by the DNA polymerase, and as a result, a double strand of the promoter sequence and a double strand of the amplification sequence are formed.
(13-4) Since the double strand of the promoter sequence is formed, RNA polymerase is repeatedly synthesized and amplified by RNA polymerase using the antisense strand of the amplification sequence downstream of the promoter sequence as a template.
(13-5) Detect the amplified RNA with a molecular beacon. In addition, starting from the double strand formation of the amplified RNA and the second primer, a DNA strand complementary to the RNA sequence is synthesized by reverse transcriptase activity.
(13-6) The RNA chain of the DNA-RNA hybrid double strand is degraded by the RNaseH activity, and the single strand DNA strand and the first primer form a double strand.
(13-7) The promoter double strand of the first primer is formed by the DNA polymerase activity, whereby RNA is repeatedly synthesized and amplified using the antisense strand of the amplification sequence downstream of the promoter sequence as a template.
The detection of the (13-8) RNA chain and the RNA amplification reaction of (13-5 to 7) using the amplified RNA as a template are repeatedly performed, and the RNA amount is amplified exponentially.

Even in the measurement performed in the homogeneous system, the target sensitivity may not be reached because the signal cannot be amplified. In the measuring method of the present invention, RNA synthesized by RNA polymerase is measured, and therefore it is possible to amplify and detect a signal. Therefore, highly sensitive measurement is possible.

It is a figure which shows the outline of the measuring method of this invention. It is a figure which shows the outline of the measuring method of this invention. FIG. 3 is a diagram showing a change in fluorescence intensity per unit time when streptavidin was detected in Example 1. It is an enlarged view of the area|region where the fluorescence intensity of FIG. 3 is small. FIG. 6 is a diagram showing an outline of a measurement method of Example 2. FIG. 5 is a diagram showing a change in fluorescence intensity per unit time when streptavidin was detected in Example 2. It is an enlarged view of the area|region where the fluorescence intensity of FIG. 6 is small. FIG. 6 is a diagram showing an outline of a measuring method of Example 3. FIG. 5 is a diagram showing changes in fluorescence intensity per unit time when streptavidin was detected in Example 3. 7 is a diagram showing an outline of a measuring method of Example 4. FIG. FIG. 5 is a diagram showing changes in fluorescence intensity per unit time when BNP was detected in Example 4. It is a figure which shows the outline of the measuring method of Examples 5 and 6. FIG. 8 is a diagram showing changes in fluorescence intensity per unit time when BNP was detected in Example 5. FIG. 6 is a diagram showing a change in fluorescence intensity per unit time when streptavidin was detected in Example 6. FIG. 8 is a diagram showing changes in fluorescence intensity per unit time when detecting MACS streptavidin microbeads in Example 7.

Hereinafter, the present invention will be specifically described with reference to Examples using biotin as an association site, but the present invention is not limited thereto.

Example 1
(1) Preparation of probe Probe A in which 5'end of single-stranded DNA (SEQ ID NO: 1) was modified with biotin (association site), and 3'end of single-stranded DNA (SEQ ID NO: 2) was modified in biotin (association site) Each of the probes B was produced by contract synthesis by Greiner. The DNA sequence in probe A is designed to include a double-stranded sequence (positions 5 to 10 of SEQ ID NO: 1), and the DNA sequence in probe B is a double-stranded sequence (positions 47 to 52 of SEQ ID NO: 2), It was designed to include a promoter sequence (positions 30 to 49 of SEQ ID NO: 2) and an amplified sequence (positions 4 to 29 of SEQ ID NO: 2).

(2) Preparation of Molecular Beacon A molecular beacon in which the 5'end of the DNA sequence of SEQ ID NO: 3 was modified with FAM and the 3'end was modified with DarkQuencher was produced by requesting Nippon Gene from the sequence of Non-Patent Document 2.

(3) Measurement of Streptavidin Streptavidin, which is known to associate with biotin, was measured using probe A, probe B, and molecular beacon. Details of the enzyme concentration and substrate concentration in the measurement solution are shown below.

T7 RNA polymerase (manufactured by Takara Bio) 144 U/assay, 96-7 DNA polymerase (manufactured by Nippon Gene) 8 U/assay, T7 RNA polymerase Buffer (manufactured by Takara Bio), DTT 5 mM, NTP 3 mM, dNTP 0.25 mM, RNase Inhibitor 0.2 U/μL, DMSO 20%, probe A 50 nM, probe B 50 nM, molecular beacon 20 nM. Reaction temperature 42°C.

Streptavidin at each concentration shown in FIG. 3 was added to the above-mentioned measurement solution, and the fluorescence intensity derived from the molecular beacon was measured with the use of TRC Rapid-160 (manufactured by Tosoh Corporation) at each elapsed time. The results are shown in FIGS. FIG. 4 shows an enlarged view of the region of FIG. 3 where the fluorescence intensity is low. As a result, it was confirmed that the amplification of the fluorescence intensity per time increased depending on the streptavidin concentration. It is considered that this is because the amount of the double strand of the promoter sequence formed by the association of streptavidin and biotin in each probe was increased.

From the above results, it was confirmed that the measurement object can be measured by the method of the present invention.

Example 2 5′-end labeled probe A schematic diagram of the measurement principle in Example 2 is shown in FIG.

(1) Preparation of probe Probe A in which 5'end of single-stranded DNA (SEQ ID NO: 4) was modified with biotin (association site), and 5'end of single-stranded DNA (SEQ ID NO: 5) was modified in biotin (association site) Each of the probes B was produced by contract synthesis by Greiner. The DNA sequence in probe A is designed to include a promoter sequence (positions 7 to 34 of SEQ ID NO: 4) and a duplex forming sequence (positions 59 to 64 of SEQ ID NO: 4), and the DNA sequence in probe B is an amplified sequence ( It was designed so as to include a duplex forming sequence (positions 28 to 47 of SEQ ID NO: 5) (positions 48 to 53 of SEQ ID NO: 5).

(2) Preparation of Molecular Beacon The molecular beacon in which the 5'end of the DNA sequence of SEQ ID NO: 6 was modified with FAM and the 3'end was modified with BHQ1 was prepared by requesting Greiner.

(3) Measurement of Streptavidin Streptavidin, which is known to associate with biotin, was measured using probe A, probe B, and molecular beacon. Details of the enzyme concentration and substrate concentration in the measurement solution are shown below.

T7 RNA polymerase (manufactured by Takara Bio) 150 U/assay, 96-7 DNA polymerase (manufactured by Nippon Gene) 8 U/assay, T7 RNA polymerase Buffer (manufactured by Takara Bio), DTT 5 mM, NTP 3 mM, dNTP 0.25 mM, RNase Inhibitor 0.2 U/μL, DMSO 15%, probe A 250 nM, probe B 250 nM, molecular beacon 300 nM. Reaction temperature 42°C.

Streptavidin at each concentration shown in FIGS. 6 and 7 was added to the above-mentioned measurement solution, and the fluorescence intensity derived from the molecular beacon was measured with the use of TRC Rapid-160 (manufactured by Tosoh Corporation) every time. The results are shown in FIGS. FIG. 7 shows an enlarged view of the region of FIG. 6 where the fluorescence intensity is low. As a result, it was confirmed that the amplification of the fluorescence intensity per time increased depending on the streptavidin concentration. It is considered that this is because the amount of the double strand of the promoter sequence formed by the association of streptavidin and biotin in each probe was increased.

From the above results, it was confirmed that the measurement target can be measured by the method of the present invention using two probes (probe A and probe B) having an association site at the DNA 5'end.

Example 3 3′-end labeled probe A schematic diagram of the measurement principle in Example 3 is shown in FIG.

(1) Preparation of Probe A probe A having biotin (association site) modified at the 3′ end of DNA and a probe B having biotin (association site) modified at the 3′ end of DNA were prepared by contract synthesis by Greiner. The DNA in the probe A is composed of the DNA of SEQ ID NO:7 (3′ end is biotin-modified) and the DNA of SEQ ID NO:8, each of which has a sequence complementary to a part thereof. DNA in probe B, which is a sequence complementary to positions -20, designed to include a promoter sequence (positions 21 to 43 of SEQ ID NO: 8) and a duplex forming sequence (positions 67 to 72 of SEQ ID NO: 8). Consists of the DNA of SEQ ID NO: 9 (biotin-modified at the 3′ end) and the DNA of SEQ ID NO: 10 each having a complementary sequence, and positions 1 to 21 of SEQ ID NO: 9 are positions 1 to 21 of SEQ ID NO: It is a complementary sequence, and the DNA sequence was designed to include an amplification sequence (positions 42 to 61 of SEQ ID NO: 10) and a duplex forming sequence (positions 62 to 67 of SEQ ID NO: 10).

(2) INAF probe As the INAF probe of SEQ ID NO: 11, the probe described in JP-A-2006-340664 was used.

(3) Measurement of streptavidin Streptavidin, which is known to associate with biotin, was measured using probe A, probe B, and INAF probe. Details of the enzyme concentration and substrate concentration in the measurement solution are shown below.

T7 RNA polymerase (manufactured by Takara Bio) 144 U/assay, 96-7 DNA polymerase (manufactured by Nippon Gene) 8 U/assay, T7 RNA polymerase Buffer (manufactured by Takara Bio), DTT 5 mM, NTP 3 mM, dNTP 0.25 mM, RNase Inhibitor 0.2 U/μL, DMSO 20%, probe A 200 nM, probe B 200 nM, INAF probe 50 nM. Reaction temperature 42°C.

Streptavidin at each concentration shown in FIG. 9 was added to the above-mentioned measurement solution, and the fluorescence intensity derived from the INAF probe was measured with the use of TRC Rapid-160 (manufactured by Tosoh Corporation) every time. The results are shown in Fig. 9. As a result, it was confirmed that the amplification of the fluorescence intensity per time increased depending on the streptavidin concentration. It is considered that this is because the amount of the double strand of the promoter sequence formed by the association of streptavidin and biotin in each probe was increased.

From the above results, it was confirmed that the measurement target can be measured by the method of the present invention using two probes (probe A and probe B) having an association site at the DNA 3'end.

Example 4 Assay System Using BNP (Brain Native Peptide)-Recognizing Antibody at Association Site A schematic diagram of the assay principle in Example 4 is shown in FIG.

(1) BNP-recognizing antibody As the C-terminal recognizing antibody of BNP, the BC23-11 antibody described in Japanese Patent No. 5810514 was used. As the antibody for recognizing the cyclic portion of BNP, the BM33-28 antibody described in JP2012-140331A was used.

(2) Preparation of probe Probe A in which BC23-11 (association site) was modified at the 5'end of single-stranded DNA (SEQ ID NO: 12), and BM33-28 (at the 5'end of single-stranded DNA (SEQ ID NO: 13)) The probe B modified at the association site) was prepared by the method described below.

After reacting 3 nmol of each antibody with 500 nmol of SM(PEG)24 (manufactured by Thermo Fisher Scientific) at room temperature for 1 hour in PBS, ultrafiltration is carried out with Amicon Ultra 0.5 mL (30 K) (manufactured by Merck Millipore). Thus, unreacted SM(PEG)24 was removed. DNAs having thiol-modified 5'ends (SEQ ID NO: 12 and SEQ ID NO: 13, custom-made by Greiner) were added thereto and reacted at room temperature for 30 minutes, followed by gel filtration purification (G3000swxl column, manufactured by Tosoh Corporation). Then, a probe A and a probe B were obtained.

The DNA sequence in probe A is designed to include a promoter sequence (positions 7 to 34 of SEQ ID NO: 12) and a duplex forming sequence (positions 59 to 64 of SEQ ID NO: 12), and the DNA sequence in probe B is an amplified sequence ( It was designed so as to include a duplex forming sequence (positions 28 to 47 of SEQ ID NO: 13) (positions 48 to 53 of SEQ ID NO: 13).

(3) Molecular beacon The same molecular beacon as SEQ ID NO: 6 described in Example 2 was used.

(4) Measurement of BNP BNP was measured using probe A, probe B, and molecular beacon. Details of the enzyme concentration and substrate concentration in the measurement solution are shown below.

T7 RNA polymerase (manufactured by Takara Bio) 144 U/assay, 96-7 DNA polymerase (manufactured by Nippon Gene) 8 U/assay, T7 RNA polymerase Buffer (manufactured by Takara Bio), DTT 5 mM, NTP 3 mM, dNTP 0.25 mM, RNase Inhibitor 0.2 U/μL, Probe A 200 nM, probe B 200 nM, molecular beacon 300 nM. Reaction temperature 42°C.

BNP of each concentration shown in FIG. 11 was added to the above-mentioned measurement solution, and the fluorescence intensity derived from the molecular beacon was measured with the use of TRC Rapid-160 (manufactured by Tosoh Corporation) at each time passage. The results are shown in Fig. 11. As a result, it was confirmed that the amplification of the fluorescence intensity per unit time increased depending on the BNP concentration. It is considered that this is because the amount of the double strand of the promoter sequence formed by the association of BNP and the antibody in each probe was increased.

From the above results, it was confirmed that the measurement target can be measured by the method of the present invention using two probes (probe A and probe B) using an antibody as an association site.

Example 5 Measurement system in which the amount of RNA is amplified exponentially (measurement system using a BNP recognition antibody at the association site)
A schematic view of the measurement principle in Example 5 is shown in FIG.
(1) Preparation of probe Probe A in which BC23-11 (association site) was modified at the 5'end of single-stranded DNA (SEQ ID NO: 12), and BM33-28 (at the 5'end of single-stranded DNA (SEQ ID NO: 13)) The probe B having a modified association site was the same as the probe used in Example 4.
(2) Molecular beacon The same molecular beacon as in Example 2 was used.
(3) Primer The first primer (SEQ ID NO: 14) and the second primer (SEQ ID NO: 15) having a promoter sequence on the 5′-terminal side were prepared by contract synthesis by Greiner. The DNA sequence in the first primer was designed to include a promoter sequence (1-28 of SEQ ID NO: 14).

(4) Measurement of BNP BNP was measured using the probe A, the probe B, the molecular beacon, the first primer, and the second primer. Details of the enzyme concentration and substrate concentration in the measurement solution are shown below.

18 mM MgCl ₂ , 60 mM Tris-HCl, 1 mM DTT, 0.25 mM dNTP, 3 mM NTP, 3.06 mM ITP, 0.12 mg/mL BSA, 2% sorbitol, 40 mM KCl, 0.2 U/μL RNase Inhibitor, 8 U/30 μL 96-7 DNA polymerase, 144 U/30 μL T7 RNA polymerase, 7.4 U/30 μL AMV (reverse transcriptase and RNase H activity), probe A 200 nM, probe B 200 nM, first primer 1 μM, second primer 1 μM. Reaction temperature 42°C.

BNP of each concentration shown in FIG. 13 was added to the above-mentioned measurement solution, and the fluorescence intensity derived from the molecular beacon was measured with the use of TRC Rapid-160 (manufactured by Tosoh Corporation) every time. The results are shown in Fig. 13. As a result, it was confirmed that the fluorescence intensity was amplified according to the BNP concentration, and it was confirmed that the amplification curve drew a sigmoid curve. It is considered that this is because the amplified RNA was used as a template to form a new RNA synthesis promoter duplex with the first primer and the second primer, and the amount of RNA was exponentially amplified.

From the above results, it was confirmed that the measurement target can be measured by the method of the present invention using the measurement system in which the RNA amount is amplified exponentially.

Example 6 Measurement system in which RNA amount is amplified exponentially (measurement system using biotin at the association site)
A schematic view of the measurement principle in Example 6 is shown in FIG.
(1) Preparation of probe Probe A in which 5'end of single-stranded DNA (SEQ ID NO: 4) was modified with biotin (association site), and 5'end of single-stranded DNA (SEQ ID NO: 5) was modified in biotin (association site) As the probe B, the same probe as that used in Example 2 was used.
(2) Molecular beacon The same molecular beacon as in Example 2 was used.
(3) Primer As the first primer (SEQ ID NO: 14) and the second primer (SEQ ID NO: 15) having a promoter sequence on the 5′-terminal side, the same ones as used in Example 5 were used.

(4) Measurement of Streptavidin Streptavidin was measured using probe A, probe B, molecular beacon, first primer, and second primer. Details of the enzyme concentration and substrate concentration in the measurement solution are shown below.

18 mM MgCl ₂ , 60 mM Tris-HCl, 1 mM DTT, 0.25 mM dNTP, 3 mM NTP, 3.06 mM ITP, 0.12 mg/mL BSA, 2% sorbitol, 40 mM KCl, 0.2 U/μL RNase Inhibitor, 8 U/30 μL 96-7 DNA polymerase, 144 U/30 μL T7 RNA polymerase, 7.4 U/30 μL AMV (reverse transcriptase and RNase H activity), probe A 2 nM, probe B 2 nM, DMSO 13%, first primer 1 μM, second primer 1 μM. Reaction temperature 43°C.

Streptavidin at each concentration shown in FIG. 14 was added to the above-mentioned measurement solution, and the fluorescence intensity derived from the molecular beacon was measured with the use of TRC Rapid-160 (manufactured by Tosoh Corporation) every time. The results are shown in Fig. 14. As a result, it was confirmed that the fluorescence intensity was amplified according to the streptavidin concentration, and it was confirmed that the amplification curve drew a sigmoid curve. It is considered that this is because the amplified RNA was used as a template to form a new RNA synthesis promoter duplex with the first primer and the second primer, and the amount of RNA was exponentially amplified.

From the above results, it was confirmed that the measurement target can be measured by the method of the present invention using the measurement system in which the RNA amount is amplified exponentially.

Example 7: Measurement system when the measurement target is fine particles The measurement principle in Example 7 is the same as in FIG.
(1) Preparation of probe Probe A in which 5'end of single-stranded DNA (SEQ ID NO: 4) was modified with biotin (association site), and 5'end of single-stranded DNA (SEQ ID NO: 5) was modified in biotin (association site) As the probe B, the same probe as that used in Example 2 was used.
(2) Molecular beacon The same molecular beacon as in Example 2 was used.
(3) Fine Particles to be Measured As fine particles to be measured, MACS streptavidin microbeads (MACS, order No. 130-048-102) having streptavidin immobilized on the surface of fine particles of 50 nm were used.

(3) Measurement of Microparticles MACS streptavidin beads were measured using probe A, probe B, and molecular beacon. Details of the enzyme concentration and substrate concentration in the measurement solution are shown below.

T7 RNA polymerase (manufactured by Takara Bio) 144 U/assay, 96-7 DNA polymerase (manufactured by Nippon Gene) 8 U/assay, T7 RNA polymerase Buffer (manufactured by Takara Bio), DTT 5 mM, NTP 3 mM, dNTP 0.25 mM, RNase Inhibitor 0.2 U/μL, Probe A 200 nM, probe B 200 nM, molecular beacon 300 nM. Reaction temperature 43°C.

Fine particles having a dilution ratio shown in FIG. 15 were added to the measurement solution, and the fluorescence intensity derived from a molecular beacon was measured with the use of TRC Rapid-160 (manufactured by Tosoh Corporation) every time. The results are shown in Fig. 15. As a result, it was confirmed that the amplification of the fluorescence intensity per unit time increased depending on the concentration of the fine particles. It is considered that this is because the amount of the double strand of the promoter sequence formed by the association of biotin in each probe with streptavidin on the surface of the microparticles increased.

From the above results, it was confirmed that the particles to be measured can be measured by the method of the present invention using two probes (probe A and probe B) having an association site at the DNA 5'end.

Claims (6)

Probe A having an association site and single-stranded DNA, and probe B having an association site and single-stranded DNA,
A method of measuring an object to be measured in a homogeneous system using
(I) the double-stranded sequence is contained in the single-stranded DNAs of probe A and probe B,
The promoter sequence and the amplified sequence are contained in the single-stranded DNA of probe A or probe B, respectively,
(Ii) associate the association site of probe A and the association site of probe B with the measurement target,
(Iii) hybridizing the double-stranded sequence of probe A and the double-stranded sequence of probe B to form a double strand of DNA,
(Iv) A double strand of the promoter sequence is formed by extending the DNA with DNA polymerase using it as a starting point,
(V) RNA is amplified by RNA polymerase using the antisense strand of the amplified sequence downstream of the promoter sequence as a template,
(Vi) detecting amplified RNA,
An assay method in which RNA amplification is performed under constant temperature conditions . In probe A and probe B, one probe has an association site bound to the 5'end side of single-stranded DNA, and the other probe has an association site bound to the 3'end side of single-stranded DNA. The method of claim 1, wherein The method according to claim 1, wherein in probe A and probe B, the association site of both probes is bound to the 5'end side of the single-stranded DNA. The method according to claim 1, wherein in probe A and probe B, the association site of both probes is bound to the 3'end side of the single-stranded DNA. The association site and single-stranded DNA probe A and / or the probe B is attached via a spacer, the method according to any one of claims 1-4. The method according to claim 5 , wherein the spacer is a DNA strand.

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