JP4779514B2 - Specimen and manufacturing method thereof - Google Patents

Description

  The present invention relates to a test piece for detecting a physiological complex reaction that can be used in a medical field, a manufacturing method thereof, and a detection method using the test piece. Specifically, the base material, the physiologically active substance is fixed, the physiologically active substance fixing region provided on the base material, and the colorimetric color provided on the base material are arranged around the physiologically active substance fixing region. The present invention relates to a test piece including a region, characterized by a color scheme of the physiologically active substance fixing region and the colorimetric region, a manufacturing method thereof, and a detection method using 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. Technology development for identification and diagnosis of diseases is 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 Document 1 discloses a line probe (LiPA: Line Probe Assay, trademark of Indogenetics) method 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 care or a part of diagnosis is required to be disposable like a test paper, the lithography technique has a problem in terms of manufacturing cost.

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, since the detection using the nucleic acid microarray disclosed in Patent Document 2 or 3 is performed using a specialized machine such as a scanner, it is difficult to make a direct visual determination. Moreover, since it is necessary to introduce a specialized machine, the burden of a medical institution is required.

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.

(ii) 該比色領域の物体色は、次式（２）の関係が成り立つ色である。

［ここで、式中のＬ^＊はＬ^＊ａ^＊ｂ^＊表色系（ＣＩＥ １９７６、またはＪＩＳ Ｚ ８７２９）における明度指数、ａ^＊およびｂ^＊はそれぞれ同表色系におけるクロマティクネス指数である］、
［２］ 生理活性物質が、核酸、抗原、抗体、リガンドまたはレセプターである［１］に記載の試験片、
［３］ 生理活性物質が、核酸である［１］に記載の試験片、
［４］ 比色領域が、該基材表面における該生理活性物質固定領域以外の領域全てである［１］に記載の試験片、
［５］ 基材と、生理活性物質が固定され、該基材上に備えた生理活性物質固定領域および該生理活性物質固定領域の周囲に配置し、該基材上に備えた比色領域を含む試験片であって、以下の(i)および(ii)の条件を満足する試験片の製造方法；
(i) 次式（１）の関係が成り立つ物体色を呈する基材を製造または準備する工程、

(ii) 次式（２）の関係が成り立つ物体色を着色することにより比色領域を設けると同時に、生理活性物質固定領域を定める工程、

(iii) 生理活性物質固定領域に生理活性物質を固定する工程
を含むことを特徴とする試験片の製造方法
［ここで、式中のＬ^＊はＬ^＊ａ^＊ｂ^＊表色系（ＣＩＥ １９７６、またはＪＩＳ Ｚ ８７２９）における明度指数、ａ^＊およびｂ^＊はそれぞれ同表色系におけるクロマティクネス指数である］、
［６］ 基材と、生理活性物質が固定され、該基材上に備えた生理活性物質固定領域および該生理活性物質固定領域の周囲に配置し、該基材上に備えた比色領域を含む試験片を用いて、発色反応により生理学的複合体形成反応を検出する方法であって、以下の(i)および(ii)の条件を満足することを特徴とする生理学的複合体形成反応検出方法；
(i) 該生理活性物質固定領域の物体色は、次式（１）の関係が成り立つ

(ii) 該比色領域の物体色は、次式（２）の関係が成り立つ

［ここで、式中のＬ^＊はＬ^＊ａ^＊ｂ^＊表色系（ＣＩＥ １９７６、またはＪＩＳ Ｚ ８７２９）における明度指数、ａ^＊およびｂ^＊はそれぞれ同表色系におけるクロマティクネス指数である］、
［７］ 発色反応が、酵素発色法によるものであることを特徴とする［６］に記載の検出方法、
および、［８］ 発色反応が、ＮＢＴ／ＢＣＩＰ発色法によるものであることを特徴とする［７］に記載の検出方法に関する。 The present invention
[1] A base material, a physiologically active substance is fixed on the base material, a bioactive substance fixing region provided on the base material, and a colorimetric region provided on the base material are arranged around the bioactive substance fixing region. A test piece that satisfies the following conditions (i) and (ii):
(i) The object color of the physiologically active substance fixing region is a color that satisfies the relationship of the following formula (1):

(ii) The object color in the colorimetric region is a color that satisfies the relationship of the following equation (2).

Here, L ^* in the formula is a lightness index in the L ^* a ^* b ^* color system (CIE 1976 or JIS Z 8729), and a ^* and b ^* are chromaticness indices in the same color system, respectively. ,
[2] The test piece according to [1], wherein the physiologically active substance is a nucleic acid, an antigen, an antibody, a ligand or a receptor,
[3] The test piece according to [1], wherein the physiologically active substance is a nucleic acid,
[4] The test piece according to [1], wherein the colorimetric regions are all regions other than the physiologically active substance fixing region on the substrate surface,
[5] A base material, a physiologically active substance fixed on the base material, a bioactive substance fixing region provided on the base material, and a colorimetric region provided on the base material are arranged around the bioactive substance fixing region. A test piece including the test piece that satisfies the following conditions (i) and (ii):
(i) producing or preparing a substrate exhibiting an object color satisfying the relationship of the following formula (1):

(ii) providing a colorimetric region by coloring an object color satisfying the relationship of the following formula (2), and simultaneously determining a physiologically active substance fixing region;

(iii) A method for producing a test piece comprising a step of fixing a physiologically active substance in a physiologically active substance fixing region [where L ^* is a L ^* a ^* b ^* color system (CIE 1976) Or brightness index in JIS Z 8729), a ^* and b ^* are chromaticness indices in the same color system],
[6] A base material, a physiologically active substance fixed on the base material, a bioactive substance fixing region provided on the base material, and a colorimetric region provided on the base material, arranged around the physiologically active substance fixing region. A method for detecting a physiological complex formation reaction by a color reaction using a test piece containing the test piece, wherein the physiological complex formation reaction detection satisfies the following conditions (i) and (ii): Method;
(i) The relationship of the following equation (1) holds for the object color of the physiologically active substance fixing region

(ii) The object color in the colorimetric region satisfies the relationship of the following equation (2)

Here, L ^* in the formula is a lightness index in the L ^* a ^* b ^* color system (CIE 1976 or JIS Z 8729), and a ^* and b ^* are chromaticness indices in the same color system, respectively. ,
[7] The detection method according to [6], wherein the color development reaction is performed by an enzyme color development method,
[8] The detection method according to [7], wherein the color development reaction is performed by an NBT / BCIP color development method.

  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.

  The test piece of the present invention refers to a test piece that can be easily determined visually by a special color scheme. 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 explanation focuses on the explanation of nucleic acid detection in view of the frequency of use, but the present invention 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 as appropriate 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 kind of the physiologically active substance immobilized on the test piece of the present invention can be appropriately set by those skilled in the art, and is not particularly limited.

  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 greater the number of nucleic acid probes and types. 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.

  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, at least 22 kinds of nucleic acid probes are required.

  In the present invention, a physiologically active substance-fixed region is a region where a physiologically active substance is fixed, and refers to a portion that develops color by a color development reaction in the detection of a physiological complex reaction, and an operator determines the presence or absence of a physiological complex reaction. This refers to a closed region provided on the surface of the substrate to be noted. The physiologically active substance fixing region can be defined at any position on the substrate. In particular, from the viewpoint of easy visual determination, it is preferable that all physiologically active substance fixing regions are on the same surface.

  Examples of the base material include organic materials such as polyethylene, polypropylene, polycarbonate, polymethyl methacrylate, polyamide, polystyrene, polyethylene terephthalate and nitrocellulose, inorganic materials such as glass and silica, and gold, silver and cylinders. Examples include metal materials. Among them, an organic material is preferable from the viewpoint of easy molding processability, and polypropylene, polycarbonate, polymethyl methacrylate, and polyamide are more preferable, but are not limited thereto. The base material and the physiologically active substance fixing region may be the same material or different materials.

Examples of the substrate include a substrate, a container, a film, and a tube. Among these, from the viewpoint of easy handling, a film and a substrate are preferable, but not limited thereto. Further, the size of the substrate is not particularly limited, but a surface to be detected needs a certain area from the viewpoint of facilitating detection. For example, when the substrate is a rectangular film or substrate, the area of the upper surface is about 40 to 1000 mm ² , preferably about 60 to 300 mm ² .

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

  The physiologically active substance fixing region in the present invention is a place where the physiologically active substance is fixed and develops color by a color development reaction, and thus exhibits an object color in a range that allows easy visual discrimination of color development. In the present invention, a color that satisfies the condition of the following formula (1) is meant as an object color in a range that allows easy visual discrimination of color development.

Here, L ^* in the above formula (1) is a lightness index in the L ^* a ^* b ^* color system (CIE 1976 or JIS Z 8729), and a ^* and b ^* are chromaticness indices in the same color system, respectively. It is.

The L ^* a ^* b ^* color system is a standard (CIE 1976 and JIS Z 8729) standardized by the International Commission on Illumination (CIE) in 1976 and adopted in JIS in Japan. Luminance) is a color system represented by L ^* , and chromaticity indicating hue and saturation is expressed by chromaticness indices a ^* and b ^* . a ^* and b ^* indicate the direction of the color, a ^* is a red direction, -a ^* is a green direction, b ^* is a yellow direction, and -b ^* is a parameter indicating a blue direction. The larger the value of a ^* and / or b ^*, the brighter the color, and the closer a ^* and b ^* are to 0, the duller the color.

FIG. 2 is a color tone diagram in which the vertical axis is L ^* and the horizontal axis is [(a ^* ) ² + (b ^* ) ² ] ^1/2 . Here, [(a ^* ) ² + (b ^* ) ² ] ^1/2 is a parameter indicating the saturation of the object color and may be expressed by C ^* . That is, the object color that satisfies the condition in the above formula (1) refers to the object color represented by the area A in FIG. 2, and the lightness (L ^* ) and saturation {[(a ^* ) ² + (b ^* ) ² ] ^1/2 or C ^* } need only be considered, and hue is not considered. In other words, it is a so-called white color. The object color of the physiologically active substance fixing region is not particularly limited as long as it is an object color that satisfies the condition of formula (1) and the color reaction can be easily determined visually. However, for example, white, reddish white, bluish white, yellowish white and greenish white in the Japanese Industrial Standard “Color Name of Object Color” (JIS Z 8102) can be mentioned. Of these, white is preferable from the viewpoint of making visual determination the easiest.

  In order to satisfy the condition of the above formula (1) for the object color of the physiologically active substance fixing region, for example, a method of forming a base material having a color that satisfies the condition of the above formula (1), the surface of the base material And a method of coating or painting a material (ink or the like) having a color satisfying the condition of the above formula (1). Among these, from the viewpoint of easy production, a method of forming the substrate itself so as to satisfy the condition of the above formula (1) is preferable, but the present invention is not limited to this.

  For example, when the base material is an organic material such as polypropylene and polycarbonate, the base material itself is molded and processed so as to satisfy the condition of the above formula (1). And a method of adding a material). Examples of the inorganic filler include titanium dioxide, but the present invention is not limited to this.

The above L ^* , a ^* and b ^* can be measured by a measuring device such as a commercially available colorimeter and spectrocolorimeter. In addition, the object color of the physiologically active substance fixing region is visually compared with a commercially available color sheet, the corresponding color is screened, and the RGB value of the color (decimal number or hexadecimal number does not matter), for example, Foundation Converted to L ^* , a ^*, and b ^* values using color system conversion software published on the contents of the Japan Color Research Institute website (URL: http://www.jcri.jp/) The method of doing is also mentioned. These methods can be appropriately selected by those skilled in the art and are not particularly limited. However, from the viewpoint of measurement sensitivity, a colorimeter and a spectrocolorimeter are preferable, and a spectrocolorimeter is more preferable. .

The shape of the physiologically active substance fixing region is not particularly limited and can be appropriately set by those skilled in the art depending on the method of fixing the physiologically active substance. For example, when a solution of a physiologically active substance is prepared and immobilized by dropping onto a substrate by a spotting method, a circular shape can be selected. Moreover, when immobilizing a physiologically active substance by an inkjet method and an extrusion method, a rectangular shape (band shape) can be selected. Further, the area of the physiologically active substance fixing region depends on the total area of the base material and the type of physiologically active substance to be fixed, and is not particularly limited, but from the viewpoint of facilitating visual determination, About 3 to 150 mm ² , preferably about 6 to 100 mm ² .

  The physiologically active substance is fixed in a physiologically active substance fixing region on the substrate surface. The physiologically active 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 dropping the physiologically active substance solution onto the physiologically active substance immobilization region. Examples of the dropping method include a spotting method, an extrusion method using a dispenser, an ink jet method, and the like. The spotting method and the extrusion method are preferable from the viewpoint of production cost, and the ink jet method is preferable from the viewpoint of immobilization accuracy.

  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, some treatment may be performed on the physiologically active substance fixing region. For example,
(A) a method of coating a surface of a physiologically active substance fixing region with a polycationic polymer such as polylysine,
(B) when the physiologically active substance fixing region is an inorganic material such as glass, a method of treating with a silane coupling agent having an amino group such as aminoethoxysilane,
And (C) when the physiologically active substance immobilization region is a metal material such as gold, a method of treating the surface of the carrier 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 a physiologically active substance fixing region at the end of a nucleic acid probe, or a terminal of a nucleic acid probe and a physiologically active substance fixing region And the like, and the like. For example,
(a) When the physiologically active substance immobilization region is an inorganic material such as glass or silicon, a functional group capable of silane coupling reaction such as trimethoxysilane or triethoxysilane is modified at the end of the probe for nucleic acid detection to A method of immobilization by a coupling reaction,
And (b) in the case where the physiologically active substance fixing region is an inorganic material such as glass or silicon, the physiological material is treated with a silane coupling agent having an amino group such as aminoethoxysilane on the inorganic material substrate. The surface of the active substance fixing region is aminated. On the other hand, the carboxylic acid is modified at the end of the nucleic acid probe. Examples thereof include a method in which an amide bond is formed and immobilized by an amino coupling reaction between the amino group on the surface of the physiologically active substance immobilization region and the carboxylic acid of the nucleic acid probe.
Even if the physiologically active substance immobilization region is a metal material such as gold or silver, those skilled in the art cannot immobilize the silane coupling bond by replacing it with a metal-thiol or metal-disulfide bond. It can be easily conceived.

  Among the above-described immobilization methods, with respect to immobilization of nucleic acids, from the viewpoint of preparation, it is preferable to add polythymine to the end of the nucleic acid probe and immobilize by UV irradiation, but the method is not limited thereto. is not.

  The test piece of the present invention includes a colorimetric region around the physiologically active substance fixing region. The colorimetric region refers to means indicated by a different color within a range visually distinguishable from the object color of the physiologically active substance fixing region. In the present invention, the color satisfying the condition of the following expression (2) is referred to as a different color within a range visually distinguishable from the object color of the physiologically active substance fixing region.

Here, L ^* in the above formula (2) is a lightness index in the L ^* a ^* b ^* color system (CIE 1976), and a ^* and b ^* are chromaticness indices in the color system.

The object color satisfying the condition of the above formula (2) refers to the object color represented by the area B in FIG. 4, and the lightness index (L ^* ) and the saturation {[(( Only a ^* ) ² + (b ^* ) ² ] ^1/2 or C ^* } needs to be considered, and hue is not considered. The object color of the physiologically active substance fixing region is not particularly limited as long as the object color satisfies the condition of the expression (2). For example, Japanese Industrial Standard “Color Name of Object Color” (JIS Z 8102), black, bluish black, dark gray, yellowish dark gray, medium gray, greenish gray, very dark purple, dark gray reddish purple, dark yellowish green, dull red, Examples include soft blue-green, dark green, strong yellow, and brilliant red.

  In the present invention, when a material having a metallic luster such as gold, silver and copper is used as the substrate, the color of the physiologically active substance fixing region is regarded as an object color that can be approximated by a color sample or the like. For example, it can be regarded as a dull yellow in the case of gold, and gray in the case of silver, and can be handled as an object color that satisfies the condition of Expression (2).

Here, the color satisfying the condition of the above formula (1) and the color satisfying the condition of the above formula (2) are color differences that can be clearly distinguished from each other by human eyes. In the L ^* a ^* b ^* color system, the color difference corresponds to a distance between a color satisfying the condition of the formula (1) and a color satisfying the condition of the formula (2). As ΔE ^* ab. The minimum color difference between the color satisfying the condition of the formula (1) and the color satisfying the condition of the formula (2) in the present invention is the same coordinate plane (for example, a ^* or b ^*) shown in FIG. It is only necessary to consider the minimum distance between the formula (1) and the formula (2) in (assuming a constant constant). This minimum distance is approximately 13.42. In general, if the color difference (ΔE ^* ab) between two different colors in the L ^* a ^* b ^* color system is 12 or more, it is said that the color difference is such that humans can clearly distinguish different colors. The present invention fully satisfies this condition. From the above, also in colorimetric logic, the color of the physiologically active substance fixing region and the colorimetric region in the present invention are color differences that can be clearly distinguished by the measurer, It is clear that the area where is fixed is clear.

The colorimetric region only needs to be large enough to identify the physiologically active substance fixing region.
For example, the region is at least 1 mm from the end of the physiologically active substance fixing region. In addition, from the viewpoint of facilitating visual determination, it is preferable to use a colorimetric region other than the physiologically active substance fixing region, but the present invention is not necessarily limited thereto.

  In order to satisfy the condition of the above formula (2) for the object color in the colorimetric area, for example, a base having a color that satisfies the condition of the formula (2) is used, for example, as in the physiologically active substance fixing area. It can be produced by a method of molding into a material, a method of coating or painting a material (ink or the like) having a color satisfying the condition of the above formula (2) on the surface of the substrate. Among them, a material having a color that satisfies the condition of the above formula (2) on the surface of the base material from the viewpoint that it is easy to form and process the base material to have an object color that satisfies the condition of the formula (1) ( A method of coating or painting an ink or the like is preferable, but the present invention is not limited thereto.

  In addition, the physiologically active substance is a base material such as polypropylene, polyamide or polycarbonate in which a hydrophilic polymer such as DNA is kneaded with an inorganic filler (a filler), that is, an object color satisfying the condition of the above formula (1) In the case of immobilization on a substrate having a colorant, a method of providing a colorimetric region by coating the surface of the substrate with a hydrophobic material (hydrophobic ink) that satisfies the condition of the above formula (2) is preferable. That is, the physiologically active substance fixing region is the base material itself, and the colorimetric region is a hydrophobic material. This is because when the DNA is immobilized, the hydrophobic colorimetric region repels the aqueous DNA solution, and thus the DNA can be immobilized only on the physiologically active substance-immobilized region.

  Hereinafter, the method for using the test piece of the present invention will be described with reference to the drawings, taking nucleic acid detection as an example. However, the following description can be appropriately set by those skilled in the art, and is limited to the nucleic acid detection method only. Is not to be done.

Test Piece FIG. 1 shows one embodiment of the test piece of the present invention. On the rectangular film 1, there is provided a physiologically active substance-fixing region 2 having a white color (L ^* = 93.7, a ^* = 0 and b ^* = 0) having four vertical and five horizontal squares. Strand DNA probes are immobilized. Further, a colorimetric region 3 having a black color (L ^* = 14.2, a ^* = 0 and b ^* = 0) is provided around the physiologically active substance fixing region 2.

Preparation of sample First, a target sample to be brought into 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.

Amplification of nucleic acid Next, a specific base sequence to be detected is preferably 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 made into a single strand by heat treatment under reaction conditions 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. These primers can be synthesized with an automatic synthesizer or the like.

  Furthermore, it is preferable to modify the detection substance at the end of the primer to further facilitate detection of the nucleic acid. Examples of the detection substance include an enzyme that enables a color development reaction (a substance that enables an enzyme color development method), a linker substance that can bind to these substances, and the like.

  Examples of the enzyme include alkaline phosphatase and peroxidase.

  The linker substance refers to a substance that can bind to the enzyme. For example, the linker substance affects a chemical bond such as a covalent bond, an ionic bond, a hydrogen bond, a disulfide bond and a coordination bond, and a physiological complex reaction in the present invention. It refers to a substance that enables biochemical binding such as antigen-antibody binding that does not affect. Among them, the test strip of the present invention is preferably a substance that enables antigen-antibody binding from the viewpoint of mainly used for detection of DNA and the viewpoint of production cost. As the form of the antigen-antibody bond, when an antigen is selected as a linker substance, it is necessary to separately prepare an enzyme modified with an antibody that can bind to the antigen. The opposite is true when an antibody is selected as the linker substance. An example of such an antigen-antibody bond is an avidin-biotin bond. Examples of the linker substance include biotin, avidin, polymyxin B, and IgG, and the NBT / BCIP method using a biotin-modified nucleic acid and a streptavidin-modified alkaline phosphatase for detection of nucleic acid is generally used. From the viewpoint of being used in common, avidin and biotin.

Hybridization Next, the amplified sample nucleic acid is brought into contact with the test piece of the present invention to perform a hybridization reaction. The conditions for the initial hybridization reaction depend on the glass transition 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.

Detection of Double-Stranded DNA DNA that has become double-stranded by the above hybridization can be visually confirmed by adding an enzyme substrate to cause color development. Examples of the substrate include a combination of p-nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (BCIP) when the enzyme is alkaline phosphatase. It is done. These chromogenic substrates satisfy the condition of the above formula (2) by enzymatic reaction.

  When the nucleic acid is detected using the test strip of FIG. 1 of the present invention as described above, the white region is filled in the region where the double-stranded DNA is formed by the color development reaction (FIG. 1 ( b)). On the other hand, the region where the double-stranded DNA was not formed remains white (FIG. 1 (c)). Thus, the test piece of the present invention can easily determine visually whether hybridization has been performed.

FIG. 3 is an embodiment different from FIG. 1 of the test piece of the present invention. A physiologically active substance immobilization region 2 having three vertical and six horizontal white colors (L ^* = 93.7, a ^* = 0 and b ^* = 0) is provided on a rectangular film 1. Further, the area around the physiologically active substance fixing area 2 is provided with a colorimetric area 3 having a dark gray color (L ^* = 32.7, a ^* = 0 and b ^* = 0). In appearance, a total of 18 circles are arranged, and the portion in circles is a physiologically active substance fixing region (FIG. 3A).

FIG. 4 is an embodiment different from that of FIGS. 1 and 3 of the test piece of the present invention. On the rectangular film 1, a bioactive substance fixing region 2 exhibiting white color (L ^* = 93.7, a ^* = 0 and b ^* = 0) and gray color (L ^* = 50.4, a ^* = The colorimetric regions 3 exhibiting 0 and b ^* = 0) are arranged alternately and are provided in the form of vertical stripes. (FIG. 4A).

  Even when the complex reaction is detected by a color development reaction using the test piece of FIG. 3 (a) or FIG. 4 (a), the physiologically active substance-fixed region 2 forming the physiological complex is visually Becomes filled with white (FIG. 3 (b) and FIG. 4 (b)). On the other hand, the physiologically active substance fixing region 2 that has not formed a physiological complex remains unfilled (FIGS. 3C and 4C). Thus, the measurer can easily determine visually whether or not a physiological complex reaction has been performed.

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

Example 1: Production of test piece DNA probe First, DNA probes each having the base sequences of SEQ ID NOs: 1 to 6 were synthesized. The DNA probes of SEQ ID NOs: 1 to 6 comprise 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: cggcgcgacc tcctctacgc
Sequence number 2: gacctcctct acgcgtggc
Sequence number 3: atgatcaacg cccgcatac
Sequence number 4: ggcgtcatgg accctatatc
Sequence number 5: caacagttca tcccggttc
Sequence number 6: gtcgttctgc acgtcgac

A polyamide membrane (200 mm × 300 mm) was selected as the substrate 1 on which the DNA probe was immobilized. This polyamide film was white (L ^* = 93.7, a ^* = 0 and b ^* = 0). Next, six square bioactive substance fixing regions and colorimetric regions were created using black (L ^* = 14.2, a ^* = 0 and b ^* = 0) oil-based ink. And the solution of said 6 types of DNA probe was apply | coated to each physiologically active substance fixed area | region 2. FIG. 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 test piece of the present invention was obtained by removing unadsorbed DNA probe by washing with 10 × SSC solution. Here, the fixing positions of the DNA probes of SEQ ID NOs: 1 to 6 correspond to 21 to 26 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: 3 corresponds to sample 2, and the probe DNA of SEQ ID NO: 6 corresponds to sample 3.

Amplification of Genomic DNA of Specimen A forward primer having the base sequence shown in SEQ ID NO: 7, a reverse primer having the base sequence shown in SEQ ID NO: 8 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 7: ggcgtcatgg accctatatc
Sequence number 8: 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. Since there was no mutation in the pncA gene of Sample 1, it hybridized with all the probes and developed a color so that all the white physiologically active substance immobilization regions were filled. On the other hand, samples 2 and 3 have no mutation at locations corresponding to the DNA probes of SEQ ID NO: 3 and SEQ ID NO: 6, respectively, so that they do not hybridize and the physiologically active substance immobilization region 2 remains white after the color development reaction. It remained. From the above, it was shown that the presence or absence of hybridization of the test piece of the present invention can be easily determined visually.

  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.

It is one embodiment of the test piece of this invention. It is a graph which shows Formula (1) and Formula (2) of this invention. It is the schematic diagram of the detection using one embodiment different from FIG. 1 of this invention, and the said test piece. It is the schematic diagram of the detection using one embodiment and the said test piece which are different from FIG. 1 and FIG. 3 of this invention. It is a figure which shows the detection result of Experimental example 1 using the test piece of Example 1 of this invention, and the said test piece.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Base material 2 Bioactive substance fixed area | region 21 DNA probe 22 which has a base sequence of sequence number 1 DNA probe 23 which has the base sequence of sequence number 2 DNA probe 24 which has the base sequence of sequence number 3 The base sequence of sequence number 4 DNA probe 25 having DNA probe 26 having the base sequence of SEQ ID NO: 5 DNA probe having the base sequence of SEQ ID NO: 6 3 Colorimetric region

Claims (1)

A base material made of polypropylene or polycarbonate kneaded with an inorganic filler, or nitrocellulose ;
Using a test piece on which a physiologically active substance is fixed and disposed around the physiologically active substance fixing region provided on the base material and the colorimetric region provided on the base material, A method for detecting a physiological complex formation reaction by NBT / BCIP color development method ,
A physiological complex detection method characterized by satisfying the following conditions (i) and (ii):
(i) The relationship of the following equation (1) holds for the object color of the physiologically active substance fixing region

(ii) The object color in the colorimetric region satisfies the relationship of the following equation (2)

Here, L ^* in the formula is a lightness index in the L ^* a ^* b ^* color system (CIE 1976 or JIS Z 8729), and a ^* and b ^* are chromaticness indices in the same color system, respectively.

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