JP5278481B2 - Qualitative reaction specimen - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To develop a cheap test plate which makes it possible to judge the detection of complex physiological reactions easily and visually. <P>SOLUTION: The test plate is used for medical care. To be concrete, the test plate includes: base material; a detection region prepared on the base material; and a physiologically active substance fixed region, which is arranged at a predetermined position in the detection region and where at least two or more kinds of physiologically active substances are respectively fixed. In the detection region, at least two different physiologically active substances are placed adjacent and/or the physiologically active substance fixed region and a test substance fixed region where a test substance is fixed are placed adjacent. And a method for producing the test plate is also provided. The test plate, when the number of fixed physiologically active substances is high, enables to detect complex physiological reactions without requiring the burden of measurer's analysis, and brings a prompt response in medical care. Moreover, the production cost of the test plate is also low. <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a test piece that can be used in a medical field. Specifically, a test piece including a base material, a detection region provided on the base material, and a physiologically active substance fixing region in which at least two or more types of physiologically active substances provided in the detection region are fixed. The present invention relates to a test piece in which at least two different types of physiologically active substance immobilization regions are adjacent to each other and / or a physiologically active substance immobilization region is adjacent to a detection substance immobilization region to which a detection substance is immobilized, and a method for manufacturing the test piece.

In recent years, with the progress of genome sequencing, the entire base sequence of the genome of each organism is being clarified, and a technique for effectively utilizing this result is desired.
Nucleic acid microarray is a technology that can analyze multiple genes simultaneously, and has been developed not only as a base sequencing method but also as a method for efficiently examining gene expression levels and polymorphisms. Applications for identification and diagnosis of diseases are being developed.

In general, nucleic acid microarrays that can be used for tailor-made medicine, as well as biological classification or variant identification, are sufficient to qualitatively detect specific base sequences and are inexpensive for disposable use such as test strips. Things are sought.
For example, Patent Literature 1 discloses a line probe assay (LiPA: Line Probe Assay, trademark of Inogenetics) as a nucleic acid microarray used in a method for identifying an HCV (hepatitis C virus) isolate. In this assay, nucleic acid probes are fixed as parallel lines on a polyamide membrane piece, and thus rapid DNA detection is possible.

  In addition, since the nucleic acid probes to be immobilized on the nucleic acid microarray are designed with a length of 10 to 40 bp from the wild type gene, there are many types of nucleic acid probes (the number of spots or lines) that can be immobilized. There are at most about 100 kinds. That is, the determination of the presence or absence of hybridization using the above-described nucleic acid microarray or the like requires the measurer to count the number of hybridized spots or lines, and the operation is complicated.

As a means for solving such a problem, a nucleic acid microarray in which unevenness is provided on a substrate by a lithography technique and a nucleic acid probe is fixed to the convex part can be cited (Patent Document 2). In these microarrays, since the position of the nucleic acid probe is clear, the presence or absence of hybridization can be easily determined from the presence or absence of color development or light emission of the convex portion.
However, since a nucleic acid microarray that can be used for tailor-made medical treatment or a part of diagnosis is required to be disposable like a test piece, the lithography technique has a problem in terms of manufacturing cost. Furthermore, it is necessary to design and manufacture the base material so that the number of probes and the number of convex portions are the same, resulting in a more expensive product.

On the other hand, Patent Document 3 discloses a technique in which a marker is provided around a spot where a nucleic acid probe is immobilized. Since this nucleic acid microarray is manufactured using an inkjet method, it is less expensive in terms of manufacturing cost than a nucleic acid microarray manufactured by a lithography technique.
However, even if a marker is provided around the spot, the greater the number of nucleic acid probes, the easier it is to make a visual illusion, and it is not easy to find an unreacted spot from a large number of spots.

  Furthermore, the nucleic acid microarray disclosed in Patent Document 2 or 3 is an array that is densely integrated, and is not intended to be directly determined visually. Since the detection is performed using a specialized machine such as a scanner after the fluorescent label is developed, it is necessary to introduce a specialized machine, which requires a burden on a medical institution.

JP 7-503143 A JP 2004-264289 A JP 2000-270896 A

  Therefore, development of an inexpensive test piece that can easily determine the detection of a physiological complex reaction visually is required.

The present invention relates to the following [1] to [7].
[1] A detection method using a test piece to detect a wild-type nucleic acid having two or more known mutations,
The specimen is
A substrate, one detection region provided on the substrate, and at least two or more types of regions disposed in the detection region to which at least two types of nucleic acid probes are respectively fixed;
The at least two types of regions to which the at least two types of nucleic acid probes are respectively fixed are adjacent to each other in the detection region,
The nucleic acid probe has a base sequence that hybridizes with a specific wild-type base sequence and does not hybridize with a base sequence having a known mutation,
The detection method includes the following steps.
(1) Amplifying the nucleic acid of a sample using a primer modified with a chromogenic enzyme at the end
(2) contacting the amplified nucleic acid with a test piece
(3) Coloring process using chromogenic substrate
(4) When the entire detection region is colored, the sample nucleic acid is a wild type, and when a part of the detection region is not colored, the sample is judged to be a mutant type [2] The shape of the test piece is rectangular,
The detection method according to [1], wherein the at least two or more types of regions to which the at least two or more types of nucleic acid probes are respectively fixed are arranged in parallel and in stripes with respect to the short side of the test piece.
[3] The detection method according to [2], wherein the width of at least two types of regions to which the at least two types of nucleic acid probes are respectively fixed is 1 to 5 mm.
[4] The detection region further includes a region in which an enzyme serving as a chromogenic substrate catalyst or a linker substance capable of binding to the enzyme serving as a chromogenic substrate catalyst is immobilized,
Between at least two or more types of regions to which each of the at least two or more types of nucleic acid probes is fixed, and a region to which an enzyme serving as a catalyst for the chromogenic substrate or a linker substance capable of binding to an enzyme serving as a catalyst for the chromogenic substrate is immobilized The detection method according to [1], wherein the space is adjacent to each other in the detection region.
[5] The shape of the test piece is a rectangle,
The at least two or more types of regions to which each of the at least two types of nucleic acid probes is respectively fixed, and the region to which the enzyme serving as a catalyst for the chromogenic substrate or a linker substance capable of binding to the enzyme serving as the catalyst for the chromogenic substrate is immobilized The detection method according to [4], which is arranged in parallel and in stripes with respect to the short side of the test piece.
[6] Fix at least two or more regions to which each of the at least two types of nucleic acid probes is fixed, and an enzyme serving as a catalyst for the chromogenic substrate or a linker substance capable of binding to the enzyme serving as the chromogenic substrate catalyst. The detection method according to [5], wherein the width of the region is 1 to 5 mm.
[7] A detection kit using a test piece for detecting a wild-type nucleic acid in which two or more known mutations exist.
The kit comprises a test piece and a primer modified with a chromogenic enzyme at the end,
The specimen is
A substrate, one detection region provided on the substrate, and at least two or more types of regions disposed in the detection region to which at least two types of nucleic acid probes are respectively fixed;
The at least two types of regions to which the at least two types of nucleic acid probes are respectively fixed are adjacent to each other in the detection region,
The nucleic acid probe has a base sequence that hybridizes with a specific wild-type base sequence and does not hybridize with a base sequence having a known mutation.

  Since the test piece of the present invention can easily detect a physiological complex reaction by visual observation, there is no burden of analysis by a measurer, and quick response at a medical site is possible. In addition, since the lithography technique is not used, the manufacturing cost can be significantly reduced.

It is one embodiment of the test piece of this invention. It is one embodiment different from FIG. 1 of the test piece of this invention. It is one embodiment different from FIG. 1 and 2 of the test piece of this invention. It is one embodiment different from FIG.1, 2, and 3 of the test piece of this invention. It is one embodiment of the manufacturing method of the test piece of this invention. It is one embodiment different from FIG. 5 of the manufacturing method of the test piece of this invention. 2 is a schematic diagram of a test piece manufactured in Example 1. FIG. It is a result of Experimental example 1.

DESCRIPTION OF SYMBOLS 1 Base material 2 Detection area | region 3 Bioactive substance fixing | fixed area | region 31 DNA probe 32 which has the base sequence of sequence number 1 DNA probe which has the base sequence of sequence number 2 4 Detection substance fixed region 41 Linker substance 5 Display means

  With the test piece of the present invention, at least two or more types of physiologically active substances are fixed in different regions within the detection region of the base material, and at least two or more types of physiological complex reactions are simultaneously performed and easily determined visually. Things that make it possible. In addition, it refers to obtaining disease diagnosis, microbial biological classification or mutant identification, and information related to an individual's constitution from the obtained results.

  The biological classification of the microorganism or identification of the mutant is not particularly limited. For example, identification of the type of HCV (hepatitis C virus) infecting a patient or the resistance of a therapeutic drug possessed by Mycobacterium tuberculosis Etc.

  In addition, the information related to the personal constitution of the individual is not particularly limited, but for example, prediction of future changes in bone density of the individual, and the sensitivity, tolerance or side effects of the therapeutic agent may occur. Prediction.

  The physiological complex reaction refers to a reaction based on complex formation between biologically derived substances, for example, DNA-DNA hybridization, DNA-RNA hybridization, DNA-PNA (peptide nucleic acid) hybridization, antigen-antibody Reaction and ligand-receptor reaction.

  Therefore, the physiologically active substance that can be immobilized on the test strip of the present invention may be any substance that can form a complex, and examples thereof include nucleic acids, antigens, antibodies, ligands, and receptors. Among them, nucleic acids are mainly used as a development for tailor-made medicine. The following description focuses on what is used for nucleic acid detection in consideration of the frequency of use, but can be applied mutatis mutandis to substances other than nucleic acids and is not necessarily limited.

  Examples of the nucleic acid include single-stranded or double-stranded DNA, RNA, and PNA. Of these, single-stranded DNA is preferred from the viewpoint of production cost and use frequency. The nucleic acid that can be fixed to these test pieces is generally called a nucleic acid probe, and when the nucleic acid is single-stranded DNA, it is called a DNA probe.

  The production of the nucleic acid probe is not particularly limited. For example, in the case of a DNA probe, it may be synthesized DNA (oligonucleotide) or cDNA reversely transcribed from mRNA. The oligonucleotide can be synthesized using a commercially available automatic nucleic acid synthesizer or the like.

  The nucleic acid is designed to have a base sequence that can hybridize with a specific base sequence of the target nucleic acid. The specific base sequence can be appropriately set by those skilled in the art depending on the purpose of use of the test strip of the present invention, and is not particularly limited. For example, genomic DNA correlated with the biological classification of the microorganism described above. The nucleotide sequence including the wild type and its mutant type.

  Furthermore, if the base sequence of the nucleic acid is within a range where it can hybridize with the specific base sequence, base insertion, mutation and deletion are performed from the base sequence completely complementary to the specific base sequence. It may be a base sequence. However, for example, when it is intended to detect a mutation at a single base level, a completely complementary base sequence is preferable from the viewpoint of reaction accuracy.

  The degree of polymerization of the nucleic acid probe is not particularly limited because it varies depending on the type of a specific base sequence to be detected. For example, when detecting a mutation at a single base level of genomic DNA extracted from an organism. Depending on the temperature at the time of detection, it is about 10 to 30 bases, preferably about 12 to 26 bases from the viewpoint of detection accuracy.

  The physiologically active substance immobilized on the test strip of the present invention may be at least two kinds depending on the purpose of use of the test strip.

  For example, when detecting a wild-type gene having two or more known mutations, it has a base sequence that hybridizes with a specific wild-type base sequence and does not hybridize with a base sequence with a known mutation. Each nucleic acid probe can be designed.

  For example, when detecting a wild-type gene in which many unknown mutations may exist, it has a base sequence that hybridizes with a specific wild-type base sequence and does not hybridize with a base sequence having any mutation. Nucleic acid probes can be designed. At this time, if the length of the nucleic acid probe is designed to be about 10 to 30 bases, preferably about 12 to 26 bases, a mutation at a single base level can be detected. That is, the longer the degree of polymerization of the wild type gene, the more types of nucleic acid probes. Furthermore, if the adjacent probes are designed in order from upstream or downstream so that about 4-7 bases overlap each other, even if an unknown mutation exists at least in the overlapping part, it can be detected by either probe. The detection accuracy is preferably improved, but the number of designed nucleic acid probes is further increased.

  As described above, the type of the physiologically active substance to be immobilized on the test piece of the present invention is appropriately set by those skilled in the art, and is not particularly limited as long as it is two or more types.

  Examples of detecting a wild type gene in which many unknown mutations may exist include detection of a wild type pncA gene of Mycobacterium tuberculosis. The pncA gene of Mycobacterium tuberculosis is said to correlate with the drug resistance of pyrazinamide of Mycobacterium tuberculosis, and the base encoding the pyramidase in the pncA gene is 580 bases. Therefore, for example, when the degree of polymerization of all nucleic acid probes is unified to 30 bases and designed to overlap 4 bases with adjacent nucleic acid probes, there are at least 22 kinds of nucleic acid probes. Such a probe design is merely an embodiment, and the present invention is not limited to this.

  The two or more types of physiologically active substances are fixed in different regions within the detection region provided on the substrate.

  The base material may be any material that can fix the physiologically active substance, for example, organic materials such as polyethylene, polypropylene, polycarbonate, polymethyl methacrylate, polyamide, polystyrene, polyethylene terephthalate and nitrocellulose, glass and silica. And inorganic materials such as gold, silver, and metal materials such as barrels. Of these, organic materials are preferable from the viewpoint of easy moldability, and polypropylene, polycarbonate, polymethyl methacrylate, and polyamide are more preferable, but are not limited thereto.

  Furthermore, when an organic material is selected as the base material, a white material is preferable from the viewpoint that detection by color development can be clearly determined, but the present invention is not limited to this.

Examples of the substrate include a substrate, a container, a film, and a tube. Among these, from the viewpoint of easy handling, a rectangular film or substrate is preferable. In addition, the size of the base material needs to have a certain area from the viewpoint of facilitating detection. For example, if the substrate is a rectangular film or substrate, the area of the upper surface, from the viewpoint it is easy to handle, about 40～1000Mm ^2, preferably about 60~300mm ^2.

  Since the said base material can be manufactured according to a condition by those skilled in the art, it is not specifically limited. For example, extrusion molding, injection molding, melt molding, compression molding and the like can be mentioned, and among them, extrusion molding is preferable from the viewpoint of manufacturing cost and ease.

  The detection area is an area where at least all physiologically active substances are fixed. On detection of a physiological complex reaction, an operator should pay attention to the presence of a physiological complex reaction on the substrate surface. A closed area provided at a predetermined position. The detection region can be defined at any position on the substrate. In particular, from the viewpoint of easy visual determination, it is preferable that all the detection regions are on the same surface. The area of the detection region can also be set from the viewpoint of the area ratio occupied by the detection region when the total surface area of the substrate is 100%. For example, it is about 10 to 90%, preferably about 25 to 75%. That is, it is preferable to provide a portion that is not a detection region on the base material, because the measurer can touch the portion to carry it and carry out operations such as measurement and the like, which improves handling.

  Further, the number of the detection regions is not particularly limited, and a plurality of the detection regions may exist. However, one is preferable from the viewpoint of easy visual determination by visual observation.

  Within the detection region of the test strip of the present invention, at least two different types of physiologically active substance fixing regions are adjacent to each other, and / or the physiologically active substance fixing region is adjacent to the detection substance fixing region to which the detection substance is fixed. Yes. The term “adjacent” in the present invention refers to a state in which two types of physiologically active substances and / or physiologically active substance immobilization areas are substantially in contact with a detection substance immobilization area on which a detection substance is immobilized. However, there may be some deviation as long as no misperception occurs in the detection. For example, there are few cases in which visual recognition makes a mistake even if the distance is about 0.01 to 0.5 mm.

  In addition, the physiologically active substance is immobilized in different areas within the detection area. That is, for example, when there are N types (N is a natural number of 2 or more) of physiologically active substances, there are at least N types of the physiologically active substance fixing regions.

  The detection substance in the present invention refers to a substance that can be visually judged or a linker substance that can be bonded to the substance that can be visually judged. Substances that can be visually judged include, for example, RI labeling (substances that enable the RI labeling method), fluorescent substances that emit visible light (substances that enable the fluorescent labeling method), and color development reactions. Enzyme (substance capable of enzyme coloring method) and the like. Among them, an enzyme that enables an enzyme coloring method is preferable from the viewpoint that a dedicated device is not required and that visual determination is easy. Examples of the enzyme include alkaline phosphatase and peroxidase.

  The linker substance refers to a substance that can bind to the substance that can be visually determined, for example, a chemical bond such as a covalent bond, an ionic bond, a hydrogen bond, a disulfide bond, and a coordination bond, and a main bond. It refers to a substance that enables biochemical binding such as antigen-antibody binding that does not affect the physiological complex reaction in the invention. Among them, the test piece of the present invention is mainly used for detection of DNA and is easily available, and the NBT / BCIP method using biotin-modified nucleic acid and streptavidin-modified alkaline phosphatase is generally used for detection of nucleic acid. From the viewpoint, an avidin-biotin bond is preferable.

  A preferred example of the detection substance is alkaline phosphatase from the viewpoint that it is generally used in nucleic acid detection methods and can be easily fixed to a substrate, but is not limited thereto.

  The shape and arrangement of the physiologically active substance fixing area and the detection substance fixing area are not particularly limited as long as there is no area where nothing is fixed in the detection area, and takes the form of a so-called jigsaw puzzle. . Examples of the shape include a triangle, a rhombus, a rectangle, and a regular hexagon. Each region may have the same shape or a different shape, but the entire detection region may be immobilized with a physiologically active substance. Design to fill in the area. Among these, from the viewpoint of easy production, the shape is rectangular (band shape), and the arrangement is preferably arranged in the form of vertical stripes (stripes). Reference may be made to FIGS. 1-4 for these embodiments.

  Further, when the number of the detection areas is one, the detection areas are all occupied by the physiologically active substance fixing area and / or the detection areas. In other words, it takes the form of a jigsaw puzzle in which the physiologically active substance fixing region and / or the detection region is a puzzle piece.

In addition, the area of each of the physiologically active substance fixing region and the detection substance fixing region depends on the area of the detection region and the number of the physiologically active substance fixed regions. ˜150 mm ² , preferably about 6 to 100 mm ² . In particular, when arranging rectangular (band-like) regions in the form of vertical stripes (stripes), the width of the band is about 1 to 15 mm, preferably about 2 to 10 mm.

  The physiologically active substance and the detection substance are fixed in a detection region on the substrate surface. The nucleic acid probe and the detection substance can be immobilized by physical or chemical treatment.

  The immobilization by the physical treatment is not particularly limited as long as it is a method of spotting a solution of the physiologically active substance or the detection substance on a base material. Examples of such spotting methods include an extrusion method using a dispenser and an inkjet method. From the viewpoint of production cost, the extrusion method is preferable, and from the viewpoint of immobilization accuracy, the ink jet method is preferable, and is not particularly limited.

  In addition, when the physiologically active substance is a nucleic acid, when immobilizing by such a physical treatment, if a base sequence unrelated to the nucleic acid probe is added, the immobilization rate on the substrate is improved by UV irradiation. It is preferable. Examples of the base sequence unrelated to the nucleic acid probe include polyadenine, polycytosine, polythymine and polyguanine, and polythymine is preferred from the viewpoint of the highest immobilization rate.

In addition, since the nucleic acid probe is a hydrophilic polymer, the substrate may be subjected to some kind of treatment. For example,
(A) A method of coating a substrate surface with a polycationic polymer such as polylysine
(B) A method of treating with a silane coupling agent having an amino group such as aminoethoxysilane when the substrate is an inorganic material such as glass
(C) When the substrate is a metal material such as gold, a method of treating the carrier surface with a thiol having an amino group such as 2-aminoethanethiol or a disulfide compound can be used.

On the other hand, as a method of immobilizing to chemistry by covalent bond, a method of modifying a functional group capable of forming a covalent bond with the base material at the end of the nucleic acid probe, or a covalent bond to the end of the nucleic acid probe and the base material, respectively. Examples thereof include a method for modifying a functional group. For example,
(a) When the base material is an inorganic material such as glass and silicon, the functional group capable of silane coupling reaction such as trimethoxysilane and triethoxysilane is modified at the end of the probe for nucleic acid detection, and the silane coupling reaction Immobilization method
(b) When the base material is an inorganic material such as glass or silicon, the surface of the base material is treated with silane coupling agent having an amino group such as aminoethoxysilane on the inorganic material base material. Aminated by Kepling bond. On the other hand, the carboxylic acid is modified at the end of the nucleic acid probe. And the method of forming and fixing an amide bond by the amino coupling reaction of the amino group of a base material, and the carboxylic acid of a nucleic acid probe etc. are mentioned.
Even if the substrate is a metal material such as gold or silver, those skilled in the art can immobilize the silane coupling bond by replacing the metal-thiol or metal-disulfide bond.

  Among the above-described immobilization methods, regarding the immobilization of nucleic acid, from the viewpoint of preparation, it is preferable to add polythymine to the end of the nucleic acid probe and immobilize it by UV irradiation.

  In the present invention, from the viewpoint of facilitating the operator to recognize the fixed position of the physiologically active substance, it is preferable to provide display means as means for specifying the detection region. The display means depends on the compatibility with the material of the base material. For example, the hydrophobic ink having a small contact angle with the base material, the seal, and the convex or concave groove when forming the base material are used. In view of easy visual determination, a hydrophobic ink is preferable, but the method is not limited to this. Further, it is not always necessary to express the detection area only by the display means, and for example, the end of the test piece of the present invention may be used together. By this display means, the operator can grasp the fixed position of the physiologically active substance and can easily detect the nucleic acid.

  From the above, the most preferable example of the test piece of the present invention is a test piece in which a physiologically active substance fixing region is arranged in stripes parallel to the short side direction of a rectangular film as shown in FIG. is there.

  For example, as shown in FIG. 5, a method for more efficiently producing the test piece of the above-described preferred form is such that the film is pulled out from a roll on which the film is wound, and the physiologically active substance and / or the detection substance is pulled out in the pulling direction. The solution is continuously applied and fixed by an extrusion method or an inkjet method. Next, a method of producing a large number of test pieces of the present invention by cutting into strips in a direction perpendicular to the drawing direction can be mentioned. For example, when manufacturing by an extrusion method, when a discharge solution is about 0.5 to 1.0 μl / min from a dispenser having a 24 gauge needle and an application speed is about 2.5 to 8.5 mm / sec, The width is about 1-2 mm. The display means may be provided at any stage before or after applying the solution of the physiologically active substance and / or the detection substance. These settings can be appropriately set by those skilled in the art, and are not particularly limited.

  Further, for example, the method shown in FIG. Specifically, the detection substance solution is applied and fixed at regular intervals, and then dried. Thereafter, a physiologically active substance solution is applied to the remaining area. Next, a method of producing a large number of test pieces of the present invention by cutting into strips in a direction perpendicular to the drawing direction can be mentioned. According to this method, since the fixation of the physiologically active substance is completed in two steps, the production cost is lower than that of the method of FIG.

  The method for using the test strip of the present invention will be described below with reference to the drawings for the method for detecting nucleic acids. Examples of the method for use described below can be appropriately set by those skilled in the art and are particularly limited. is not.

  FIG. 1 is one embodiment of the test piece of the present invention. On the rectangular film 1, DNA probes are immobilized in stripes parallel to the short side direction of the rectangle. In the detection area 2, the areas 3 where all the DNA probes are immobilized are densely packed. Yes. For example, the manufacturing method of FIG. 5 or 6 can be adopted as the method of manufacturing the test piece.

  Next, a sample for contact with the test piece of the present invention is prepared. First, genomic DNA is extracted from an animal biological sample. The biological sample includes blood, saliva, hair, and the like, and preferably various human cells such as blood cells, epidermal cells, and mucosal cells. A method for extracting DNA from a biological sample can be performed by a known method, such as a phenol extraction method, a guanidine thiocinate extraction method, and a vanadyl ribonucleoside complex extraction method.

  Next, preferably, a specific base sequence to be detected is amplified from the extracted genomic DNA. Examples of the method for amplifying a nucleic acid include a PCR method, a LAMP method, and an ICAN method. Of these, the PCR method is preferred from the viewpoint of reagent cost.

  The PCR method includes, for example, (1) a denaturation step in which a double-stranded genomic DNA is converted to a single strand by heat treatment under a reaction condition of about 92 to 95 ° C. for about 30 seconds to 1 minute, (2) An annealing step in which a double-stranded portion serving as a PCR reaction start point is prepared by binding at least two kinds of amplification primers under reaction conditions of about 50 to 65 ° C. for about 20 seconds to 1 minute, (3) Steps (1) to (3) of the chain extension step of reacting with DNA polymerase under a reaction condition of about 70 to 75 ° C. for about 20 seconds to 5 minutes are repeated 20 to 40 times by a usual method. To amplify the nucleic acid.

  The primer pair used in the PCR method has a base sequence that can hybridize with the extracted genomic DNA and can amplify a nucleic acid containing the specific base sequence, and has a polymerization degree of about 15 What is about ~ 40 bases is sufficient. The primer can be synthesized by an automatic synthesizer or the like, similar to the nucleic acid probe.

  Furthermore, it is preferable to modify the detection substance at the end of the primer to further facilitate detection of the nucleic acid. As the detection substance, as described above, for example, an RI label (a substance that enables the RI labeling method), a fluorescent substance that emits visible light (a substance that enables the fluorescent labeling method), and a color reaction. Enzymes that can be used (substances that enable enzyme coloring methods), linker substances that can bind to these substances, and the like. Here, when modifying a linker substance, it is necessary to prepare the detection substance itself couple | bonded with the said linker substance separately. For example, when biotin is modified as a linker substance, streptavidin-modified alkaline phosphatase needs to be prepared separately. Moreover, it is preferable to use the substance similar to the detection substance fixed to the said base material from a viewpoint which unifies a detection reaction and makes a detection easy for the substance which modifies these primer ends.

  Next, the amplified nucleic acid is brought into contact with the test piece of the present invention to perform a hybridization reaction. The conditions for the pre-hybridization reaction depend on the heat denaturation temperature of the DNA probe immobilized on the substrate. For example, when the heat denaturation temperature of the DNA probe is about 55.0 to 75.0 ° C, it is generally about 61.5 to 62.5 ° C.

  Then, a hybridization reaction is detected with the DNA probe immobilized on the substrate. For example, when the end of the primer is labeled with RI, it can be detected by autoradiography. When a fluorescent substance that emits visible light is modified, light having an excitation wavelength suitable for each fluorescent label is irradiated, and the emitted light (visible light) can be visually confirmed. Furthermore, in the case of an enzyme label that enables a color development reaction, it can be visually confirmed by adding a substrate for each enzyme to cause color development. Among the detection methods, color development using an enzyme (enzyme color development method) is preferable from the viewpoint of easy measurement.

  When used in the enzyme coloring method, examples of the substance that modifies the primer include alkaline phosphatase, and p-nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-yne are used as the substrate for the alkaline phosphatase. The combined use of p-toluidine salt of drill phosphate (BCIP) is mentioned. The degree of color development can be determined by visual observation, a CCD camera, a densitometer, or the like, but visual determination is sufficient unless there are special circumstances.

  When nucleic acids are detected using the test strip of the present invention as described above, if there is no mutation in the specific base sequence, the entire detection region emits light or colors. On the other hand, when there is a mutation in the specific base sequence, even one portion does not emit light or develop color. Therefore, by looking at the detection portion, it can be instantaneously determined whether or not there is a mutation in the base sequence of the target nucleic acid. For example, when the test piece of the present invention is produced for detecting mutations in the pncA gene of Mycobacterium tuberculosis, if even one of the pncA genes has a mutation, a part of the detection region does not emit light or develop color at the time of detection. It can be determined instantaneously that the Mycobacterium tuberculosis has pyramidase resistance. As a result, a therapeutic agent other than pyramidase can be selected, and the burden on the patient is greatly reduced.

  Examples of the present invention are shown below, but the present invention is not limited thereto.

Example 1: Production of test piece DNA probe As a DNA probe, the DNA probes shown in SEQ ID NOs: 1 and 2 were synthesized and used. The DNA probes of SEQ ID NOs: 1 and 2 have sequences that can hybridize at different positions to the pncA wild type gene of Mycobacterium tuberculosis. Polythymine was added to these DNA probes using a terminal transferase (manufactured by New England Biolab) at the 5 ′ end. Specifically, 4 μl of terminal transferase (20 units / μl), 10 μl of deoxythymidine triphosphate (10 pmol / μl), 4 μl of the DNA probes (50 mM) of SEQ ID NOs: 1 and 2, 5 μl of NEBuffer 4 attached to the product, and After preparing 50 μl of purified water containing 5 μl of the cacodylate buffer attached to the product, reacting at 37 ° C. for 4 hours, and adding 50 μl of 20 × SSC buffer attached to the product, the polythymine-added nucleic acid probe (average about 400 bp long) 2 pmol / μl were obtained.

Sequence number 1: atgatcaacg cccgcatac
Sequence number 2: gtcgttctgc acgtcgac

Linker substance As the linker substance, DNA having the base sequence shown in SEQ ID NO: 3 and modified with biotin at the 5 'end was used. A solution to be applied to the substrate was prepared to have a concentration of 0.1 pmol / μl. This DNA is also used as a reverse primer described later.

Sequence number 3: caacagttca tcccggttc

Manufacture of test piece A polyamide membrane was selected as a substrate on which the DNA probe was immobilized. On the polyamide membrane (10 mm × 300 mm), a solution of the above-mentioned two types of DNA probe and linker substance is used in a 300 mm size according to the method of FIG. It was applied continuously in the direction parallel to the sides. In particular,
(i) Linker substance
(ii) DNA probe of SEQ ID NO: 1
(iii) Linker substance
(ii) DNA probe of SEQ ID NO: 2
(iii) It applied continuously in the order of the linker substance.
The coating conditions were a discharge speed of about 0.7 μl / sec and a coating speed of about 2.5 mm / sec. After drying at about 25 ° C., the DNA probe was immobilized on the polyamide membrane by irradiating with 312 nm ultraviolet rays for 2 minutes. After immobilization, the unadsorbed DNA probe was removed by washing with a 10 × SSC solution. Next, a display means was provided by marking both ends of the detection region with hydrophobic ink, and a test piece of the present invention was obtained by cutting every 5 mm. The width | variety of the area | region where the DNA probe was fixed in the obtained test piece was about 2 mm. A schematic diagram thereof is shown in FIG.

Experimental Example 1: Extraction of genomic DNA from nucleic acid detection specimens The genomic DNA of M. tuberculosis samples 1 to 3 shown in Table 1 was extracted by the phenol extraction method. Table 1 shows the positions of mutations in the pncA gene. Here, the probe DNA of SEQ ID NO: 1 corresponds to sample 2, and the probe DNA of SEQ ID NO: 2 corresponds to sample 3.

Amplification of Genomic DNA of Specimen A forward primer having the base sequence shown in SEQ ID NO: 4, a reverse primer having the base sequence shown in SEQ ID NO: 3 and having biotin bound to the 5 ′ end, and Taq DNA polymerase (Roche Diagnostics) The base sequence containing the pncA gene was amplified by a PCR method using Styx. In the amplification reaction, the denaturation process was performed at 95 ° C. for 1 minute, the annealing process was performed at 55 ° C. for 1 minute, the chain extension process was performed at 72 ° C., and one cycle for 30 minutes.

Sequence number 4: ggcgtcatgg accctatatc
Sequence number 3: caacagttca tcccggttc

Detection of Nucleic Acid Sodium hydroxide (20%) (10 μL) was added to 10 μL of the amplified DNA solution and stirred well. The amplified DNA was denatured into single strands by leaving for 5 minutes. A 10% aqueous solution of sodium lauryl sulfate (1 mL) was added to the solution containing denatured DNA, the test piece of Example 1 was immersed, and the mixture was shaken at a reaction temperature of 62 ° C. for 30 minutes for hybridization. Thereafter, alkaline phosphatase-labeled streptavidin was added, and p-nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) were added to bind to each probe. A color reaction with alkaline phosphatase bound to the sample was performed.

  The result is shown in FIG. It was clear that the presence or absence of the hybridization reaction could be easily determined instantaneously by looking at the substrate.

  When the number of immobilized physiologically active substances reaches a large number, the test piece of the present invention can detect physiological complex reactions without requiring the burden of analysis by the measurer, and can respond quickly in the medical field. And its manufacture is also inexpensive.

Claims (2)

A detection method using a test piece to detect a wild-type nucleic acid in which two or more known mutations exist in a biological sample,
The specimen is
A substrate, one detection region provided on the substrate, and at least two or more types of regions disposed in the detection region to which at least two types of nucleic acid probes are respectively fixed;
The base material has a rectangular shape,
The at least two or more types of regions to which the at least two or more types of nucleic acid probes are respectively fixed are arranged in parallel and in stripes with respect to the short side of the substrate,
The area of at least two types of regions to which the at least two types of nucleic acid probes are respectively fixed is 3 to 150 mm ² , and the width is 1 to 5 mm,
The at least two or more kinds of regions to which the at least two kinds of nucleic acid probes are respectively fixed are separated by 0.01 to 0.5 mm ,
The nucleic acid probe has a base sequence that hybridizes with a specific wild-type base sequence and does not hybridize with a base sequence having a known mutation,
Detection method, detection method, which comprises the following steps.
(1) Amplifying a sample nucleic acid using a primer modified with a chromogenic enzyme at the end or a primer modified with a linker capable of binding to a chromogenic enzyme at the end
(2) contacting the amplified nucleic acid with a test piece
(3) Coloring process using chromogenic substrate
(4) A step of determining that the nucleic acid of the specimen is wild type when the entire detection region is colored, and that the nucleic acid of the specimen is mutated when a part of the detection area is not colored A detection kit using a test piece for detecting a wild-type nucleic acid having two or more known mutations,
The kit comprises a test piece and a primer modified with a chromogenic enzyme at the end,
The specimen is
A substrate, one detection region provided on the substrate, and at least two or more types of regions disposed in the detection region to which at least two types of nucleic acid probes are respectively fixed;
The base material has a rectangular shape,
The at least two or more types of regions to which the at least two or more types of nucleic acid probes are respectively fixed are arranged in parallel and in stripes with respect to the short side of the substrate,
The area of at least two types of regions to which the at least two types of nucleic acid probes are respectively fixed is 3 to 150 mm ² , and the width is 1 to 5 mm,
The at least two or more kinds of regions to which the at least two kinds of nucleic acid probes are respectively fixed are separated by 0.01 to 0.5 mm ,
The kit , wherein the nucleic acid probe has a base sequence that hybridizes with a specific wild-type base sequence and does not hybridize with a base sequence having a known mutation.

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