JP2003052396A - Method for detecting mutation in test nucleic acid using mismatch-recognition protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for simply and rapidly detecting a mutation in a test nucleic acid. SOLUTION: This method for detecting the mutation in the test nucleic acid comprises preliminarily labelling at least one selected from the group consisting of the test nucleic acid, a control nucleic acid, and a mismatch-recognition protein with a labelling substance, (1) mixing the test nucleic acid with the control nucleic acid to hybridize the test nucleic acid with the control nucleic acid, (2) reacting the hybridization product obtained in (1) with the mismatch protein, (3) at least measuring the labelled substance as the reaction result with the mismatch-recognition protein described in (2), (4) carrying out such an analysis as at least judging whether the labelled substance is liberated or not, and (5) detecting the mutation in the test nucleic acid on the basis of the analysis result of (4).

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a mutation in a test nucleic acid.

[0002]

2. Description of the Related Art For detecting polymorphisms and mutations in nucleobases, for example, a control nucleic acid having no mutation is hybridized with a test nucleic acid suspected of having a mutation, and a mismatch recognition protein is reacted therewith. There is a method of detecting a mismatch by using the above method.

As the mismatch recognition protein (generally referred to as a mismatch binding protein), MutS protein (described in Japanese Patent Publication No. 9-504699) and Mut are mentioned.
M protein (described in Japanese Patent Laid-Open No. 2000-300265), MutS protein to which GFP (Green Fluorescence Protein) is bound (International Application No. PCT / JP98 /
The use of (described in 03413) has been reported.

As an example of means for detecting a mismatch recognition protein bound to a mismatch between a control nucleic acid and a test nucleic acid, any nucleic acid or mismatch recognition protein is labeled with a labeling substance and contained in a binding product generated by the reaction. A method for separating a labeled substance to be bound to a support by bound / free separation and detecting the labeled substance (described in JP 2000-300265 A), for a mismatch recognition protein immobilized on the support. By reacting a double-stranded nucleic acid containing a mismatch by detecting a labeling substance contained in any of them (described in Japanese Patent Publication No. 9-50469) and immobilized on a support. A method for binding a labeled mismatch recognition protein to a double-stranded nucleic acid containing the identified mismatch and detecting it (International Application No. PCT / JP98 / 03413), and a mismatch recognition protein labeled to a double-stranded nucleic acid containing a mismatch, which is immobilized on a substrate and observed using an atomic force microscope. Method to detect mismatches (Tanigawa, M., et al,
Nucleic Aced Res. 28, E38 (2000)) has been reported.

In these conventional methods, in order to detect a mismatch using a mismatch recognition protein, a complicated operation such as fixing to a support or a substrate and bound / free separation is required. In addition, when observing with an atomic force microscope, it is necessary to prepare a complicated sample that requires specialized techniques, and at the same time, use multiple mismatched proteins to be observed and detect them while distinguishing them. It is difficult.

[0006]

In view of such circumstances, an object of the present invention is to provide a method for simply and quickly detecting a mutation in a test nucleic acid.

[0007]

As a result of earnest research, the inventors have found means for achieving the above object. That is, a method of detecting a mutation in a test nucleic acid, wherein at least one selected from the group consisting of a test nucleic acid, a control nucleic acid and a mismatch recognition protein is labeled with a labeling substance in advance, and (1) Mixing a test nucleic acid and a control nucleic acid and performing hybridization; (2)
Reacting the mismatching protein with the hybridization product obtained in (1), (3) measuring a labeling substance as a reaction result of the mismatching protein according to at least (2), (4) labeling substance of (3) An analysis to at least determine whether or not the labeled substance is free based on the measurement data of (5)
A method for detecting a mutation in a test nucleic acid, comprising detecting a mutation in the test nucleic acid based on the analysis result of (4).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION 1. Overview According to the present invention, a method for detecting a mutation in a test nucleic acid using a mismatch recognition protein is provided.

[0009] The mismatch recognition protein is generally called a mismatch binding protein and is a protein having the following functions. Most of the erroneous base pairing (ie, mismatches) that occur during the replication of DNA in organisms are calibrated by the proofreading function of the replicator, and in addition, the organism is a system for removing the slight mismatches that remain after calibration. have. That is, it is the mismatch recognition protein that functions in mismatch repair as a system for removing such slight mismatches.
The mismatch recognition protein that can be used in the present invention may be any mismatch recognition protein known per se capable of recognizing a mismatch in a double-stranded nucleic acid and binding to the mismatch site.

In an embodiment of the present invention, hybridization is performed between a test nucleic acid and a control nucleic acid, and the above-mentioned mismatch generated in the double strand composed of the hybridized test nucleic acid and the control nucleic acid is treated as described above. Such a mismatch recognition protein is reacted. At this time, the test nucleic acid,
At least one selected from the control nucleic acid and the mismatch recognition protein is labeled with an optically detectable labeling substance in advance. Therefore, by detecting a detectable optical signal from the labeling substance, the positional change of the labeling substance at a plurality of elapsed time points can be measured and analyzed to detect a mutation in the test nucleic acid. It will be possible.

2. The term "test nucleic acid" as used herein refers to a nucleic acid whose presence or absence of a polymorphism or mutation is to be detected. The term "mutation" as used herein refers to various polymorphisms and mutations.

As used herein, the term "control nucleic acid" refers to a standard base sequence, for example, a standard genotype, to a wild type (ie w
ild type; also referred to as a normal type). The base sequence of the control nucleic acid is complementary to the base sequence of the test nucleic acid excluding the sequence of the mutation site.

“Nucleic acid” including test nucleic acid and control nucleic acid
May be a nucleic acid derived from a natural product or an artificially synthesized nucleic acid. Here, the term "nucleic acid" may be a polynucleotide containing any simple and / or modified nucleotides. These nucleic acids are typically D, such as cDNA, genomic DNA, and synthetic DNA.
NA and RNA such as mRNA, total RNA, hnRNA, and synthetic RNA. Further, in the present invention, the term “polynucleotide” refers to polynucleotides and oligonucleotides, and artificially synthesized nucleic acids such as peptide nucleic acid, morpholino nucleic acid, methylphosphonate nucleic acid, and S-oligonucleic acid for convenience. The test nucleic acid and the control nucleic acid can be freely selected by the tester. Furthermore, these nucleic acids may be mixed when performing detection.

When the test nucleic acid is extracted from a subject which is an animal including human, the nucleic acid may be extracted from a desired tissue, organ or cell sample from the subject by a method known per se, After extraction, if necessary, conditions such as size of nucleic acid fragment and purification purity may be adjusted to an appropriate state, and then amplification reaction by general polymerase chain reaction (ie, PCR) is performed. You may make it amplify.

The test nucleic acid and the control nucleic acid may be single-stranded or double-stranded. In the case of a single strand, hybridization may be carried out under suitable conditions after mixing. In the case of a double strand, after mixing, denaturation may be carried out by a means known per se such as heating or a chemical treatment such as alkali treatment to form a single strand and then hybridization.

As described above, the mismatch recognition protein that can be used in accordance with the present invention may be any of those known to those skilled in the art. For example, such mismatch recognition proteins include MutM, MutS, and analogs thereof, which have already been disclosed in the following documents; Radman, Me.
t al., Annu. Rev. Genet. 20: 523-538 (1986); Radaman, M.et
al., Sci.Amer., August 1988, pp40-46; Modrich, P., J.Bi
ol.Chem.264: 6597-6600 (1989); Lahue, RS et al., S
cience 245: 160-164 (1988); Jiricny, J. et al ,. Nucl. Aci
ds Res. 16: 7843-7853 (1988); Su, S Set al., J. Biol. Che
m.263; 6829-6835 (1988); Lahue, R Set al., Mutat.Res.1
98: 37-43 (1988); Dohet, C. et al., Mol. Gen. Gent. 206: 181.
-184 (1987); Jones, M. et al., Gentics 115: 605-610 (19
87); Muts of Salmonella typhimurium (Lu, AL, G
enetics 118: 593-600 (1988); Haber L.T. et al., J. Bacte
riol. 170: 197-202 (1988); Pang, PP et al., J. Bacterio.
l.163: 1007-1015 (1985)); and Priebe SD et al., J.
Bacterilo. 170: 190-196 (1988).

The optically detectable labeling substance used in the embodiment of the present invention includes a fluorescent substance and a luminescent substance which generate an optical signal such as fluorescence or luminescence by laser irradiation or energy of itself.

The optical signal detecting device used in the embodiment of the present invention may be any device capable of optically measuring the positional change of the labeling substance at a plurality of time points. For example, when a fluorescent substance is used as a labeling substance, fluorescence correlation spectroscopy (hereinafter abbreviated as FCS; Fluorescence Correlation Spectroscopy)
scopy), or fluorescence cross-correlation spectroscopy (FluorescenceC
It is preferable to use ross Correlation Spectroscopy).

3. Fluorescence Correlation Spectroscopy Fluorescence Correlation Spectroscopy (ie FC
S) is a measurement of the fluctuation motion of a fluorescently labeled target molecule in a medium, and the autocorrelation function
is a technique for accurately measuring the micromotion of individual target molecules by using (ion) (Reference: D. Magde and E.
Elson, “Fluorescence correlation spectroscopy. I
I. An experimental realization ”, Biopolymers 1974
13 (1) 29-61).

The FCS used according to the present invention analyzes the Brownian motion of fluorescent molecules in a solution in a minute region by a laser confocal microscope system, analyzes the diffusion time from the fluctuation of the fluorescence intensity, and analyzes the physical quantity (number of molecules). ,size)
Is carried out by measuring Analysis by FCS that captures molecular fluctuations in such a minute region has high sensitivity,
It is effective in specifically detecting intermolecular interactions.

The principle of FCS detection implemented in accordance with the present invention will be described in more detail. The FCS detects and analyzes the fluorescence signal generated from the microscopic field region in the sample. At this time, the fluorescently labeled target molecule in the medium is constantly moving (that is, Brownian motion). Therefore, the detected fluorescence intensity changes depending on the frequency with which the target molecule enters this microscopic visual field region and the time for which the target molecule remains in the microscopic visual field region. For example, if dimerization occurs and the apparent molecular weight increases, the movement of the target molecule slows down and the apparent number of molecules decreases.
As a result, the frequency of entering the microscopic field area decreases,
The observed fluorescence intensity changes. By monitoring such changes in fluorescence intensity, changes in the apparent molecular weight of the target molecule can be tracked.

4. Fluorescence Correlation Analyzer An example of a fluorescence correlation analyzer that can be used in the method of the present invention will be described below with reference to FIG. As shown in FIG. 1, the fluorescence correlation spectroscopy apparatus includes a laser light source 1, a light intensity adjusting means (an ND filter here) 2 for reducing the intensity of a light beam from the laser light source 1, and an appropriate light intensity adjustment. A light attenuation selection device (here, an ND filter changer) 3 for setting the means 2, and optical systems 4 and 5 for converging the light beam from the laser light source 1 on the sample to form a confocal region, and fluorescent molecules 6 with a sample containing
And optical systems 7 to 11 for collecting fluorescence from the sample,
It comprises a photodetector 12 for detecting the collected fluorescence and a fluorescence intensity recording means for recording the change in the fluorescence intensity. As described above, the fluorescence correlation analyzer is an application of the confocal laser microscope system. In FIG. 1, the laser light source (1)
May be an argon laser, a helium-neon laser, a krypton, a helium-cadmium, or the like.

In FIG. 1, the optical systems 4 and 5 for converging the light beam from the laser light source on the sample to form a confocal region specifically mean the dichroic mirror 4 and the objective lens 5. The light beam from the laser light source 1 is first attenuated according to the attenuation degree of the fluorescence intensity adjusting means (here, the ND filter) 2 along the path shown by the arrow in FIG. 1, and then the dichroic mirror 4 is used.
Refracts in the direction of the stage at 90 degrees to the incident light,
The sample on the stage 6 is irradiated through the objective lens 5. In this way, the light beam is focused on the sample at one minute point to form a confocal region.

In the present invention, the stage 6 sample is a solution containing a test nucleic acid, a control nucleic acid and a mismatch recognition protein. Here, if desired, at least one selected from the group consisting of a test nucleic acid, a control nucleic acid, and a mismatch recognition protein is fluorescently labeled.

In FIG. 1, the optical systems 7 to 11 for collecting the fluorescence emitted from the fluorescent molecules in the confocal region are, specifically, the filter 7, the tube lens 8 and the reflecting mirror 9.
After being refracted by the lens and focused on the pinhole 10, the lens 1
The light passes through 1 and is focused on the photodetector 12.

A photodetector (here, avalanche photodiode) 12 for detecting the collected fluorescence converts the received light signal into an electric signal and sends it to a fluorescence intensity recording means (here, computer) 13. To do.

The fluorescence intensity recording means 13 for recording the change in fluorescence intensity records and analyzes the transmitted fluorescence intensity data. Specifically, the autocorrelation function is set by analyzing the fluorescence intensity data. Increase in molecular weight and decrease in number of molecules due to movement of fluorescent molecules (eg, fluorescent-labeled test nucleic acid, fluorescent-labeled control nucleic acid and fluorescent-labeled mismatch recognition protein), or number of molecules due to binding of fluorescent molecule to specific DNA region Can be detected by a change in the autocorrelation function.

An apparatus for performing the above-mentioned FCS is also included in the scope of the present invention.

5. EXAMPLES Hereinafter, the embodiments of the present invention will be described with reference to examples. However, these examples are for illustrative purposes. Therefore, these examples should not be construed as limiting the invention.

Example (1) Method for labeling mismatch recognition protein The first aspect of the present invention will be described with reference to FIG.

First, the control nucleic acid 21 extracted from the sample
Is prepared in a large amount by an amplification reaction (FIG. 2 (A)). Similarly, a large amount of the test nucleic acid 22 extracted from the sample is prepared by an amplification reaction (FIG. 2 (B)).

After the control nucleic acid 21 and the test nucleic acid 22 are mixed, each is made into a single strand by heating or chemical treatment. Subsequently, the control nucleic acid 21 and the test nucleic acid 22 are cross-hybridized by cooling or returning to a solvent that forms a double strand again (FIG. 2).
(C)). As a result of cross-hybridization, if there is no difference in the base sequence between the control nucleic acid and the test nucleic acid, there is no mismatched hybridization product 23; if there is a difference in the base sequence between the control nucleic acid and the test nucleic acid, then there is a mismatched hybridization product 24 Occurs (FIG. 2 (C)).

While observing with FCS, the mismatch recognition protein 2 labeled with the fluorescent substance 26 in advance for the product generated by cross-hybridization
5 is added, and the reaction is carried out under the appropriate conditions described in the aforementioned patent publications and documents or other appropriate conditions known per se (FIG. 2 (D)). For example, such a reaction is conducted at about 25 to about 37 ° C. As a result, when there is a mismatch site, the mismatch recognition protein binds to the site, the movement of the fluorescent molecule observed by FCS is changed, and the mutation in the test nucleic acid is detected.

(2) Variation of Marking Another embodiment of the present invention will be described with reference to FIG. Detection can be performed in the same manner as in the method described in (1), except for the molecule serving as a label control and the reaction product.

3 (A) to 3 (D) show different modes. They respectively represent the molecules present in the reaction solution obtained after the hybridization and the binding reaction of the mismatch recognition protein, ie a, b and c.

In the embodiment shown in FIG. 3A, the control nucleic acid 32
On the other hand, the fluorescent substance 34 is used for labeling, and the test nucleic acid 31 and the mismatch recognition protein 33 are not labeled. In one detection system, a plurality of types of control nucleic acids 32 having different sequences are present, and a fluorescent substance having a different fluorescence wavelength is labeled for each type of the control nucleic acids 32, whereby mutations of the test nucleic acid related thereto are detected simultaneously. It is possible to Further, it is possible to simultaneously perform a plurality of items by using one kind of mismatch recognition protein.

In the embodiment shown in FIG. 3B, the test nucleic acid 31
However, the control nucleic acid 32 and the mismatch recognition protein 33 are not labeled with the fluorescent substance 34.

In the embodiment shown in FIG. 3C, the control nucleic acid 32
With the fluorescent substance 34, and with the fluorescent substance 35 having a fluorescence wavelength different from that of the fluorescent substance 34, the mismatch recognition protein 33 is also labeled.
No. 1 is not labeled.

In the embodiment shown in FIG. 3D, the test nucleic acid 31
Is labeled with a fluorescent substance 34, and the mismatch recognition protein 33 is also labeled with a fluorescent substance 35 having a fluorescence wavelength different from that of the fluorescent substance 34.
No. 2 is not labeled.

By simultaneously labeling a plurality of molecules as described above, it is possible to simultaneously analyze a plurality of items, for example, mutations at different loci and different genotypes. Further, analysis by fluorescence cross correlation spectroscopy is also possible.

Further, the products obtained by carrying out the main detection with labeling as described above are as shown in FIG. When no mismatch exists, a double strand of the test nucleic acid 31 and the control nucleic acid 32 is obtained as shown in a of FIG. 3 (A). If a mismatch exists, as shown in FIG. 3 (b), a complex in which a mismatch recognition protein is bound to a mismatch site where a double strand of the test nucleic acid 31 and the control nucleic acid 32 is obtained. Is obtained.

As a further application, if a plurality of control nucleic acids are used in one detection system and labeled with fluorescent substances having different fluorescence wavelengths for each type, a plurality of mutation sites can be detected simultaneously. It is possible.

When the measurement is carried out using fluorescence correlation spectroscopy or fluorescence cross-correlation spectroscopy, operations such as washing operation and fixing to a support are not required, and the solution in the container remains inviable. It is possible to handle the sample while maintaining the state, and the time required for the measurement is shorter than that of the conventional method.

(3) Automatic detection device Furthermore, the present invention provides each reaction sample in a state in which the sample is subjected to two or more stepwise reactions under the control of a computer for mutation in the test nucleic acid by the optical signal detection device. There is also provided an automatic detection device that automatically performs from the dispensing of reaction samples of various fluids into the reaction container 46 to the analysis of data obtained by continuous and optical observation.

The optical signal detection device used in the automatic detection device according to the embodiment of the present invention is a liquid phase detector in which any of the above-mentioned nucleic acid molecules to be measured is labeled based on the measurement data of the labeled substance. It is important that the measurement and analysis be performed so that it can at least determine whether it is free in the phase or hybridizes to form a complex. For such a measuring device, it is preferable to use kinematic measurement of the molecular weight of the labeling substance, and as an example, a plurality of devices, that is,
Any device can be used as long as it can optically measure the change in the position of the labeling substance at the elapsed time. In the following description, FC
An example using S will be described, but the present invention is not limited to FCS.

An example of such an automatic detection device is as shown in FIG. That is, such a device is provided with commonly used components (for example, various memories such as a storage unit and a RAM, programs stored in the memory, an input unit such as a keyboard, and a control unit such as a CPU). A computer 42 having a display unit such as a monitor for displaying results, an FCS measuring device 41 for performing FCS measurement, and an analyzing device for analyzing the data obtained by the FCS measuring device 41. , A dispenser 45 for adding a fluid to a reaction container, a reaction container 46 capable of sequentially performing two or more reactions in the reaction container, and maintaining a sample to be subjected to FCS measurement, A computer having a temperature controller 42 for controlling the temperature, the components of which are connected to each other, and the operation of each is stored in the computer. It is controlled in accordance with various programs more available.

Here, the temperature control device is controlled so as to provide the reaction container with a temperature suitable for at least the hybridization reaction between the test nucleic acid and the control nucleic acid and the binding reaction due to the mismatch recognition protein. In particular, when the first reaction step, that is, the hybridization reaction (including the nucleic acid amplification reaction if necessary) is carried out at a plurality of different temperatures, the temperature controller controls the heating to a high temperature and the heating to a low temperature. Both the cooling and the cooling are controlled so as to be switched at an appropriate time setting, and the binding by the mismatch recognition protein is controlled to be kept at a predetermined reaction temperature. On the other hand, when the first reaction step, that is, the hybridization reaction (including the nucleic acid amplification reaction if necessary) is performed by a plurality of different chemical treatment solutions, the temperature control device determines that the hybridization reaction and the mismatch recognition protein are different. Is controlled so as to keep the temperature constant at a predetermined reaction temperature suitable for each reaction, and the dispensing device applies the chemical treatment liquid required for the hybridization reaction to the mixture of the test nucleic acid and the control nucleic acid. It is controlled to perform such dispensing. In this way, a nucleic acid test including two different reactions can be conveniently and quickly performed under appropriate control.

For example, the FCS measuring device 41 may be the FCS measuring device shown in FIG. 1 and its stage 6
Further, the temperature control device 44 may be provided. Thereby, the hybridization reaction and the mismatch recognition protein binding reaction carried out therein are carried out, and then F
It is possible to make CS measurements. Alternatively, after the dispensing operation to the container 46 and the desired reaction or treatment are performed outside the FCS measuring device 41, the container 46 may be transported to the measuring section of the FCS measuring device 41. Such an automatic detection device is also included in the scope of the present invention.

In FIG. 4, one dispenser is provided as the dispenser 45 in the automatic detection device, and each reaction component is dispensed by the dispenser 45. However, the dispenser 45 may include two or more dispensing means. For example, the dispensing means used in the body may be any commonly used dispensing means. For example, the reagents used, the nucleic acids 21 and 3 to be tested
It is preferable to assign separate dispensing means to 1, the control nucleic acids 22 and 32, and the mismatch recognition proteins 25 and 33, respectively, because it is possible to prevent contamination. Further, for the dispensing process, the dispenser 45 can be used to sequentially add a fluid containing a reaction sample necessary for two or more reactions performed in the container 46.

Further, in the container 46, two or more reactions, such as a hybridization reaction and a mismatch recognition protein binding reaction, can be sequentially performed under appropriate conditions (for example, the above-mentioned temperature). Is.

[0051]

Industrial Applicability According to the present invention, there is provided a method for detecting a mutation in a test nucleic acid simply and quickly.

[Brief description of drawings]

FIG. 1 is a diagram showing an example of an FCS that can be used in the present invention.

FIG. 2 is a scheme showing the reaction principle of the present invention.

FIG. 3 is a diagram showing an embodiment of the present invention.

FIG. 4 is a diagram showing an example of a measuring apparatus of the present invention.

[Explanation of symbols]

21. Control nucleic acid 22. Test nucleic acid 23. Hybridization product 24. Hybridization product 25. Mismatch recognition protein binding 26. Fluorescent substance 31. Test nucleic acid 32. Control nucleic acid 33. Mismatch recognition protein 34. Fluorescent substance 35. Fluorescent substance 41. FC
S measuring device 42. Computer 43. Reaction analyzer 44. Heating control unit 45. Dispensing means 4
6. container

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/566 G01N 33/566 33/58 33/58 A // C12N 15/09 C12N 15/00 AF Terms (reference) 2G045 AA35 AA40 BA11 BB50 DA12 DA13 DA14 FB02 4B024 AA11 CA04 CA09 CA11 HA12 4B029 AA07 AA23 BB20 FA15 4B063 QA12 QA17 QA18 QQ42 QQ52 QR48 QR56 QS34 QS39 QX02

Claims (7)

[Claims] 1. A method for detecting a mutation in a test nucleic acid, wherein at least one selected from the group consisting of a test nucleic acid, a control nucleic acid and a mismatch recognition protein comprises:
Preliminarily labeled with a labeling substance, and (1) mixing a test nucleic acid and a control nucleic acid to carry out hybridization; (2) reacting the mismatching protein with the hybridization product obtained in (1) (3) At least measuring a labeling substance as a reaction result by the mismatch protein according to (2), (4) Whether or not the labeling substance is free based on the measurement data of the labeling substance in (3). A detection method for detecting a mutation in a test nucleic acid, which comprises performing an analysis for determining at least the above, and detecting a mutation in the test nucleic acid based on the analysis results of (5) and (4). 2. A method for detecting a mutation in a test nucleic acid, wherein at least one selected from the group consisting of a test nucleic acid, a control nucleic acid and a mismatch recognition protein comprises:
Preliminarily labeled with an optically detectable labeling substance, and (1) mixing a test nucleic acid and a control nucleic acid to carry out hybridization; (2) mismatch with the hybridization product obtained in (1) Reacting a protein, (3) during the hybridization according to (1) and the reaction described in (2), or during the reaction described in (2), a labeling substance at a plurality of elapsed time points Of the mutation in the test nucleic acid by optically measuring and analyzing the change in (4) the measurement with a labeling substance in the reaction with the mismatch protein according to at least (2) based on the optical measurement data. Analyzing whether or not the data has changed, and detecting a mutation in the test nucleic acid based on the analysis results of (5) and (4). Detection method for detecting a mutation in a test nucleic acid that. 3. A method for detecting a mutation in the test nucleic acid according to claim 1, wherein the test nucleic acid and the control nucleic acid are double-stranded, and (1) above.
The detection method, wherein the hybridization is cross-hybridization between the test nucleic acid and the control nucleic acid that are denatured and single-stranded after mixing the test nucleic acid and the control nucleic acid. 4. A method for detecting a mutation in the test nucleic acid according to claim 2 or 3, wherein the optical measurement and analysis in (4) above is performed by fluorescence correlation spectroscopy and fluorescence cross A detection method selected from the group consisting of correlation spectroscopy. 5. A method for detecting a mutation in the test nucleic acid according to any one of claims 1 to 4, wherein the labeling of (1) is performed on a plurality of types of control nucleic acids. And a detection method in which a different type of labeling substance is labeled for each type of control nucleic acid. 6. A method for detecting a mutation in the test nucleic acid according to any one of claims 1 to 4, further comprising: before the step (1), the test nucleic acid is extracted from a sample and amplified. A detection method comprising reacting and preparing. 7. An automatic inspection device using a computer for carrying out the detection method according to claim 1, wherein an optical signal detection device for a labeling substance and two or more in a reaction container are provided. 2 required for sequentially performing the two or more reactions in a reaction container capable of sequentially performing the above reactions and capable of maintaining the sample to be subjected to the optical signal detection measurement.
A dispensing device for sequentially adding a plurality of types of fluids so as to form different reaction samples, a temperature control device capable of controlling the temperature of the reaction, and a device controlling the temperature control device and / or the dispensing device. And an analyzer for analyzing the data measured by the optical signal detector.