JP2008125383A - Method for detecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for easily detecting a nucleic acid in high accuracy even in the case of minute amounts thereof. <P>SOLUTION: The method for detecting the presence/absence of a double-stranded nucleic acid having a specific target sequence is provided, comprising (A) the step of replicating, for a nucleic acid in a specimen, a double-stranded nucleic acid having a target sequence joined with a pair of different kinds of ligands, (B) the step of preparing a solid-phase carrier immobilized with a receptor specifically binding to either one of the pair of ligands and optically discriminable microparticles immobilized with a receptor specifically binding to the other ligand, (C) the step of inserting the region immobilized with the former receptor into the specimen after subjected to the replication, (D) the step of adding the microparticles while the above region is left inserted in the specimen, (E) the step of extracting the solid-phase carrier from the specimen, and (F) the step of detecting the optical properties of the above region. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for detecting a nucleic acid that can be detected easily and with high precision even when the amount of nucleic acid to be detected is small.

With the recent development of genetic engineering, genetic diagnosis is frequently used not only in the detection of pathogenic bacteria and viruses in the medical field, but also in various fields such as criminal investigation, parentage testing, archeological research, biology, etc. Yes. And when performing a genetic diagnosis, it is calculated | required that the nucleic acid which is a target can be detected with high precision.
Conventional genetic diagnosis has been performed, for example, by preparing a target DNA as a specimen and a probe DNA complementary thereto, annealing them under conditions that allow hybridization, and testing for the presence or absence of hybridization. . In the initial method, the specimen was fixed to a solid phase such as a nylon membrane, and the binding of a gene complementary to the target gene sequence was examined. However, in the case of detecting single nucleotide polymorphism (SNP) mutation or the like, a problem has been pointed out that the accuracy is not necessarily high. In addition, the diagnosis takes a long time of several hours to one day, and in order to detect gene binding, a radioisotope element or a complicated chemical reaction using an enzyme reaction is used, so that the operation is complicated. However, there is a problem that the accuracy is not necessarily high. In particular, when a radioisotope element is used, there is a safety problem. Therefore, a safe, simpler and more accurate detection method has been demanded.

On the other hand, recently, a genetic diagnosis method using a gene chip fixed on a probe DNA base has been studied and reported. However, this method still does not solve the problem of accuracy in detecting single nucleotide polymorphism mutations.
Furthermore, genetic diagnosis methods utilizing aggregation of complexes of latex microparticles or gold colloidal particles and DNA have been reported (see Non-Patent Documents 1 and 2 and Patent Document 1) and have attracted attention. However, in such agglomeration of the complex, since the DNA added as the sample crosslinks the plurality of fine particles, the aggregation is promoted. Therefore, a plurality of types of DNA-immobilized fine particles that bind to a plurality of locations of the sample DNA are used. There was a problem that it was necessary to prepare and the operation was complicated.

To solve such problems, a highly accurate and versatile genetic diagnosis method using a DNA conjugate substance has been proposed (see Patent Document 2). In this method, gene DNA is added to an aqueous solution containing a DNA conjugated substance, which is a complex of single-stranded DNA and a hydrophobic substance, and a metal cation, and either the light scattering intensity or light transmittance of the aqueous solution is added. It is a genetic diagnosis method characterized by measuring such changes. As the hydrophobic substance, a polymer of isopropylacrylamide is usually used.
In addition, a method for detecting a PCR product in which both ends are hapten-modified using an immunochromatography method is disclosed (see Patent Document 3). In this method, target DNA can be detected with high specificity even at room temperature by performing detection using an antigen-antibody reaction between a hapten and an antibody. Further, this method applies an immunochromatography method, and B / F separation is performed by a capillary phenomenon without using a special apparatus.
Special table 2003-503699 gazette JP 2001-252098 A JP 2003-194817 A Chemistry Let. 1999, 1041-1042. J. et al. Am. Chem. Soc. 1998, 120, 1959-1964.

  However, in the method described in Patent Document 2, it is possible to detect single nucleotide polymorphism mutations in genetic DNA by using one kind of DNA conjugate substance. However, the preparation of this DNA conjugate substance is complicated in operation, and it has been necessary to heat to a temperature of 30 ° C. or higher in order to obtain a micelle structure during analysis. Further, as in the example of Patent Document 2, the temperature is maintained at 40 ° C. In this method, in order to detect single nucleotide polymorphism variation, hybridization of DNA of the DNA conjugate substance and gene DNA is performed. It was necessary to precisely control the temperature. As described above, there are many restrictions on the temperature condition, and the operation is complicated.

  In the method described in Patent Document 3, colloidal gold is used, and the colloidal gold is monochromatic. For example, when SNP typing is performed, different antibodies are bound to two different locations on the solid phase. It is necessary to keep. And in the principle of immunochromatography, if a reaction takes place at a certain site and the hapten-labeled PCR product is captured, the flow may be hindered and the reaction efficiency upstream may be reduced. When detecting a multiplex to detect the error, there was a problem that the accuracy was not good. In addition, the immunochromatography method utilizes a capillary phenomenon and has a problem that the antigen-antibody reaction between the hapten and the antibody cannot be promoted by stirring or the like. Furthermore, it is necessary to introduce the sample solution into the solid phase to which the antibody is attached, and a dispensing operation of the sample solution is always required. And especially when processing a plurality of specimens, this dispensing operation is not only complicated and troublesome, but also, for example, a container containing a sample solution after a PCR reaction and a solid phase subjected to the dispensing operation. There was a problem that the correspondence relationship with was easily lost.

As described above, in the conventional genetic diagnosis methods, there is no simple and highly accurate method for detecting nucleic acids.
This invention is made | formed in view of the said situation, and makes it a subject to provide the detection method of the nucleic acid which can be detected simply and with high precision even if the nucleic acid of detection object is trace amount.

That is, to solve the above problem,
The invention according to claim 1 is a method for detecting the presence or absence of a double-stranded nucleic acid having a specific target sequence, wherein (A) a ligand pair consisting of different types of ligands binds to a nucleic acid in a sample. A step of replicating a double-stranded nucleic acid having the target sequence, (B) a solid phase carrier on which a receptor that specifically binds to one of the ligand pairs is immobilized, and the other ligand A step of preparing optically distinguishable microparticles on which a specifically binding receptor is immobilized; and (C) a step of inserting a region on which the receptor of the solid phase carrier is immobilized in the sample. (D) adding the fine particles to the sample while the receptor-immobilized region is inserted into the sample; (E) removing the solid phase carrier from the sample; ) Optical of the region where the receptor of the solid phase carrier is immobilized And detecting the quality, a method for detecting nucleic acids, which comprises a.
The invention according to claim 2 is a method for detecting the presence or absence of a double-stranded nucleic acid having a specific target sequence, wherein (a) a ligand pair consisting of different types of ligands binds to a nucleic acid in a sample. A step of replicating a double-stranded nucleic acid having a target sequence, and (b) preparing an optically distinguishable microparticle on which a receptor that specifically binds to one of the ligand pairs is immobilized And (c) preparing a solid phase carrier in which a receptor that specifically binds to the other of the ligand pair is immobilized, and the microparticles are attached to the outside of the immobilized region, and (d) A region where the receptor of the solid phase carrier is immobilized is inserted into the sample, and the region where the solid phase carrier fine particles are attached is inserted into the sample while the receptor immobilized region is inserted into the sample. Insert into the sample and add the microparticles into the sample And (e) removing the solid phase carrier from the sample; and (f) detecting the optical properties of the region where the receptor of the solid phase carrier is immobilized. A method for detecting nucleic acid
The invention according to claim 3 is characterized in that the sample is agitated using the solid phase carrier inserted into the sample after the addition of the fine particles into the sample. The method for detecting a nucleic acid described in 1. above.
The invention according to claim 4 is the method for detecting a nucleic acid according to any one of claims 1 to 3, wherein the target sequence is a plurality of types, and a different ligand pair is used for each target sequence. is there.
In the invention according to claim 5, the receptor immobilized on the microparticle and the ligand that specifically binds to the receptor are the same regardless of the target sequence, and are immobilized on the solid phase carrier. The receptor and the ligand that specifically binds to the receptor are different for each target sequence, and the receptor on the solid phase carrier is classified and immobilized by type. A method for detecting a nucleic acid according to claim 4.
The invention according to claim 6 is characterized in that the receptor immobilized on the microparticle, the ligand that specifically binds to the receptor, and the optical properties of the microparticle are different for each target sequence, and the solid phase carrier. The method for detecting a nucleic acid according to claim 4, wherein the receptor immobilized on the ligand and the ligand that specifically binds to the receptor are the same regardless of the target sequence.
The invention according to claim 7 is characterized in that the receptor immobilized on the microparticle, the ligand that specifically binds to the receptor, and the optical properties of the microparticle are different for each target sequence, and the solid phase carrier The receptor immobilized on the ligand and the ligand that specifically binds to the receptor are different for each target sequence, and the receptor on the solid support is classified and immobilized by type. The method for detecting a nucleic acid according to claim 4, wherein:
The invention according to claim 8 is the nucleic acid detection method according to any one of claims 1 to 7, wherein the replication operation of the target sequence is performed by a polymerase chain reaction method or a ligation method. .

According to the present invention, even a small amount of nucleic acid to be detected can be detected easily and with high accuracy.
Specifically, since the detection of the target nucleic acid is based on specific binding between the ligand and the receptor, even a trace amount of the target nucleic acid can be detected with high accuracy. Further, the dispensing operation of the sample solution is unnecessary, and the correspondence between the container containing the sample solution and the solid phase alone is clear. In addition, since the sample solution can be stirred while the solid phase is inserted into the sample solution and the fine particles are added, the formation of specific binding can be further promoted. Furthermore, a plurality of target nucleic acids can be detected at the same time, and detection can be performed inexpensively and rapidly.

Hereinafter, the present invention will be described in detail.
The nucleic acid detection method of the present invention is a method for detecting the presence or absence of a double-stranded nucleic acid having a specific target sequence,
(A) A step of performing an operation of replicating a double-stranded nucleic acid having a target sequence to which a ligand pair consisting of different types of ligands is bound to a nucleic acid in a sample;
(B) A solid phase carrier on which a receptor that specifically binds to one of the ligand pairs is immobilized, and an optically distinguishable on which a receptor that specifically binds to the other ligand is immobilized A step of preparing fine particles;
(C) inserting the region where the receptor of the solid phase carrier is immobilized into the sample after the replication operation;
(D) adding the fine particles to the sample while the receptor-immobilized region is inserted into the sample;
(E) removing the solid phase carrier from the sample;
(F) detecting the optical property of the region where the receptor of the solid phase carrier is immobilized;
It is characterized by including.
Or
(A) performing a process of replicating a double-stranded nucleic acid having a target sequence to which a ligand pair consisting of different types of ligands is bound to a nucleic acid in a sample;
(B) preparing an optically distinguishable microparticle on which a receptor that specifically binds to any one of the ligand pairs is immobilized;
(C) preparing a solid phase carrier in which a receptor that specifically binds to the other of the ligand pair is immobilized, and the fine particles are attached outside the immobilization region;
(D) The region where the receptor of the solid phase carrier is immobilized is inserted into the sample after the replication operation, and the solid phase carrier fine particles are further inserted while the receptor immobilized region is inserted into the sample. Inserting the attached region into the sample and adding the microparticles to the sample;
(E) removing the solid phase carrier from the sample;
(F) detecting an optical property of a region where the receptor of the solid phase carrier is immobilized;
It is characterized by including.
Hereinafter, the present invention will be described in detail for each step.

[When there is only one target sequence]
First, the following description will be focused on the case where there is only one type of target sequence.
<Step of performing an operation of replicating a double-stranded target nucleic acid>
(Target sequence, target nucleic acid)
In the present invention, the target sequence refers to a sequence containing the base sequence to be detected, and at the stage of applying the detection method of the present invention, the presence or absence of the presence in the sample to be detected may not be known. . The present invention does not attempt to detect the target sequence itself originally contained in the sample, but uses a sample to form a double-stranded target nucleic acid (hereinafter referred to as a target nucleic acid) having a target sequence to which a ligand pair is bound. (Which may be abbreviated) and attempts to detect the target nucleic acid. Thus, no target nucleic acid is detected unless the target sequence is originally present in the sample. That is, in the present invention, “detection of nucleic acid” also means “confirmation of presence or absence of target nucleic acid”. In the following, “a sample that has been subjected to an operation for replicating a target nucleic acid with respect to a nucleic acid in a sample” will be referred to as a “sample that may contain a target nucleic acid”.

(Operation to replicate the target nucleic acid)
The method for performing the operation of replicating the target nucleic acid on the nucleic acid in the sample is not particularly limited, and various conventionally known methods can be applied. For example, the presence or absence of the target sequence may be unknown, the target sequence can be bound directly or indirectly by electrostatic bond, hydrogen bond, covalent bond, hydrophobic bond, etc. Prepare a solution in which a nucleic acid synthesis enzyme and a substrate necessary for the reaction of the enzyme are added to a buffer solution according to the information on the compound and target sequence, and incubate at the optimum temperature of the enzyme to sufficiently perform the enzyme reaction. good. If the target sequence is present in the sample, the target nucleic acid is produced by the enzyme reaction. In addition, the said compound with which the ligand was couple | bonded can be obtained by a conventionally well-known method, and if there exists a commercial item, this may be used.

  The sample to be subjected to the enzyme reaction is not particularly limited, and examples thereof include a patient-derived sample itself and a sample containing a nucleic acid such as gDNA extracted from a patient-derived sample. The patient-derived sample is not particularly limited, and blood can be exemplified as a preferable sample. The method for extracting nucleic acid such as gDNA from a patient-derived sample is not particularly limited. For example, when the patient-derived sample is blood, an automated nucleic acid such as EZ1 (trade name) manufactured by QIAGEN. An extraction device may be used, or a commercially available nucleic acid extraction kit may be used. If Ampdirect (trade name) manufactured by Shimadzu Corporation is used, PCR described later can be performed as it is after nucleic acid extraction. And what was mentioned here is an example and what is necessary is just to select suitably according to a detection environment. In addition, RNA can be extracted by changing only the reagent in the same manner, and thus RNA may be used as necessary.

Specific examples of such a method include a polymerase chain reaction (hereinafter abbreviated as PCR) method and a ligation method.
In the PCR method, an operation for amplifying a target sequence may be performed using a primer pair to which a ligand is bound.
In the ligation method, a probe pair to which a ligand is bound may be used to perform a ligation reaction on the target sequence.
Among these, the PCR method is preferable because a very small amount of nucleic acid can be replicated.

FIG. 1 illustrates a schematic diagram of how a target nucleic acid is produced by a PCR method.
A target sequence 10 contained in a patient sample is extracted, and a forward primer 11 to which a first ligand 110 is bound and a reverse primer 12 to which a second ligand 120 is bound are prepared, and PCR amplification is performed using these. To obtain the target nucleic acid 1 to which the first ligand 110 and the second ligand 120 are bound.

In the PCR method, the primer to be used preferably has a length of 16 to 32 mer. For example, when the target nucleic acid has a gene polymorphism mutation, a sequence suitable for the mutation is selected. Primers are available from Sigma Aldrich Japan Co., Ltd. Two types of primers, a forward primer and a reverse primer, are required, and each binds a different ligand. The binding position of the ligand can be arbitrarily selected from the 5 ′ end, the 3 ′ end and between them, but since PCR is extended from the 3 ′ end of the primer, it is not preferable to bind near the 3 ′ end. . And since it is the easiest and economical, it is preferable to make it couple | bond with 5 'terminal.
As the primer, a commercially available product in which a ligand is bound can be easily obtained.

There are many commercially available PCR reagents and they are easily available. For example, KOD Plus (trade name) manufactured by Toyobo Co., Ltd. includes PCR enzyme, dNTP, Mg2 ⁺ , buffer, and the like as one kit. In addition, Applied Biosystems' AmpliTaq Gold PCR Master Mix (trade name) and the like are not only in a state in which a reaction solution containing an enzyme is mixed, but also can be stored in a refrigerator. However, when the purpose is to detect a gene polymorphism, it is preferable to select a high fidelity enzyme with few errors. PCR reagents may be selected in view of availability, economy, fidelity, and the like.

  The PCR apparatus is preferably a commercially available product such as GeneAmp PCR System 9700 (trade name) manufactured by Applied Biosystems or Thermal Cycler PTC-200 (trade name) manufactured by MJ Research. However, since the efficiency of the reaction differs slightly depending on each PCR apparatus, it may be necessary to correct them. For detection that does not require quantitativeness such as gene polymorphism, correction is not necessary. However, when gene expression is observed, correction is required.

  Appropriate primer concentrations, template DNA concentrations, PCR temperature cycle conditions, and the like are selected according to the target nucleic acid. An example of these conditions is shown in Tables 1 and 2 for the case where KOD Plus (trade name) manufactured by Toyobo Co., Ltd. is used. These conditions take the form of competitive PCR in which two target sequences having single nucleotide polymorphism mutations are simultaneously detected in one well.

Next, FIG. 2 illustrates a schematic diagram of how the target nucleic acid is produced by the ligation method.
A target sequence 10 contained in a patient sample is extracted, and a first probe 21 to which a first ligand 110 is bound and a second probe 22 to which a second ligand 120 is bound are prepared. The target nucleic acid 2 to which the first ligand 110 and the second ligand 120 are bound is obtained by performing a ligation reaction on the target sequence 10.

  In the ligation method, the sample to be provided is the same as in the PCR method. However, since the ligation reaction is not an amplification reaction and it is difficult to ensure S / N, it is preferable to extract the nucleic acid from the sample before subjecting it to the reaction. The nucleic acid extraction method is not particularly limited, and may be appropriately selected depending on the detection environment.

  The probe used in the ligation reaction preferably has a length of 12 to 32 mer. For example, when the target nucleic acid has a gene polymorphism mutation, a sequence suitable for the mutation is selected. Similar to the primer used in the PCR method, a commercially available product in which a ligand is bound can be easily obtained.

In the ligation method, since a reaction for linking adjacent probes is performed, in the present invention, a ligand is not bound to the vicinity of the 3 ′ end of the probe located downstream and the vicinity of the 5 ′ end of the probe located upstream. To do. Further, the 5 ′ end of the probe located upstream is originally a hydroxyl group, but it is necessary to bind a phosphate group here. The binding of the phosphate group can be performed by a conventionally known method, or a commercially available probe in a state where the phosphate group is bound may be used.
Therefore, the ligand is preferably bound to the upstream probe at or near the 3 ′ end. The ligand to be bound to the probe may be the same as that used in the PCR method, and may be appropriately selected according to the type of probe.

  There are many commercially available ligation reagents, and they are readily available. For example, Taq DNA Ligase (trade name) manufactured by New England BioLabs includes ligase (enzyme), buffer solution, and the like as one kit. In addition, Amplase Thermostable DNA Ligase (trade name) manufactured by EPCENTRE is an improved heat-resistant ligase that is not easily deactivated even after being exposed to high temperature for a long time, and is an LCR (Ligation Chain) that repeatedly performs ligation and heat denaturation. It is effective in (Reaction). As described above, the ligation reagent may be selected in consideration of the purpose of use, availability, economy, and the like, and is not particularly limited.

  Since the ligation reaction is performed by incubation under a constant temperature condition of about 40 ° C. to 65 ° C., it can be performed using a constant temperature bath. However, a thermal cycler or the like used in the PCR method may be used when heat denaturation or LCR is performed before incubation.

  The probe concentration, template DNA concentration, ligation temperature conditions, and the like are selected appropriately according to the target nucleic acid. An example of these conditions is shown in Tables 3 and 4 in the case of using Amplase Thermostable DNA Ligase (trade name) manufactured by EPCENTRE. These conditions take the form of competitive ligation in which two target sequences having single nucleotide polymorphism mutations are simultaneously detected in one well.

  In the present invention, the target nucleic acid to be detected is double stranded. For example, in both the PCR method and the ligation method, two pairs of primers and probes are always used. As a result, a double-stranded target nucleic acid cannot be obtained unless both of the paired primers or probes function normally. Due to the effect of enhancing specificity, an enzyme reaction product can be obtained with high accuracy. The obtained enzyme reaction product is in a double-stranded state unless special post-treatment such as heat denaturation or alkali denaturation is performed.

Nucleic acids are difficult to take a straight-chain form in a single strand due to their structure, and often have some three-dimensional structure unless the sequence is specifically designed. However, in the present invention, since the target nucleic acid is a double-stranded nucleic acid, the single-stranded nucleic acid supports each other so that it has a substantially linear structure and can be arranged on a solid phase or fine particles at high density. .
Further, in the single-stranded state, hybridization with other single-stranded nucleic acids may occur near room temperature. In this case, the target nucleic acid is entangled with each other and the detection efficiency of the target nucleic acid is significantly reduced. However, in the present invention, such a decrease in detection efficiency does not occur.

(Ligand)
The ligand used in the present invention is not particularly limited as long as it can bind to a nucleic acid and can specifically bind to a receptor. For example, hapten, biotin, FITC, DNP, Cy, DIG, FAM, HEX, TET, Examples include FLC, Rhodamine Green, TAMRA, Texas Red, Alexa, peptides, proteins, antigen / antibody substances, sugar chains, and the like. Then, it may be selected in consideration of availability and economy.

  In the present invention, specific binding means binding based on intermolecular force with high specificity, which is selectively formed between specific substances. Specific examples include, but are not limited to, binding by an immunological reaction between an antigen and an antibody, binding between biotin and avidin, binding between a sugar and a lectin, and the like.

  In the present invention, a target nucleic acid is prepared using two different types of ligands (ligand pairs) for one target sequence.

<Step of preparing solid support and fine particles>
(Receptor)
In the present invention, the receptor refers to all substances that can specifically bind to the ligand. By utilizing specific binding in this way, high-precision detection is possible even with a small amount of target nucleic acid.

(Fine particles)
The fine particles used in the present invention are optically distinguishable. Specifically, optically distinguishable means that, for example, the colors are different, that is, the wavelengths of light absorption / reflection / scattering, light emission, fluorescence, etc. are different, the particle diameters are different, or the shape is different. Is different, and the material is different. For example, even if the color of the fine particles is the same, if the particle size of the fine particles is different, the light transmittance is different, so that these fine particles can be optically distinguished. Further, since the degree of light scattering varies depending on the shape of the fine particles, these fine particles can be optically distinguished. Also, if the material of the fine particles is different, the optical properties are different due to the different surface properties of the fine particles, so that these fine particles can be optically distinguished. The fine particles used in the present invention may be those having a plurality of optically distinguishable elements, including those listed here.
The particle size, material, shape, and the like of these fine particles are appropriately selected so as to impart desired optical properties.
These fine particles may be produced by a conventionally known method, and commercially available products may be used.

  Among these, those that can visually recognize the difference in color are preferable. If such a thing is used, it will be unnecessary to use the apparatus for optical property detection, and nucleic acid can be detected cheaply and quickly. And manufacture or acquisition of a commercial item is also easy. In the case of producing fine particles having different colors visually, for example, the surface of the formed fine particles may be colored with a dye or the like, or may be formed into fine particles using a colored material.

  When the target nucleic acid is prepared by the PCR method, a large amount of the target nucleic acid can be obtained. However, when the target nucleic acid is prepared by a ligation method not involving an amplification reaction, the amount of the target nucleic acid to be obtained is limited. Therefore, the detection method of the present invention using optically distinguishable fine particles is an excellent method in that detection can be performed by visual observation.

The material of the fine particles whose color difference can be visually recognized is not particularly limited as long as the effects of the present invention are not impaired, and may be appropriately selected according to the detection conditions. For example, fine particles made of a polymer material such as latex beads and polystyrene beads are preferable in that a commercially available product is easily available, and latex beads are particularly preferable.
Colored latex beads are commercially available. For example, Color-Rich (Dyed Carboxylate-Modified) (trade name) manufactured by Seradyn is red, highly visible, and coated with a carboxyl group. Various receptors can be immobilized depending on the application, which is preferable.
In addition to fine particles made of these polymer materials, metal colloids, non-metal colloids, and the like can also be used.

  Moreover, the particle size of such colored beads is preferably about 0.2 to 3 μm, and the particle size is not necessarily uniform.

  The color of the colored beads is not particularly limited, but the closer to the primary color, the better the visual visibility. However, it is preferable to select appropriately according to the color of the solid phase carrier that is the background color.

A receptor that specifically binds to one of the ligand pairs is immobilized on the microparticle. The number of immobilized receptors is not particularly limited, but is preferably as long as the effects of the present invention are not hindered. Usually, 1 μg to 110 μg of receptors are preferably immobilized per 1 mg of fine particles. . With such an immobilized amount of receptor, for example, as described later, it is easy to immobilize the receptor on fine particles having a functional group such as a carboxyl group on the surface.
The position of the receptor immobilization on the fine particle is not particularly limited, but when the number of receptors is fixed to a specific value and compared, the distance between the receptors may be as large as possible. preferable. In this way, specific binding with a ligand in the target nucleic acid is further facilitated.

  Since such optically distinguishable fine particles having the receptor immobilized thereon are commercially available, they may be used as they are or may be prepared by a conventionally known method. Examples of the preparation method include a method of bonding a functional group in the receptor to a functional group such as a carboxyl group or an amino group exposed on the surface of the fine particles. As a method for bonding these functional groups, conventionally known methods can be applied. For example, EDAC method in which a carboxyl group and an amino group are bonded with water-soluble carbodiimide, 1-ethyl-3- (3-dimethylaminopropyl), ) Carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are mixed in advance to bond the carboxyl group and amino group, the method of cross-linking amino groups using a bipolar linker, activation And a method of bonding a functional group with an aldehyde group or tosyl group.

  For example, in a conventional nucleic acid detection method using agglomeration reaction of microparticles, it is necessary to previously bind a primer or probe having a specific base sequence to the microparticles. Such microparticles each time the target sequence changes. It had to be re-prepared. However, according to the present invention, since the primer or probe is not bound to the microparticles in advance, it is sufficient to prepare only the primer or probe, which is highly versatile and simplifies detection and reduces costs.

(Solid phase carrier)
A receptor that specifically binds to the other of the ligand pair is immobilized on the solid phase carrier. The number of immobilized receptors is not particularly limited, but is preferably as long as the effects of the present invention are not hindered, and usually 1 μg to 110 μg of receptors are immobilized per 1 mg of the solid phase carrier. Is preferred. With such an amount of immobilized receptor, for example, as described later, it is easy to immobilize the receptor on a solid phase carrier having a functional group such as a carboxyl group on the surface.

  The material of the solid phase carrier is not particularly limited, and examples thereof include various plastics. Of these, polystyrene is preferred. A solid phase carrier made of polystyrene is excellent in durability, portability, and ease of immobilizing fine particles. In addition, there are various methods and kits for immobilizing antibodies, such as those used as materials for ELISA (enzyme immunoassay) plates, and it is easy to immobilize receptors such as antibodies. But it ’s excellent. For example, IMMUNO-TEK ELISA Construction System (trade name) manufactured by Funakoshi Co., Ltd. is suitable.

The shape of the solid phase carrier is not particularly limited, and may be appropriately selected in consideration of ease of production, ease of holding, ease of insertion into a sample, and the like. A preferable shape is a strip shape.
More specific examples of the strip-shaped material are as follows. When inserted into a PCR tube, the normal 0.2 ml PCR tube has an inner diameter of 5.5 mm and a thin tip, so the width and thickness are 2 to 2. About 3 mm is preferable. Moreover, since it reaches the bottom of a PCR tube and it is easy to hold it with a finger, the length is preferably about 5 to 10 cm.

  The receptor immobilization region on the solid phase carrier is not particularly limited as long as the receptor can contact the sample. For example, when a sample is filled in a 0.2 ml PCR tube, or when a solution after PCR reaction having a total liquid volume of about 50 μl is used as it is as shown in Table 1, the sample is inserted into a solid phase carrier. It is preferable that an area of 1 to 2 mm from the end is a fixing area of the receptor. When the sample is 50 μl or more, for example, 100 μl, a region farther from the sample insertion end of the solid phase carrier may be used as a receptor-immobilized region. However, the region of 1 to 2 mm from the sample insertion end of the solid phase carrier not only can accommodate various amounts of the sample, but is also advantageous when attaching microparticles to the solid phase carrier as will be described later.

  The number of receptor immobilization regions on the solid phase carrier is not necessarily one, and may be plural.

  The distribution of receptor immobilization positions in the receptor immobilization region is not particularly limited, but when the number of receptors is fixed to a specific value and compared, the distance between the receptors is as large as possible. It is preferable to do so. In this way, specific binding with a ligand in the target nucleic acid is further facilitated.

  The method for immobilizing the receptor on the solid phase carrier is not particularly limited. For example, the above-mentioned immobilized commercial kit may be used, or a method of binding the functional group on the surface of the solid support to the functional group of the receptor in the same manner as the method of immobilizing the receptor to the fine particles. Also good. When functional groups are bonded to each other in this manner, for example, a reaction liquid containing a receptor is attached to a predetermined region on a solid phase carrier to bond the functional groups, and then a washing solution such as a buffer solution is used. Use and clean.

  The method for attaching the reaction solution onto the solid phase carrier is not particularly limited, and may be appropriately selected according to the shape and number of desired receptor-immobilized regions. Examples thereof include a method of spotting the reaction solution using a needle, a method of spotting the reaction solution by an ink jet method, and a method of applying the reaction solution using a thread.

In the present invention, the receptor can be immobilized on a solid phase carrier, and the fine particles can be further adhered. That is, the fine particle can be added to the sample by inserting the fine particle adhesion region of the solid phase carrier into the sample and releasing the fine particle into the sample.
Here, adhesion is different from immobilization, and the fine particles are held on the solid phase carrier so that the fine particles are detached from the solid phase carrier when the fine particle adhesion region of the solid phase carrier is inserted into the sample. Refers to being.

  The fine particles are attached to a region other than the receptor-immobilized region on the solid phase carrier. Adhering the fine particles in the receptor-immobilized region on the solid phase carrier means that the retention density of the receptor and the fine particles in the region is increased. In this case, it is preferable not only to fix the receptor and attach the microparticles at such a high density in this way, but also to bind the target nucleic acid sufficiently to the solid phase carrier and then bind the microparticles to the target nucleic acid. Since it becomes difficult to go through the procedure, it is not preferable. This “preferred procedure” will be described in detail later.

The fine particle adhesion region on the solid phase carrier is preferably located above the receptor immobilization region with reference to the sample insertion end of the solid phase carrier.
Further, as described above, when a region of 1 to 2 mm from the sample insertion end of the solid phase carrier is used as the receptor immobilization region, the fine particle adhesion region is at least a region of 5 mm or less from the sample insertion end of the solid phase carrier. It is preferable that

If the positional relationship between the receptor and the fine particles on the solid phase carrier is set in this way, as described later, first, the receptor-immobilized region is inserted into the sample, whereby the ligand and the receptor are placed on the solid phase carrier. The target nucleic acid is bound to the target nucleic acid, and then the microparticle attachment region is inserted into the sample, so that the microparticle released into the sample can be bound to the target nucleic acid via the ligand and the receptor. The target nucleic acid can be detected by the procedure.
In addition, when the container filled with the sample is, for example, in a tube shape and is narrower as it is closer to the bottom, the shape of the solid phase carrier is made narrower as it is closer to the sample insertion end. If so, the solid phase carrier can be inserted to the vicinity of the bottom of the container. By doing so, it becomes easier to perform stepwise insertion of the solid phase carrier as described above.

  Examples of the method of attaching the fine particles to the solid phase carrier include a method of attaching the solution containing the fine particles to a predetermined region of the solid phase carrier and then removing the solvent by freeze drying, vacuum drying, air drying or the like. In this case, it is preferable that the solution containing the fine particles contains a substance that assists the adhesion of the fine particles. For example, when an aqueous solution that is widely used as the sample is used, it is preferable that a water-soluble polymer compound, specifically, a polysaccharide such as trehalose or starch is contained.

In the present invention, as described later, it is preferable that the fine particles are added to the sample after the specific binding between the receptor immobilized on the solid phase carrier and the ligand in the target nucleic acid has sufficiently progressed. . Therefore, the release of the fine particles attached to the solid phase carrier into the sample preferably proceeds gradually, and for that purpose, it is preferable to adjust the adhesion force of the fine particles to the solid phase carrier. Examples of the method for adjusting the adhesion force include a method for adjusting the concentration of trehalose or the like that assists the adhesion of the fine particles. The concentration of the fine particles can be increased by increasing the concentration in the solution without impairing the effects of the present invention. The method of making it discharge | release gradually is mentioned.
The fine particles can be gradually released by adjusting the form of attachment so that the fine particles adhere to the surface of the solid support in a thin film form.
For this purpose, a solution containing fine particles may be thinly applied to a predetermined region on the surface of the solid phase carrier in a flat shape.

  The method for adhering the solution containing the fine particles to the solid phase carrier is not particularly limited, and for example, a method for adhering the reaction liquid containing the receptor as described above to the solid phase carrier can also be applied.

  The number of fine particle adhesion regions on the solid phase carrier is not necessarily one, and may be plural.

<Step of inserting the receptor-immobilized region of the solid phase carrier into the sample>
When a receptor-immobilized region of a solid phase carrier is inserted into a sample that may contain a target nucleic acid, if the target nucleic acid is present in the sample, one of the ligand pairs in the target nucleic acid is a solid phase carrier. It binds specifically to the above receptors. Thus, the target nucleic acid is bound to the solid phase carrier through specific binding of the ligand and the receptor. In the present invention, hereinafter, “binding of target nucleic acid and solid phase carrier through specific binding of ligand and receptor” is simply referred to as “binding of target nucleic acid and solid phase carrier”. There is. At this time, there are no microparticles in the sample, the target nucleic acid can move freely in the sample, and there is nothing that prevents specific binding between the ligand and the receptor other than the steric hindrance of the target nucleic acid itself. The specific bond formation is most likely to proceed.
Then, after the solid phase carrier is inserted into the sample, when the sample is stirred, the binding of the target nucleic acid to the solid phase carrier proceeds more rapidly and uniformly. Stirring may be performed in any manner, for example, by stirring the solid support itself.

  In order to insert the receptor-immobilized region of the solid phase carrier into the sample and allow the target nucleic acid to sufficiently bind to the solid phase carrier, it is usually sufficient to leave it inserted for 1 minute at room temperature. It is preferable to leave it inserted at 5 ° C. for 5 to 15 minutes.

  A conventional immunochromatography method or the like is characterized in that liquid feeding is performed using a capillary phenomenon, and means for promoting the formation of specific bonds have been limited. However, in the present invention, since the solid phase carrier is directly inserted into the sample in this way, the formation of specific binding can be promoted uniformly by stirring the sample as necessary after the insertion.

  Also, by inserting the solid phase carrier directly into the sample, the sample dispensing operation, which has been a problem of the conventional methods using immunochromatography and agglutination, can be omitted. Thereby, not only can the nucleic acid be easily detected, but also the correspondence between the container containing the sample and the solid phase alone is clarified, thereby avoiding the risk of erroneous information processing.

<Step of adding fine particles to the sample>
When the microparticles are added to the sample while the solid phase receptor-immobilized region is inserted into the sample, the other ligand pair in the target nucleic acid bound to the solid phase carrier and the microparticle are immobilized. It specifically binds to the receptor. In this way, the microparticles are bound to the target nucleic acid through specific binding of the ligand and receptor. In the present invention, hereinafter, “binding of microparticles to target nucleic acid through specific binding of ligand and receptor” may be simply referred to as “binding of microparticles to target nucleic acid”. At this time, the microparticles can move freely in the sample, and there is nothing to prevent the specific binding between the ligand and the receptor other than the steric hindrance of the microparticles and the target nucleic acid itself, and the specific binding formation proceeds most. Easy to use. Then, after adding the microparticles to the sample, when the sample is stirred, the binding of the microparticles to the target nucleic acid proceeds more rapidly. Stirring may be performed in any manner, for example, by stirring the solid support itself.

  In order to add microparticles to the sample and allow them to bind sufficiently to the target nucleic acid, it is usually sufficient to leave the solid support inserted in the sample for 1 minute after adding the microparticles at room temperature. It is preferable to leave the solid support inserted in the sample at 25 to 37 ° C. for 5 to 15 minutes after the addition.

FIGS. 3 and 4 schematically illustrate how the target nucleic acid is bound to the solid phase carrier and the fine particles are bound to the target nucleic acid so far. FIG. 3 shows the case where the target nucleic acid prepared by the PCR method is used, and FIG. 4 shows the case where the target nucleic acid prepared by the ligation method is used. In both cases, the same reference numerals are used for the same components used in FIGS.
In any case, except that the target nucleic acid 1 prepared by the PCR method and the target nucleic acid 2 prepared by the ligation method are used, the first receptor 140 immobilized on the solid phase carrier 30 is the same. When the second receptor 130 that specifically binds to the first ligand 110 and is immobilized on the microparticle 13 specifically binds to the second ligand 120, the target nucleic acid 1 or 2 is optically coupled. It is bound to the solid phase simple substance 30 in a distinguishable state.

As described above, the order in which the receptor-immobilized region of the solid-phase carrier is inserted into the sample and the fine particles are added is important, and this will be described.
When a specific bond between a ligand and a receptor is formed by a procedure different from that of the present invention, for example, consider a case where a target nucleic acid and a microparticle are bound and then contacted with a solid phase carrier. First, the target nucleic acid and the microparticles specifically bind with high efficiency. This is because both the target nucleic acid and the microparticle can move freely in the sample, and the collision probability between them is high. When the target nucleic acid and the microparticle collide, although depending on the number of receptors immobilized on the microparticle, a complex in which the surface of the microparticle is covered with the target nucleic acid is formed. The ligand in this complex that is not involved in the binding of the target nucleic acid and the microparticle, ie, the ligand in the target nucleic acid that is to specifically bind to the receptor on the solid support is then the receptor on the solid support. When it comes into contact, the complex is bound to the solid support. However, not all target nucleic acids on the surface of the microparticles bind to the solid phase carrier, and depending on the amount of target nucleic acid on the surface of the microparticles, some or most of them do not form a bond, so-called waste. turn into.
Further, the complex is larger in size than the fine particles in a state where the target nucleic acid is not bound, and the electrostatic repulsive force is enhanced by the negative charge of the target nucleic acid. Therefore, when one complex binds to the solid support, the combined complex generates a strong repulsive force around it, and remarkably prevents other complexes from approaching the receptor-immobilized region of the solid support. It will be. For this reason, the target nucleic acid can only be bound to the solid support with much lower efficiency than when using the procedure of the present invention.

  On the other hand, according to the procedure of the present invention, the target nucleic acid hardly binds to the microparticles and can move freely in the sample, so that it binds to the solid support with high efficiency and is wasted. There is almost no. Fine particles added after the target nucleic acid is sufficiently bound to the solid phase carrier can move freely in the sample and are not subject to repelling by the repulsive force on the surface of the solid phase carrier. Join. Therefore, the detection sensitivity of the target nucleic acid is the highest.

  When adding the microparticles to the sample, as described above, the microparticles are attached to the solid phase carrier, the receptor-immobilized region of the solid phase carrier is inserted into the sample, and the target nucleic acid is immobilized. The solid phase carrier may be inserted into the sample while the immobilized region is sufficiently inserted into the sample and the immobilized region is inserted into the sample, and the microparticles may be released into the sample and bound to the target nucleic acid. good. Although this method can also increase the detection sensitivity of the target nucleic acid, the detection sensitivity can be further increased if the release of fine particles into the sample gradually proceeds. Therefore, as described above, it is preferable to adjust the adhesion force and the adhesion form of the fine particles to the solid phase carrier.

<Step of removing solid phase carrier>
By removing the solid support from the sample after the addition of the fine particles, the excess can be easily separated (B / F separation). At this time, if necessary, the solid phase alone after removal may be washed with a washing solution such as a buffer solution. The cleaning liquid to be used is not particularly limited as long as the effects of the present invention are not hindered.

<Step of detecting optical properties of receptor-immobilized region of solid phase carrier>
If the target nucleic acid is present in the sample, an optical characteristic indicating the presence of the target nucleic acid is shown in the receptor-immobilized region of the solid phase carrier. The optical property detection method may be appropriately selected by using a detection device according to the optical characteristics of the used fine particles. For example, if fine particles that can be visually recognized are used, special detection is performed. Since no device is required, detection can be performed at the lowest cost, in a quick and simple manner.
In addition, since the solid phase carrier taken out from the sample has a clear correspondence with the container containing the sample, erroneous information processing such as erroneously recording the detection result can be avoided.

  For example, the solid phase carrier taken out from the sample does not fall off unless an operation such as vigorous rubbing is applied. Therefore, the solid phase carrier may be subjected to drying or may be stored. In addition, when the target nucleic acid is detected, it can be amplified again by the PCR method or the like, and more detailed analysis can be performed.

The state until the detection of the target nucleic acid up to this point is illustrated in schematic diagrams in FIGS. 5 and 6 in the case of using a solid phase carrier to which fine particles are attached. FIG. 5 shows the case where the target nucleic acid prepared by the PCR method is used, and FIG. 6 shows the case where the target nucleic acid prepared by the ligation method is used. In both cases, the same reference numerals are used for the same components used in FIGS. Reference numeral 35 denotes a receptor immobilization region, and reference numeral 36 denotes a fine particle adhesion region.
In either case, the same applies except that the target nucleic acid 1 prepared by the PCR method and the target nucleic acid 2 prepared by the ligation method are used, and the target nucleic acid 1 or 2 and the fine particles 13 are present in the receptor-immobilized region 35. Are combined.

[Detection method when there are multiple types of target sequences]
In the present invention, even when there are a plurality of types of target sequences, a plurality of corresponding types of target nucleic acids can be detected simultaneously. Here, "when there are multiple types of target sequences" means (i) when there are multiple types of nucleic acids having one type of target sequence, and (ii) when there is one type of nucleic acids having multiple types of target sequences (Iii) This refers to the case where there are a plurality of types of nucleic acids having a plurality of types of target sequences.
For example, an operation for simultaneously obtaining a plurality of types of target nucleic acids, such as performing an enzyme reaction for each target sequence, can be performed. Examples of such a method include a method using multiplex PCR in which a plurality of PCR amplifications are simultaneously performed, or a multiplex ligation reaction in which a plurality of ligation reactions are simultaneously performed.

  When there are a plurality of target sequences, different ligand pairs are used for each target sequence. At this time, different ligands may be used, and either one of the ligand that binds to the receptor immobilized on the microparticles and the ligand that binds to the receptor immobilized on the solid phase carrier, all ligand pairs May be the same.

  For example, when the ligands that bind to the receptors immobilized on the microparticles are made the same regardless of the target sequence, all the receptors that are immobilized on the microparticles are made the same. In order to detect multiple types of target nucleic acids simultaneously, the ligand that binds to the receptor immobilized on the solid phase carrier is different for each target sequence, and is immobilized on the solid phase carrier accordingly. The receptors to be processed are also different for each target sequence, and further, these receptors are immobilized on a solid phase carrier according to type (first embodiment).

  In this first embodiment, the optical properties of the fine particles may all be the same. In this case, the optical properties detected on the solid phase carrier are all the same, but since the relationship between the type of receptor immobilized on the solid phase carrier and the immobilized region is clear, The type of the detected target nucleic acid can be determined based on the difference.

  When the ligands that bind to the receptor immobilized on the solid phase carrier are the same regardless of the target sequence, all the receptors that are immobilized on the solid phase carrier are also made the same. In order to detect multiple types of target nucleic acids at the same time, the ligand that binds to the receptor immobilized on the microparticles is different for each target sequence, and there are also receptors that are immobilized on the microparticles accordingly. Each target sequence is different, and the optical properties of the microparticles are also different for each previous target sequence (second embodiment).

  Thereby, in the receptor immobilization region of the solid phase carrier, a plurality of types of target nucleic acids are detected in a state where the optical properties of all the corresponding microparticles are superimposed. For example, when using fine particles whose color difference can be visually recognized, selecting the combination of fine particles so that the color superposition can be emphasized by utilizing the complementary color relationship makes it easy to check the detection result. Is.

  Furthermore, the form which combined 1st and 2nd embodiment may be sufficient. That is, the receptor immobilized on the microparticles, the ligand that binds to the receptor, and the optical properties of the microparticles differ for each target sequence, and the receptor immobilized on a solid support and the receptor These receptors are immobilized by differentiating each of the ligands that bind to each target sequence and by classifying them on the solid phase carrier (third embodiment).

According to the first and second embodiments, the types of ligands to be prepared can be reduced. Therefore, when the number of target sequences is large, the detection can be simplified and the cost can be reduced.
On the other hand, with the third embodiment, more accurate detection is possible.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
[Example 1] Detection of double-stranded target nucleic acid prepared by PCR (preparation of double-stranded target nucleic acid)
A reaction solution having the composition shown in Table 5 was prepared, and PCR amplification was performed in the PCR cycle shown in Table 6 using the materials, devices, and the like shown below.
Template nucleic acid: human gDNA having the base sequence shown in SEQ ID NO: 3
Primer: Primer in which DIG is bound to the 5 ′ end of the base sequence shown in SEQ ID NO: 1, Primer in which biotin is bound to the 5 ′ end of the base sequence shown in SEQ ID NO: 2 Sample container: 200 μl microplate with removable lid Tube (made by Assist)
Thermal cycler: DNA Engine PTC-200 (trade name, manufactured by MJ Research (currently BioLad))

(Solid phase carrier)
A solid support was prepared as follows.
That is, as shown in FIG. 7, among polystyrene rods (Φ2 mm, length 10 cm) 701, about 2 mm from the sample insertion end 705 is coated with a carboxyl group, and the anti-DIG antibody 702 is made of ethylenediaminocarbodiimide (Pierce). ). In addition, anti-biotin antibody-added red produced by reacting anti-biotin antibody (goat immunity, manufactured by SIGMA) and red latex beads coated with carboxyl groups (Φ0.3 μm, manufactured by Seradyn) using the recommended protocol of Seradyn The latex bead 703 solution is attached to the vicinity of 3 mm to 5 mm from the sample insertion end 705 of the rod 701 and dried, so that the beads 703 are adhered to a predetermined portion of the rod 701, and a solid phase carrier for detection and did.

(Formation of specific binding)
The solid phase carrier was inserted into a microtube subjected to PCR amplification, and was manually shaken at 25 ° C. (room temperature) for 1 minute and mixed. After mixing, it was left at room temperature for about 5 minutes.

(Detection and judgment)
When the solid support is slowly pulled up after mixing and leaving, the red latex beads 703 are attached to the anti-DIG antibody attachment region 704 about 2 mm from the sample insertion end 705 as shown in FIG. 7B. This was clearly observed visually. FIG. 7A is a diagram in which the anti-DIG antibody 702 is emphasized to show that the beads are attached to the anti-DIG antibody attachment region 704.
This is because the DNA fragment having the base sequence shown in SEQ ID NO: 3 to be detected is PCR-amplified with a primer with a ligand consisting of the base sequences shown in SEQ ID NOS: 1 and 2, and the amplified products are anti-DIG antibody and anti-biotin antibody It was confirmed that the base sequence shown in SEQ ID NO: 3 was indeed present in the template nucleic acid used.

[Example 2]
Detection was carried out in the same manner as in Example 1 except that human gDNA having the base sequence shown in SEQ ID NO: 4 was used instead of human gDNA having the base sequence shown in SEQ ID NO: 3.
As a result, as shown in FIG. 7C, the red latex beads did not adhere to the solid phase carrier. That is, it was confirmed that the base sequence shown in SEQ ID NO: 4 is a SNP of the base sequence shown in SEQ ID NO: 3, and was not PCR-amplified due to the C → G mutation.

[Example 3] Detection of two types of double-stranded target nucleic acids prepared by multiplex PCR
(Preparation of two types of double-stranded target nucleic acids)
A reaction solution having the composition shown in Table 7 was prepared, and PCR amplification was performed in the PCR cycle shown in Table 6 using the materials, devices, and the like shown below.
Template nucleic acid: human gDNA having the base sequence shown in SEQ ID NO: 3 and not having the base sequence shown in SEQ ID NO: 7
Primer: primer in which DIG is bound to the 5 ′ end of the base sequence shown in SEQ ID NO: 1, primer in which biotin is bound to the 5 ′ end of the base sequence shown in SEQ ID NO: 2, and 5 ′ of the base sequence shown in SEQ ID NO: 5. Primer with FITC bound to the end, primer with biotin bound to the 5 ′ end of the base sequence shown in SEQ ID NO: 6 Sample container: 200 μl microtube with removable lid (manufactured by Assist)
Thermal cycler: DNA Engine PTC-200 (trade name, manufactured by MJ Research (currently BioLad))

(Solid phase carrier)
A solid support was prepared as follows.
That is, as shown in FIG. 8, among the polystyrene rod (Φ2 mm, length 10 cm) 801, about 2 mm from the sample insertion end 807 is coated with a carboxyl group, and anti-DIG antibody 802 and anti-FITC antibody 803 are treated with ethylenediamino It was divided into two sections using carbodiimide (Pierce) and immobilized. In addition, an anti-biotin antibody-added red color produced by reacting an anti-biotin antibody (goat immunity, manufactured by SIGMA) and a red latex bead coated with a carboxyl group (Φ0.3 μm, manufactured by Seradyn) using the recommended protocol of Seradyn The latex bead 804 solution is attached to the vicinity of 3 mm to 5 mm from the sample insertion end 807 of the rod 801 and dried, so that the beads 804 are attached to a predetermined portion of the rod 801, and a solid phase carrier for detection and did.

(Formation of specific binding)
The solid phase carrier was inserted into a microtube subjected to PCR amplification, and was manually shaken at 25 ° C. (room temperature) for 1 minute and mixed. After mixing, it was left at room temperature for about 5 minutes.

(Detection and judgment)
When the solid support is slowly pulled up after mixing and standing, the red latex beads 804 are attached to the anti-DIG antibody attachment region 806 about 2 mm from the sample insertion end 807 as shown in FIG. This was clearly observed visually. On the other hand, the red latex beads 804 were not attached to the anti-FITC antibody attachment region 805 in the vicinity of 3 mm to 5 mm from the sample insertion end 807. That is, the pattern A in FIG. 8 is shown. FIG. 8A is a diagram in which the anti-DIG antibody 802 and the anti-FITC antibody 803 are emphasized to show that the beads are attached to the anti-DIG antibody attachment region 806.
This is because a DNA fragment having the base sequence shown in SEQ ID NO: 3 among the DNA fragments to be detected is PCR-amplified with a primer with a ligand consisting of the base sequences shown in SEQ ID NOS: 1 and 2, and the amplified product is an anti-DIG antibody And that the DNA fragment having the base sequence shown in SEQ ID NO: 7 was not PCR-amplified with a primer with a ligand consisting of the base sequences shown in SEQ ID NOs: 5 and 6. Is. From the above, it was confirmed that the base sequence shown in SEQ ID NO: 3 was present in the template nucleic acid used, but the base sequence shown in SEQ ID NO: 7 was not present.

[Example 4]
Detection was performed in the same manner as in Example 3 except that human gDNA having the base sequence shown in SEQ ID NO: 7 and not having the base sequence shown in SEQ ID NO: 3 was used instead of the human gDNA used in Example 3. Went.
As a result, as shown in FIG. 8C, it is clearly observed visually that the red latex beads 804 are attached to the anti-FITC antibody attachment region 805 in the vicinity of 3 mm to 5 mm from the sample insertion end 807. It was. On the other hand, the adhesion of the red latex beads 804 was not observed in the anti-DIG antibody adhesion region 806 about 2 mm from the sample insertion end 807. That is, the pattern B of FIG. 8 is shown.
This is because a DNA fragment having the base sequence shown in SEQ ID NO: 7 among the DNA fragments to be detected is PCR-amplified with a primer with a ligand consisting of the base sequences shown in SEQ ID NOs: 5 and 6, and the amplified product is anti-FITC antibody And that the DNA fragment having the base sequence shown in SEQ ID NO: 3 was not PCR-amplified with the liganded primer consisting of the base sequences shown in SEQ ID NOS: 1 and 2. Is. From the above, it was confirmed that the base sequence shown in SEQ ID NO: 7 was present in the template nucleic acid used, but the base sequence shown in SEQ ID NO: 3 was not present.

  From the results of Examples 1 to 4, it was confirmed that the presence or absence of two kinds of DNA fragments can be simultaneously detected with high sensitivity when the detection region is divided on the solid phase carrier.

[Example 5] Detection of two types of double-stranded target nucleic acids prepared by multiplex PCR-No division-
(Preparation of two types of double-stranded target nucleic acids)
Two kinds of double-stranded target nucleic acids were prepared in the same manner as in Example 3.

(Solid phase carrier)
A solid support was prepared as follows.
That is, as shown in FIG. 9, among the polystyrene rods (Φ2 mm, length 10 cm) 901, about 2 mm from the sample insertion end 906 is coated with a carboxyl group, and the anti-biotin antibody 902 is ethylenediaminocarbodiimide (manufactured by Pierce). ). Anti-DIG antibody (goat immunity, manufactured by SIGMA) and red latex beads coated with carboxyl groups (Φ0.3 μm, manufactured by Seradyn) were reacted according to the recommended protocol of Seradyn, and the manufactured anti-DIG antibody-added red color The latex beads 904 solution, anti-FITC antibody (goat immunity, manufactured by SIGMA) and blue latex beads coated with carboxyl groups (Φ0.3 μm, manufactured by Seradyn) were reacted using the recommended protocol of Seradyn. The FITC antibody-added blue latex bead 903 solution is attached to the vicinity of 3 mm to 5 mm from the sample insertion end 906 of the rod 901 and dried, so that the beads 903 and 904 are adhered to predetermined positions of the rod 901 and detected. A solid support for use.

(Formation of specific binding)
The solid phase carrier was inserted into a microtube subjected to PCR amplification, and was manually shaken at 25 ° C. (room temperature) for 1 minute and mixed. After mixing, it was left at room temperature for about 5 minutes.

(Detection and judgment)
When the detection carrier is slowly pulled up after mixing and leaving, the red latex beads 904 are attached to the anti-biotin antibody attachment region 905 about 2 mm from the sample insertion end 906 as shown in FIG. 9B. Was clearly observed visually. On the other hand, the blue latex beads 903 did not adhere to the same region. That is, the pattern A in FIG. 9 is shown.
This is because a DNA fragment having the base sequence shown in SEQ ID NO: 3 among the DNA fragments to be detected is PCR-amplified with a primer with a ligand consisting of the base sequences shown in SEQ ID NOS: 1 and 2, and the amplified product is an anti-DIG antibody And that the DNA fragment having the base sequence shown in SEQ ID NO: 7 was not PCR-amplified with a primer with a ligand consisting of the base sequences shown in SEQ ID NOs: 5 and 6. Is. From the above, it was confirmed that the base sequence shown in SEQ ID NO: 3 was present in the template nucleic acid used, but the base sequence shown in SEQ ID NO: 7 was not present.

[Example 6]
Detection was carried out in the same manner as in Example 5 except that two types of double-stranded target nucleic acids were prepared in the same manner as in Example 4.
As a result, as shown in FIG. 9C, it was clearly observed visually that the blue latex beads 903 were attached to the anti-biotin antibody attachment region 905 about 2 mm from the sample insertion end 906. On the other hand, the red latex beads 904 did not adhere to the same region. That is, the pattern B of FIG. 9 is shown.
This is because a DNA fragment having the base sequence shown in SEQ ID NO: 7 among the DNA fragments to be detected is PCR-amplified with a primer with a ligand consisting of the base sequences shown in SEQ ID NOs: 5 and 6, and the amplified product is an anti-FITC antibody And that the DNA fragment having the base sequence shown in SEQ ID NO: 3 was not PCR-amplified with the liganded primer consisting of the base sequences shown in SEQ ID NOS: 1 and 2. Is. From the above, it was confirmed that the base sequence shown in SEQ ID NO: 7 was present in the template nucleic acid used, but the base sequence shown in SEQ ID NO: 3 was not present.

[Example 7]
Detection was performed in the same manner as in Example 5 except that human gDNA having both the base sequence shown in SEQ ID NO: 3 and the base sequence shown in SEQ ID NO: 7 was used as the template nucleic acid.
As a result, as shown in FIG. 9D, the anti-biotin antibody adhesion region 905 about 2 mm from the sample insertion end 906 is colored purple, and both the red latex beads 904 and the blue latex beads 903 are in this region. The adhesion was clearly observed visually. That is, the pattern C in FIG. 9 is shown. FIG. 9A is a diagram in which the anti-biotin antibody 902 is emphasized to show that the beads are attached to the anti-biotin antibody attachment region 905.
This is because a DNA fragment having the base sequence shown in SEQ ID NO: 3 was PCR amplified with a primer with a ligand consisting of the base sequences shown in SEQ ID NOs: 1 and 2, and the amplified product was specifically detected with anti-DIG antibody and anti-biotin antibody. In addition, a DNA fragment having the base sequence shown in SEQ ID NO: 7 was PCR amplified with a primer with a ligand consisting of the base sequences shown in SEQ ID NOs: 5 and 6, and the amplified products were anti-FITC antibody and anti-biotin This indicates that the antibody specifically binds. From the above, it was confirmed that all of the base sequences shown in SEQ ID NOs: 3 and 7 were present in the used template nucleic acid.

  From the results of Examples 5 to 7, it was confirmed that the presence or absence of two types of DNA fragments can be simultaneously detected with high sensitivity without dividing the detection region on the solid phase carrier.

  The present invention can be used not only for detection of pathogenic bacteria and viruses in the medical field, but also for genetic investigations in criminal investigations, parentage testing, archeological research, biology, and the like.

It is a schematic diagram which illustrates a mode that target nucleic acid produces by PCR method. It is a schematic diagram which illustrates a mode that a target nucleic acid produces by the ligation method. It is a schematic diagram which illustrates a mode until the target nucleic acid obtained by PCR method couple | bonds with a solid-phase carrier, and microparticles | fine-particles couple | bond with this target nucleic acid. It is a schematic diagram which illustrates a mode until the target nucleic acid obtained by the ligation method couple | bonds with a solid-phase carrier, and microparticles | fine-particles couple | bond with this target nucleic acid. It is a schematic diagram which illustrates a mode until it detects the target nucleic acid prepared by PCR method using the solid-phase support | carrier to which microparticles | fine-particles were attached. It is a schematic diagram which illustrates a mode until it detects the target nucleic acid prepared by the ligation method using the solid-phase support | carrier to which microparticles | fine-particles were attached. 3 is a conceptual diagram illustrating a solid phase carrier during detection in Examples 1 and 2. FIG. 6 is a conceptual diagram illustrating a solid phase carrier at the time of detection in Examples 3 and 4. FIG. It is a conceptual diagram which illustrates the solid-phase carrier at the time of the detection in Examples 5-7.

Explanation of symbols

1, 2 ... target nucleic acid, 10 ... target sequence, 11 ... forward primer, 12 ... reverse primer, 13 ... fine particles, 21 ... first probe, 22 ... first Two probes, 30 ... solid phase carrier, 35 ... receptor immobilization region, 36 ... fine particle adhesion region, 110 ... first ligand, 120 ... second ligand, 130 .... Second receptor, 140 ... first receptor

Claims (8)

A method for detecting the presence or absence of a double-stranded nucleic acid having a specific target sequence,
(A) A step of performing an operation of replicating a double-stranded nucleic acid having a target sequence to which a ligand pair consisting of different types of ligands is bound to a nucleic acid in a sample;
(B) A solid phase carrier on which a receptor that specifically binds to one of the ligand pairs is immobilized, and an optically distinguishable on which a receptor that specifically binds to the other ligand is immobilized A step of preparing fine particles;
(C) inserting the region where the receptor of the solid phase carrier is immobilized into the sample after the replication operation;
(D) adding the fine particles to the sample while the receptor-immobilized region is inserted into the sample;
(E) removing the solid phase carrier from the sample;
(F) detecting the optical property of the region where the receptor of the solid phase carrier is immobilized;
A method for detecting a nucleic acid, comprising: A method for detecting the presence or absence of a double-stranded nucleic acid having a specific target sequence,
(A) performing a process of replicating a double-stranded nucleic acid having a target sequence to which a ligand pair consisting of different types of ligands is bound to a nucleic acid in a sample;
(B) preparing an optically distinguishable microparticle on which a receptor that specifically binds to any one of the ligand pairs is immobilized;
(C) preparing a solid phase carrier in which a receptor that specifically binds to the other of the ligand pair is immobilized, and the fine particles are attached outside the immobilization region;
(D) The region where the receptor of the solid phase carrier is immobilized is inserted into the sample after the replication operation, and the solid phase carrier fine particles are further inserted while the receptor immobilized region is inserted into the sample. Inserting the attached region into the sample and adding the microparticles to the sample;
(E) removing the solid phase carrier from the sample;
(F) detecting an optical property of a region where the receptor of the solid phase carrier is immobilized;
A method for detecting a nucleic acid, comprising:   The method for detecting a nucleic acid according to claim 1 or 2, wherein the sample is agitated using the solid phase carrier inserted in the sample after the addition of the microparticles to the sample.   The method for detecting a nucleic acid according to any one of claims 1 to 3, wherein a plurality of types of the target sequences are used, and different ligand pairs are used for each target sequence.   The receptor immobilized on the microparticle and the ligand that specifically binds to the receptor are the same regardless of the target sequence, and the receptor immobilized on the solid phase carrier and specific to the receptor The nucleic acid detection according to claim 4, wherein ligands that bind to each other are different for each of the target sequences, and the receptors on the solid phase carrier are classified and immobilized for each type. Method.   The receptor immobilized on the microparticle, the ligand that specifically binds to the receptor, and the optical properties of the microparticle are different for each target sequence, and the receptor immobilized on the solid phase carrier and the receptor The method for detecting a nucleic acid according to claim 4, wherein ligands that specifically bind to a receptor are the same regardless of the target sequence.   The receptor immobilized on the microparticle, the ligand that specifically binds to the receptor, and the optical properties of the microparticle are different for each target sequence, and the receptor immobilized on the solid phase carrier and the receptor 5. The ligand that specifically binds to a receptor is different for each target sequence, and the receptor on a solid phase carrier is classified and immobilized by type. Method for detecting nucleic acid of   The method for detecting a nucleic acid according to any one of claims 1 to 7, wherein the replication operation of the target sequence is performed by a polymerase chain reaction method or a ligation method.

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