JP2006217818A - Micro reactor for inspecting gene and method for inspecting gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a DNA inspection device which can cope many purpose gene inspections by suitably changing a DNA amplification primer loaded on a micro reactor for inspecting genes, a probe hybridizing with an amplification gene. <P>SOLUTION: This micro reactor can analyze highly sensitively, because of incorporating a DNA amplification process for detection, and can detect an objective gene in good accuracy with a probe DNA specifically hybridizing with an amplified gene. The DNA inspection device hardly causes acute problems such as cross contamination or carryover contamination for microanalysis, amplification analysis, because system-constituting separates a reagent/solution-sending system element-loading component and a control/detection component for each specimen. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a gene testing microreactor and a DNA testing device including the microreactor.

In recent years, by making full use of micromachine technology and ultrafine processing technology, devices and means (for example, pumps, valves, flow paths, sensors, etc.) for performing conventional sample preparation, chemical analysis, chemical synthesis, etc. have been miniaturized. Systems integrated on a chip have been developed. this is,
It is also called μ-TAS (Micro total Analysis System), bioreactor, Lab-on-chips, biochip, and its application is expected in medical examination / diagnosis field, environmental measurement field, agricultural production field Has been. In particular, as seen in genetic testing, when skilled procedures and equipment operations are required as well as complicated processes, automated, high-speed and simplified microanalysis systems are cost effective , Not only the required time but also any time and place can be analyzed. Therefore, it can be said that the benefits are great.

  In the field of various tests including clinical tests, importance is attached to the quantitativeness of analysis, the accuracy of analysis, and the economy of these analytical chips that produce results quickly regardless of location. Since the analysis chip is severely restricted by its size and form, it is a challenge to establish a highly reliable liquid delivery system with a simple configuration. Therefore, there is a need for a microfluidic control element that has high accuracy and excellent reliability. The present inventors have already proposed a micropump system suitable for this (Patent Documents 1 and 2 and Non-Patent Document 1).

  In addition, minimizing the amount of sample required and reducing the amount of reagent required is the greatest challenge for microreactors. In order to realize this, it is necessary to efficiently mix liquids (specimens and reagents) in the mixing channel or the reaction chamber. In addition, a mechanism capable of detecting or quantifying a minute amount of reaction product with high sensitivity and simply needs to be provided on the chip.

  Furthermore, it is desirable for chips to target a large number of samples to be measured, especially clinical samples that are at risk of contamination and infection. In addition, problems such as support for multi-item analysis and manufacturing costs are overcome. There is a need to.

The microreactor that provides simple and quick inspection means as described above has raised specific problems that should still be solved in practice, and the solution is desired.
JP 2001-322099 JP 2004-108285 A "DNA chip technology and its applications", "Protein Nucleic Acid Enzyme", Volume 43, No. 13 (1998), published by Fumio Kimizuka, Akinoshitsu Kato, Kyoritsu Publishing Co., Ltd.

  The microreactor for gene testing and the DNA testing device including the same according to the present invention have been made in view of the above-described circumstances, and incorporate a simple configuration and a high-accuracy liquid feeding system and can perform highly accurate detection. The purpose is to provide.

  An object of the present invention is to provide a disposable type microreactor and a genetic test apparatus equipped with control, detection and processing means for its function.

The present invention is a microreactor having a fine channel and at least a detection portion being a transparent plastic,
In order to trap the sample gene amplified using the biotin-labeled primer in the microchannel, the biotin affinity protein is located downstream of the gene amplification reaction site in the microchannel and is detected as polystyrene as the detection site. A microreactor for genetic testing characterized by being adsorbed.

The probe that hybridizes with the amplified gene is preferably labeled with peroxidase.
Furthermore, the probe is reacted with a coloring reagent.

The coloring reagent is preferably 3,3 ′, 5,5′-tetramethylbenzidine, 3,3′-diaminobenzidine, p-phenylenediamine, 5-aminosalicylic acid, 3-amino-9-ethylcarbazole as a coloring substance. 4-chloro-1-naphthol, 4-aminoantipyrine or o-
Contains dianisidine.

The present invention also provides the gene testing microreactor,
A micropump,
A micropump and a device for controlling the temperature, and a detection device for optically detecting the gene amplification reaction, and automatically detecting the gene amplification reaction and the amplification reaction by mounting the microreactor. It is a characteristic DNA testing device.

The micropump is
A first channel in which the channel resistance changes according to the differential pressure;
A second flow path in which a ratio of a change in flow path resistance to a change in differential pressure is smaller than the first flow path;
A pressurizing chamber connected to the first flow path and the second flow path;
A piezo pump provided with an actuator for changing the pressure inside the pressurizing chamber is desirable.

The detection device includes:
After the gene amplified in the microchannel is bound to the biotin-affinity protein adsorbed to the detection site of the microchannel, the hybridization product is obtained by hybridization with the probe DNA, or the microchannel The hybridization product obtained by hybridizing the amplified gene and probe DNA is bound to the biotin-affinity protein adsorbed on the detection site of the microchannel, and then the probe DNA is converted into a colored substance. It is the device which detects optically by doing.

The method for producing a detection site of a microreactor for genetic testing according to the present invention comprises a biotin-affinity protein for trapping a gene amplified in a microreactor channel, downstream of the gene amplification reaction site in the channel. To immobilize the amplified gene as a detection site, biotin-affinity protein is dissolved in SSC buffer or physiological saline to prepare a solution with a concentration of 10 to 35 μg / mL. The solution is applied to the channel to adsorb the biotin-affinity protein onto the fine channel.

The method for detecting an amplified gene of the present invention comprises:
Mixing the reaction solution containing the gene amplified at the gene amplification reaction site in the microchannel of the microreactor with the denaturation solution, and denaturing the amplified gene into a single strand;
The treatment solution obtained by denaturing the amplified gene into a single strand is sent to the detection site of the microchannel where the biotin-affinity protein is adsorbed to polystyrene, and the amplified gene is sent to the detection site. Trapping process;
Sending a probe DNA modified with peroxidase to the detection site, and hybridizing the amplified gene and the probe DNA;
Then, a solution of a coloring reagent containing a coloring substance is sent to the detection site where the amplified gene is trapped, and the coloring substance is colored by a reaction catalyzed by the peroxidase;
And a step of optically measuring the color development at the detection site.

  The microreactor of the present invention enables highly sensitive gene analysis because it incorporates a DNA amplification step prior to detection, and uses a probe DNA that specifically hybridizes to the amplified gene to accurately detect the target gene. can do.

  In addition, the DNA testing device of the present invention has a system configuration in which a chip component for each specimen, on which elements for reagents and a liquid feeding system are mounted, and a control / detection component as a DNA testing device main body are separated. Serious problems such as cross-contamination and carry-over contamination are less likely to occur in analysis and amplification reactions. Since it is easy to wash and remove non-specific binding substances other than the binding (or interaction) between the sample DNA and the primers and probes, it is possible to provide a microreactor chip with a low background.

The microreactor of the present invention has a configuration suitable for mass production including materials and components, and is compatible with multi-item measurement. The amplified target gene trap and its detection method are simple techniques that have already been established, and the probes and reagents used for detection are easily available. Can be manufactured at low cost.
Detailed Description of the Invention
Hereinafter, a microreactor of the present invention, a DNA test device comprising the microreactor, each control device, and a detection device, and a gene test method including gene amplification and detection using the device and the microreactor will be described. In the present specification, “gene” refers to DNA or RNA carrying genetic information that expresses some function, but may also be simply referred to as DNA or RNA that is a chemical entity. The “base” refers to a nucleotide nucleobase.
Overview of Microreactor and DNA Testing Device First, the microreactor and DNA testing device of the present invention will be outlined. FIG. 1 is a schematic diagram of a microreactor for gene testing in one embodiment of the present invention, and FIG. 2 is a DNA testing device (also referred to as “gene testing apparatus”) comprising the microreactor and the apparatus main body in one embodiment of the present invention. FIG.

The microreactor shown in FIGS. 1 and 2 is composed of a single chip manufactured by appropriately combining members such as plastic resin, glass, silicon, and ceramics. Preferably, the fine flow path and the casing of the microreactor are formed of a plastic resin that is easy to process and inexpensive, and easy to dispose of by incineration. Among them, polystyrene resin is desirable because it has excellent moldability, has a strong tendency to adsorb streptavidin and the like as will be described later, and can easily form a detection site on a fine channel. Further, in such a microreactor, in order to optically detect the product of the hybridization reaction, at least the detection portion covering the detection site on the fine channel on the surface of the microreactor is transparent, preferably a transparent plastic. It is necessary to be.

  The chip includes a sample storage unit, a reagent storage unit, a probe DNA storage unit, a control storage unit, a flow path, a pump connection unit, a liquid feed control unit, a backflow prevention unit, a reagent quantitative unit, and a mixing unit. It is installed at an appropriate position by microfabrication technology. If necessary, a reverse transcriptase part may be provided. The sample storage unit communicates with the sample injection unit, and temporarily stores the sample and supplies the sample to the mixing unit. In some cases, the sample storage unit may be provided with a pretreatment function such as viscosity adjustment of the sample liquid or blood cell separation. The mixing of the reagent and the reagent and the mixing of the sample and the reagent may be performed at a desired ratio in a single mixing section, or a plurality of confluence sections are provided by dividing one or both, and finally You may mix so that it may become a desired mixing ratio.

  The microreactor for gene testing of the present invention injects a sample such as blood or sputum into a sample container and attaches it to a DNA testing device body, thereby performing a gene amplification reaction and processing necessary for detecting the amplification reaction in the chip. It is configured to automatically perform genetic testing for multiple items at the same time and in a short time. In a preferred embodiment of the DNA testing device of the present invention, a predetermined amount of reagents to be used in the microreactor are encapsulated in advance, and as a unit for detecting a sample DNA or RNA, a predetermined amplification reaction, and an amplification product, A microreactor is used for each specimen.

  On the other hand, a micropump, a control system related to temperature and reaction control, an amplification reaction detection, data collection and processing apparatus, together with a micropump and a detection apparatus for optically detecting an amplification reaction, are in accordance with the present invention. The apparatus main body in which the DNA testing device is integrated is constructed. This device main body is commonly used for the sample group by mounting the microreactor thereon. Therefore, even if there are a large number of samples, they can be processed efficiently and quickly. In the prior art, when analyzing or synthesizing different contents, it is necessary to configure a microchannel device corresponding to the contents to be changed each time. Unlike this, in the present invention, only the removable chip needs to be replaced. If it is necessary to change the control of each device element, the control program stored in the apparatus main body may be appropriately modified.

  In the DNA testing device of the present invention, all components are miniaturized and are in a form that is convenient to carry. Therefore, the DNA testing device is not restricted by the place and time of use, and has good workability and operability. Since the specimen can be analyzed promptly regardless of the place and time, it can be used for emergency medical care or for personal use at home medical care. Since a large number of micropump units and the like used for liquid feeding are also incorporated in the apparatus main body, the chip has a relatively simple configuration and can be used as a disposable type.

The outlines of the microreactor and DNA test device (gene test apparatus) for genetic testing of the present invention have been described above, but the present invention can be arbitrarily modified and changed in various embodiments in accordance with the spirit of the present invention. And they are included in the present invention. That is, the structure, configuration, arrangement, shape, dimensions, material, method, method, etc. of the microreactor and the inspection apparatus of the present invention may be variously modified as long as they meet the spirit of the present invention. it can.
Preferred embodiment of microreactor for genetic testing A preferred embodiment of the microreactor for genetic testing of the present invention is as follows.
A sample container into which a sample or DNA extracted from the sample is injected;
A reagent container for storing a reagent used in the gene amplification reaction;
A positive control accommodating part for accommodating a positive control;
A negative control accommodating portion for accommodating a negative control;
A probe DNA containing portion that contains probe DNA that hybridizes to a gene to be detected amplified by a gene amplification reaction;
A flow path communicating with each of these accommodating portions,
A pump connection unit that can be connected to a micropump unit of a separate device that feeds the liquid in each storage unit and the flow path, and is provided.
A micropump is connected to the chip via a pump connection unit, and the sample stored in the sample storage unit or the DNA extracted from the sample and the reagent stored in the reagent storage unit are fed to the flow channel. After mixing and amplification reaction at the gene amplification reaction site, the processing solution treated with this amplification reaction product is sent to the downstream flow path where the detection site is located, and the probe accommodated in the probe DNA accommodating portion DNA is mixed and hybridized in the flow path, amplification reaction is detected for this hybridization product,
Similarly, for the positive control housed in the positive control housing section and the negative control housed in the negative control housing section, after amplification reaction in the flow path with the reagent housed in the reagent housing section, It is characterized in that it is configured to hybridize with the accommodated probe DNA in the flow path, and to detect the amplification reaction for this hybridization product.

The gene testing microreactor further injects a sample or RNA extracted from the sample into the sample storage unit, and stores reverse transcriptase for synthesizing cDNA from the RNA contained therein by a reverse transcription reaction. A photoenzyme storage unit is provided,
After the sample contained in the sample storage unit or the RNA extracted from the sample and the reverse transcriptase stored in the reverse transcriptase storage unit are fed to the flow channel and mixed in the flow channel to synthesize cDNA, You may be comprised so that the said amplification reaction and its detection may be performed.
Amplification of the gene amplification reaction site specimen gene is performed as follows at a predetermined position in the microchannel in the microreactor of the present invention, that is, the gene amplification reaction site. A specimen storage section in which the DNA solution is stored and a reagent storage section in which the reagent liquid is stored in each flow path upstream of the merging section that joins the DNA solution containing the DNA to be amplified and the reagent liquid. And are provided. A pump connection part is provided on the upstream side of each of these accommodating parts, and a separate micropump of the apparatus is connected to these pump connecting parts, and the driving solution is supplied from each micropump to supply the DNA solution in each of the accommodating parts. The gene amplification reaction is started by extruding the reagent liquid and joining them together. The form of such an amplification reaction site is not particularly limited, and various forms and modes are conceivable. Basically, at least two kinds of liquids containing the reaction reagent are sent together by a micropump and joined together,
A fine channel provided in advance from the merging portion and in which each of the liquids is diffusively mixed;
A liquid reservoir portion that is provided first from the downstream end of the fine flow path, and has a space wider than the fine flow path, and stores a mixed liquid that is diffusely mixed in the fine flow path to perform a reaction; It is preferable to provide.
-Sample In the case of genetic testing, the sample to be measured according to the present invention is a gene, DNA or RNA as a nucleic acid that serves as a template for an amplification reaction. It may be prepared or isolated from a sample that may contain nucleic acid (DNA or RNA) of such an analyte. A method for preparing a gene, DNA or RNA from such a sample is not particularly limited, and conventional techniques can be used. Recently, a technique for preparing a gene, DNA or RNA from a biological sample for DNA amplification has been developed and can be used in the form of a kit or the like.

There are no particular restrictions on the sample itself, but most biological samples such as whole blood, serum, buffy coat, urine, feces, saliva, sputum; cell cultures; viruses, bacteria, molds, yeasts, plants, animals, etc. This includes nucleic acid-containing samples; samples that may contain or contain microorganisms, and other samples that may contain nucleic acids.

  DNA can be separated and purified from the above sample by phenol / chloroform extraction and ethanol precipitation according to a conventional method. It is generally known to use high concentration chaotropic reagents such as guanidine hydrochloride, isothiocyanate close to saturation to release nucleic acids. Instead of applying the above-mentioned phenol-chloroform extraction method and the like, a method of directly treating a specimen with a proteolytic enzyme solution containing a surfactant (Takashi Saito, “PCR Experiment Manual” HBJ Publishing Bureau, 1991, p309) It is a simple and quick method. When the obtained genomic DNA or gene is large, it may be fragmented according to a conventional method using an appropriate restriction enzyme such as BamHI, BgLII, DraI, EcoRI, EcoRV, HindIII, PvuII and the like. In this way, an aggregate of DNA for a specimen and fragments thereof can be prepared.

  In the case of RNA, there is no particular limitation as long as a primer used for the reverse transcription reaction can be prepared. For example, in addition to total RNA in a sample, RNA molecules such as retrovirus RNA functioning as a gene, mRNA that is a direct information carrier of the expressed gene, and rRNA are targeted. These RNAs may be analyzed after being converted to cDNA using an appropriate reverse transcriptase. The method for preparing mRNA can be performed based on a known technique, and reverse transcriptase can be easily obtained.

In the microreactor of the present invention, the amount of sample required is extremely small compared to the case of manual work performed using a conventional inspection apparatus. For example, in the case of a gene, the DNA is 0.001 to 100 ng. For this reason, the microreactor according to the present invention includes a case where only a very small amount of sample can be obtained, and there are few restrictions on the specimen surface, and the amount of reagents is inevitably small, and the test cost is reduced. The sample is introduced from the injection part of the “sample storage part”.
-Amplification method The amplification method of DNA is not limited in the microreactor of the present invention. For example, a DNA amplification technique can use a PCR amplification method which is currently widely used in various fields. Various conditions for implementing the amplification technique have been examined in detail and described in various documents including improvements. In PCR amplification, it is necessary to manage the temperature by raising and lowering between three temperatures. However, a flow channel device capable of controlling temperature suitable for a microchip has already been proposed by the present inventors (Japanese Patent Laid-Open No. 2005-260260). 2004-108285). This device system may be applied to the amplification channel of the chip of the present invention. As a result, the heat cycle can be switched at high speed, and the microchannel is made into a micro reaction cell with a small heat capacity, so that DNA amplification can be carried out in a much shorter time than the conventional method that is performed manually in microtubes, microvials, etc. It can be carried out.

The recently developed ICAN® (Isothermal chimera primer initiated nucleic acid amplification) method does not require complicated temperature control as in the conventional PCR reaction. That is, DNA amplification can be performed in a short time at an arbitrary constant temperature of 50 to 65 ° C. (Japanese Patent No. 3433929). Therefore, the ICAN (registered trademark) method, which requires simple temperature control, is a particularly suitable amplification technique in the microreactor of the present invention. By hand, the method, which takes one hour, ends with the analysis of collected data in 10-20 minutes, preferably 15 minutes, in the bioreactor of the present invention.

The DNA amplification reaction may be another PCR improvement method or PCR modification method, and the microreactor of the present invention is flexible enough to cope with any change in the design of the flow path. When using any DNA amplification reaction, details of the technique are disclosed, and those skilled in the art can easily introduce them.
・ Reagents
(i) Primers PCR primers are two kinds of oligonucleotides complementary to both ends of a specific site of a DNA strand to be amplified. Dedicated application software has already been developed for the design, and those skilled in the art can easily create the design using a DNA synthesizer, chemical synthesis, or the like. The primer for the ICAN method is a chimeric primer of DNA and RNA, but the preparation method thereof has already been technically established (Japanese Patent No. 3433929). It is important to use the most appropriate primer design and selection to determine the success and efficiency of the amplification reaction.

In addition, when biotin is bound as a label to the 5 ′ end of the primer, the amplification product D
NA can be immobilized on the substrate via binding to streptavidin adsorbed on the chip substrate, and can be used for quantification of the amplification product. Examples of other primer labeling substances include digoxigenin and various fluorescent dyes.
(ii) Reagents for amplification reaction Reagents including enzymes used for the amplification reaction can be easily obtained for both PCR and ICAN methods.

Reagents for PCR include at least 2′-deoxynucleoside 5′-triphosphate, Taq DNA polymerase, Vent DNA polymerase, or Pfu DNA polymerase.

Reagents in the ICAN method include at least 2′-deoxynucleoside 5′-triphosphate, a chimeric primer that can specifically hybridize to a gene to be detected, a DNA polymerase having strand displacement activity, and an RNase of endonuclease.
(iii) Control The internal control is used as an internal standard substance for monitoring or quantifying amplification of the target nucleic acid (DNA, RNA). The internal control sequence can be amplified in the same manner as the sample because it has sequences that can hybridize to the same primer as the sample primer on both sides of the sequence different from the sample DNA. The sequence of the positive control is a specific sequence for detecting the sample, and the portion where the primer hybridizes and the sequence between them are the same as the sample. Nucleic acids (DNA, RNA) used for control may be those described in known technical literature. The negative control includes all reagents other than nucleic acids (DNA, RNA) and is used for checking for the presence of contamination and for background correction.
(iv) Reagent for reverse transcription In the case of an RNA sample, there are a reverse transcriptase and a reverse transcription primer for synthesizing cDNA from RNA, and these are also commercially available and can be easily obtained.

A predetermined amount of each of these amplification substrates (2′-deoxynucleoside 5′-triphosphate), gene amplification reagents, and the like is enclosed in advance in the reagent storage section of one microreactor. Therefore, the microreactor of the present invention does not need to be filled with a necessary amount of reagent each time it is used, and is ready for immediate use.
In general, detection methods such as visible spectrophotometry, fluorescence measurement, and luminescence luminescence are used as methods for detecting the amplified DNA of the target gene. Furthermore, techniques such as an electrochemical method, surface plasmon resonance, and quartz crystal microbalance are also included.

In the microreactor for gene testing of the present invention, a detection site for detecting the amplified gene is provided on the downstream side of the gene amplification reaction site in the fine channel. At least the detection part of the microreactor is transparent, preferably transparent plastic, to allow optical measurements. Furthermore, a biotin affinity protein for trapping the amplified gene is adsorbed to a detection site on the fine channel. It is desirable that at least the fine channel is made of polystyrene.

  The DNA testing device of the present invention detects the presence / absence, scale, etc. of the amplification reaction for the hybridization product resulting from the hybridization of the amplified gene and the probe DNA in conjunction with the process at the detection site of the microreactor. A detection device is provided. By appropriately selecting the base sequence of the probe DNA, it becomes possible to detect with high sensitivity without being affected by other DNA and background that specifically bind to the target gene and coexist.

The method for detecting the amplified DNA of the target gene is not particularly limited, but is preferably carried out in the following steps as a preferred embodiment. That is, using the microreactor,
(1) A sample or a DNA extracted from a sample, or a cDNA synthesized from a sample or RNA extracted by a reverse transcription reaction, and a biotin-modified primer at the 5 ′ position are sent from these containers to a flow path. And a step of amplifying the gene at the gene amplification reaction site in the fine channel,
(2) a step of mixing an amplification reaction solution containing a gene amplified in a fine channel and a denaturing solution, and denaturing the amplified gene into a single strand;
(3) A treatment solution obtained by denaturing the amplified gene into a single strand is sent to a detection site in a microchannel where biotin-affinity protein (preferably streptavidin) is adsorbed to polystyrene, and the amplified solution is amplified as described above. Binding the gene to a biotin affinity protein and trapping it,
(4) A step of flowing a probe DNA fluorescently labeled with FITC (fluorescein isothiocyanate) to the portion where the amplified gene is trapped and hybridizing to the gene, (5) trapped at the detection site in the microchannel Flowing a colloidal gold solution whose surface has been modified with an anti-FITC antibody that specifically binds to FITC to the amplified gene, thereby adsorbing the colloidal gold to the FITC-modified probe hybridized to the immobilized gene;
(6) a step of optically measuring the concentration of colloidal gold in the fine channel,
A method including:

Examples of the biotin affinity protein include avidin, streptavidin, and extraavidin (R). These avidins have four biotin binding sites, and streptavidin is particularly desirable because it exhibits a high degree of binding specificity to biotin and can form a strong bond. The solution obtained by dissolving streptavidin in a buffer solution is adsorbed by applying it to the portion to be immobilized in the fine channel. The present inventors have clarified suitable conditions for adsorbing the protein derived from Streptomyces avidinii in the microchannel. Details thereof are described in Examples below. Surprisingly, according to the method, no special chemical treatment is required when immobilizing streptavidin in the microchannel formed in the polystyrene substrate. Simply dissolve the biotin-affinity protein in SSC buffer or physiological saline to prepare a solution with a concentration of 10-35 μg / mL, preferably 20-30 μg / mL, and place it on the microchannel downstream of the amplification reaction site. It is only necessary to adsorb the biotin-affinity protein on the flow path provided on the polystyrene substrate. By immobilizing streptavidin in this way, a detection site for trapping the amplified gene can be provided very simply. In order to increase the amount of streptavidin adsorbed, the surface area of the detection site may be enlarged by providing fine irregularities, for example, filaments, in the portion where polystyrene is adsorbed. Alternatively, a fine polystyrene strip on which streptavidin is separately adsorbed or immobilized may be laid, or a porous material may be used.

As the probe DNA, a fluorescently labeled oligodeoxynucleotide is preferably used. As the DNA base sequence, a sequence that is complementary to a part of the gene base sequence to be detected is selected. As the fluorescent dye, a known fluorescent dye can be used. For example, fluorescent materials such as general FITC, RITC (rhodamine isothiocyanate), NBD, Cy3, and Cy5 are shown. In particular, FITC is desirable because anti-FITC antibodies such as gold colloid anti-mouse IgG are available. The probe DNA may be labeled with steroid hapten digoxigenin (DIG) instead of the fluorescent dye. In this case, an anti-DIG-alkaline phosphatase labeled antibody is used as an alternative to the anti-FITC antibody.

  In the above method, biotinylated DNA, immobilization by biotin-streptavidin bond, FITC fluorescent labeling, anti-FITC antibody, primer and probe design techniques, primer and probe preparation techniques, etc. are known techniques. Nucleic acid hybridization is also a conventional technique, but its scale and efficiency vary greatly depending on various conditions. A method was described in which the amplified gene was trapped on streptavidin via biotin labeled on the primer, and then hybridized with this. In some cases, the amplified gene and probe DNA were hybridized in the reverse order. Thereafter, the obtained hybridization product may be trapped on the streptavidin via biotin labeled on the primer. If the specific base sequence of the amplified gene trapped in streptavidin is complementary to the base sequence of the probe DNA, it will hybridize suitably. In any case, the probe DNA is converted into a colored substance and optically detected by the detection apparatus of the DNA test device of the present invention.

It is also possible to measure the fluorescence of the fluorescent dye FITC. However, it is necessary to consider light fading of the fluorescent dye, background noise, and the like. For this reason, a method capable of finally measuring with high sensitivity by visible light is preferable. The DNA test device of the present invention utilizes optical detection of colloidal gold using colloidal gold anti-mouse IgG. Alternatively, the probe DNA may be labeled with horseradish peroxidase (HRP) instead of the fluorescent dye. A coloring reaction catalyzed by the HRP enzyme can also be used. As typical coloring substances for that purpose, 3,3 ′, 5,5′-tetramethylbenzidine (TMB), 3,3′-diaminobenzidine (DAB),
Known are p-phenylenediamine (OPD), 5-aminosalicylic acid (5AS), 3-amino-9-ethylcarbazole (AEC), 4-chloro-1-naphthol (4C1N), 4-aminoantipyrine, o-dianisidine, etc. It has been. A coloring reagent containing any one of these coloring substances is reacted with the peroxidase of the probe to color the substance.

In addition to peroxidase, enzyme / coloring systems such as alkaline phosphatase and galactosidase can also be used.
Such visible light absorption analysis is superior to fluorescence photometry because the devices used are more general, and there are few interference factors and data processing is easy. In the DNA testing device of the present invention, an optical detection device for this purpose is incorporated and integrated with a liquid feeding means including the micropump and a temperature control device for controlling the reaction temperature of each reaction in the flow path of the microreactor. It is desirable that

  In addition, it is desirable that a step of feeding a cleaning liquid into the flow path in which streptavidin is adsorbed as necessary is included between the steps. As such a cleaning solution, for example, various buffer solutions, salt aqueous solutions, organic solvents and the like are suitable. In the above step, the denaturing solution is a reagent for making the gene DNA into a single strand, and examples thereof include sodium hydroxide and potassium hydroxide.

From the above, the following method is also included in the present invention.
As a method for preparing a detection site of a microreactor for genetic testing, a biotin-affinity protein for trapping a gene amplified in a microreactor channel is used, and a detection site for an amplified gene is located downstream from the amplification reaction site in the channel. In order to immobilize, biotin-affinity protein is dissolved in SSC buffer or physiological saline to prepare a solution having a concentration of 10 to 35 μg / mL, and the solution is put into a fine channel formed using polystyrene. It is a production method including applying and adsorbing a biotin-affinity protein onto the channel.

The method for detecting an amplified gene of the present invention includes:
Mixing the reaction solution containing the gene amplified at the gene amplification reaction site in the microchannel of the microreactor with the denaturation solution, and denaturing the amplified gene into a single strand;
The treatment solution obtained by denaturing the amplified gene into a single strand is sent to the detection site of the microchannel where the biotin-affinity protein is adsorbed to polystyrene, and the amplified gene is sent to the detection site. Trapping process;
Sending a probe DNA modified with peroxidase to the detection site, and hybridizing the amplified gene and the probe DNA;
Then, a solution of a coloring reagent containing a coloring substance is sent to the detection site where the amplified gene is trapped, and the coloring substance is colored by a reaction catalyzed by the peroxidase;
And a step of optically measuring the color development at the detection site.

In the above amplification and detection, the program stored in the software stored in the DNA testing device of the present invention has various conditions set in advance with respect to the liquid delivery sequence, volume, timing, etc., together with the micropump and temperature control. . When the DNA testing device body in which such software, the micropump, the detection device, and the temperature control device are integrated with the microreactor detachable from the device body, the flow path of the microreactor is also in an operating state. It becomes. Analysis is preferably started automatically by sample injection, and sample and reagent feeding, mixing-based gene amplification reaction, gene detection reaction and optical measurement are automatically performed as a series of continuous steps. As a result, the measurement data is stored in a file together with necessary conditions and recorded items.
Genetic Testing In the genetic testing microreactor and DNA testing device of the present invention, two genetic testing aspects are mainly formed by the gene amplification technology and the hybridization technology employed as detection methods. First, by using a primer having a specific sequence for a specific gene, usually a target gene, as a primer to be used in a gene amplification reaction, the presence or absence of amplification or the efficiency of amplification can be measured to derive the gene from the sample. Can be used for determining whether the target DNA is the same or different from the target gene. In particular, it is effective for rapidly identifying or determining a virus or bacterium causing an infectious disease from a gene. When performed as allele-specific PCR using allele-specific oligonucleotides as PCR oligomers, even slight mutations between alleles on homologous chromosomes can be detected.

  By designing the nucleotide sequence of the probe DNA that hybridizes to the amplified gene DNA so as to be complementary to the target gene, specific binding of the probe is achieved and detection accuracy is improved. Alternatively, the present invention can be applied to detection of gene mutations using a mismatch with a synthetic probe in hybridization as an index.

  By the genetic test according to the present invention, data for diagnosing the degree of expression of oncogenes, hereditary hypertension genes and the like can be obtained. Specifically, it is based on analysis of the kind and expression level of mRNA that is evidence of such gene expression.

Alternatively, determination of genetic predisposition indicating susceptibility to specific diseases, gene mutations involved in side effects on drugs, and other mutations in the coding region as well as in the promoter region of regulatory genes are also detected by genetic testing using the microreactor of the present invention can do. In that case, a primer having a nucleobase sequence containing a mutation portion is used. The gene mutation means a mutation in the nucleotide base of the gene. Furthermore, the analysis of gene polymorphism by using the DNA test device of the present invention is useful for identification of disease susceptibility genes.

  Compared with conventional nucleic acid sequence analysis, restriction enzyme analysis, and nucleic acid hybridization analysis, the genetic test method using the DNA test device of the present invention obtains a highly accurate result with a much smaller sample amount, a little effort, and a simple apparatus. It is clear from the principle of the device configuration and the analysis method used.

  The gene testing microreactor, gene testing device, etc. of the present invention include gene expression analysis, gene function analysis, single gene polymorphism analysis (SNP), clinical testing / diagnosis, pharmaceutical screening, pharmaceuticals, pesticides, or the safety of various chemical substances. -It can be used in the fields of toxicity testing, environmental analysis, food testing, forensic medicine, chemistry, brewing, fishery, livestock, agricultural production, agriculture and forestry.

  The present invention will be further described below with reference to the drawings shown as examples of preferred embodiments of the present invention. The present invention is not limited to such an aspect, and can be implemented in various other aspects.

The microreactor consisting of a plastic resin chip shown in the flow chart of FIG. 1 injects a gene sample extracted from blood and sputum, thereby allowing gene amplification reaction and detection thereof by the ICAN (registered trademark) method in the chip. It is configured so that genetic diagnosis can be performed on multiple items simultaneously. For example, an amplification reaction and its detection can be performed by mounting a chip on the apparatus main body 2 of FIG. 2 simply by dropping a blood sample of about 2 to 3 μL onto a chip having a length and width of several centimeters. .

The sample injected into the sample storage unit 20 of FIG. 1 and a reagent used in a gene amplification reaction previously enclosed in the reagent storage units 18a to 18c (a biotin-modified chimeric primer that specifically hybridizes to a gene to be detected, A DNA polymerase having a strand displacement activity and an endonuclease) are fed to a flow path communicating with each housing portion by a micropump (not shown) incorporated in the apparatus main body of FIG. The sample and the reagent are mixed in the flow path via the and the amplification reaction is performed. The flow path is formed to have a width of several tens to several hundreds μm, preferably 100 to 200 μm, and a depth of 25 to 200 μm, preferably 50 to 100 μm. The DNA amplified in this way is detected by optically measuring the concentration of colloidal gold. Specifically, it is detected by an optical detection device (not shown) incorporated in the apparatus main body 2 of FIG. For example, measurement light is irradiated from a LED or the like to a detection site on the flow path for each inspection item, and transmitted light or reflected light is detected by light detection means such as a photodiode, a CCD camera, or a photomultiplier tube. Thus, the amplified DNA (gene) labeled through the hybridized probe DNA can be detected.

  The apparatus main body 2 also incorporates a temperature control device for controlling the reaction temperature, and a chip in which a reagent is previously sealed is attached to a small unit in which a liquid feed pump, an optical detection device, and a temperature control device are integrated. A simple genetic diagnosis can be performed.

In the present embodiment, the microreactor is configured in the following manner in order to perform highly accurate and quick reliable genetic testing with a single chip.
First, all controls are integrated on a single chip, and internal control, positive control and negative control are enclosed in a microreactor in advance, and the amplification reaction of these controls is performed simultaneously with the sample amplification reaction and detection operation. And a detection operation is performed. As a result, a highly reliable genetic test can be performed quickly.

  Secondly, at each predetermined position of the flow path, the liquid is blocked from passing until the liquid supply pressure in the positive direction reaches a preset pressure. In order to allow the passage of the liquid, a liquid feed control unit that controls the passage of the liquid by the pump pressure of the micropump and a backflow prevention unit that prevents the backflow of the liquid in the flow path are provided. As will be described later, the liquid feed in the flow path is controlled by the micropump, the liquid feed control unit, and the backflow prevention unit, and the reagent can be quantitatively fed with high accuracy and introduced from the branch flow channel. In addition, a plurality of reagents can be mixed rapidly.

Next, an amplification reaction using the microreactor of the present embodiment and a detection operation thereof will be specifically described in association with main components of the microreactor.
Reagent storage unit The microreactor 1 is provided with a plurality of reagent storage units 18 for storing each reagent, and is used for a gene amplification reaction, a denaturing solution for denaturing the amplified gene, and hybridizing with the amplified gene. The probe DNA to be stored is accommodated.

  It is desirable that a reagent is stored in the reagent storage unit 18 in advance so that the inspection can be performed quickly regardless of the place and time. Reagents contained in the chip are sealed on the surface of the reagent storage portion to prevent evaporation, leakage, mixing of bubbles, contamination, denaturation, and the like. Further, when the microreactor is stored, the reagent is sealed with a sealing material in order to prevent the reagent from leaking into the fine flow path from the reagent container and reacting with the reagent. When a reagent is stored in advance in the microreactor, it is desirable to store the microreactor in a refrigerator for the stability of the reagent. This sealant is solidified or gelled under refrigerated conditions in which the microreactor is stored before use, and melts into a fluid state at room temperature during use. It is preferable to seal the reagent in the reagent container by filling a sealant between the reagent and the flow path 15 communicating with the reagent container 18. Note that air may be interposed between the sealant and the reagent, but it is preferable that the amount of air interposed is sufficiently small (relative to the reagent amount) from the viewpoint of quantitative liquid feeding.

As such a sealant, a plastic material that is hardly soluble in water can be used, and an oil or fat having a solubility in water of 1% or less is preferable. Such fats and oils can be examined with a handbook of fats and oils. In addition, you may fill with a sealing agent similarly between each accommodating part which accommodates positive control and negative control, and the flow path connected to this.
-Pump connection part In this embodiment, about each of the sample storage part 20, the reagent storage part 18, the positive control storage part 21h, and the negative control storage part 21i, the micropump 11 which sends the content liquid of these storage parts is used. The microreactor is provided as a separate device. The micropump 11 is connected to the upstream side of the reagent storage unit 18, and supplies the driving liquid to the reagent storage unit side by the micropump 11, thereby pushing out the reagent to the flow path and feeding the reagent. Preferably, the micropump unit (pump unit of the liquid feeding operation unit including the pump drive unit, the electric control unit, and the pump chamber) is incorporated in an apparatus body (DNA testing device) separate from the microreactor, and the microreactor is installed in the apparatus. By attaching to the main body, the pump connection unit 12 is connected to the microreactor. Among the micropump units, at least the pump drive unit and the electric control unit are incorporated in an apparatus main body separate from the microreactor, and are connected to the microreactor from the pump connection unit 12 by attaching the microreactor to the apparatus main body. It is like that. As the micropump element, in addition to the pump connection portion 12, a pump liquid supply operation unit including a pump chamber can also be provided in the microchannel of the microreactor. In this case, the micropump element Includes a pump connection portion 12 and the liquid feeding operation portion.

In this embodiment, a piezo pump is used as the micro pump. That is,
A first channel in which the channel resistance changes according to the differential pressure;
A second flow path in which a ratio of a change in flow path resistance to a change in differential pressure is smaller than the first flow path;
A pressurizing chamber connected to the first flow path and the second flow path;
A piezo pump provided with an actuator for changing the pressure inside the pressurizing chamber. The details are described in Patent Documents 1 and 2 above.
-Liquid-feeding control part and backflow prevention part In the microreactor of this embodiment, many liquid-feeding control parts 13 are provided in the flow path. The liquid feeding control unit blocks the passage of the liquid until the liquid feeding pressure in the positive direction reaches a predetermined pressure, and allows the liquid to pass by applying a liquid feeding pressure equal to or higher than the predetermined pressure.

A large number of backflow prevention sections 16 for preventing backflow of liquid are provided in the flow path of the microreactor. The backflow prevention unit 16 is a check valve in which the valve body closes the flow path opening by backflow pressure, or an active valve that closes the opening by pressing the valve body to the flow path opening by the valve body deforming means. Consists of.
-Reagent fixed_quantity | quantitative_assay part A reagent can be quantitatively sent using said liquid feeding control part and a backflow prevention part. As a configuration of the reagent quantification unit, a predetermined amount of reagent is filled in the channel (reagent filling channel 15a) between the backflow prevention unit 16 and the liquid feeding control unit 13a. Further, a branch channel that branches from the reagent filling channel 15a and communicates with the micropump 11 that feeds the driving liquid is provided. In addition, by providing the large-volume storage portion 17a in the reagent filling channel 15a, variation in fixed quantity is reduced.

The reagent is mixed by mixing two reagents through a Y-shaped channel. In this case, even if each reagent is fed simultaneously, the mixing ratio is not stable at the beginning of the liquid. Therefore, it is desirable to cut off the leading portion so that the mixed solution is fed to the next step after the mixing ratio is stabilized.
-Gene amplification reaction site Reagents such as biotin-modified chimeric primers that specifically hybridize to the gene to be detected, DNA polymerase having strand displacement activity, and endonuclease are stored in the reagent storage units 18a, 18b, and 18c of FIG. A piezo pump 11 incorporated in the apparatus main body separate from the microreactor is connected to the upstream side of each reagent storage unit by a pump connection unit 12, and these pumps are connected from the reagent storage units to the downstream flow path 15 a. The reagent is fed by the pump.

The flow path 15a, the flow path to the next process branched from the flow path 15a, and the liquid feeding control units 13a and 13b cut off the tip of the mixed liquid of the reagent fed from each reagent storage unit, and the mixed state is A flow path is formed so that the reagent mixture is sent to the next step after stabilization. In each reagent storage unit, a total of more than 7.5 μL of reagent is stored, and a total of 7.5 μL of the reagent mixed solution with the tip cut off is divided into three channels 15 b, 15 c, 2.5 μL each. The liquid is fed to 15d. The flow path 15b reacts with the analyte, to the detection system 22 (FIGS. 1 and 3), the flow path 15c reacts with the positive control, and to the detection system 22 (FIG. 1), and the flow path 15d reacts with the negative control and detection. Each communicates with the system 22 (FIG. 1).

The mixed reagent sent to the flow path 15b is filled in the reservoir 17a of FIG. In addition, a reagent filling flow path is configured between the check valve 16 on the upstream side of the storage unit 17a and the liquid supply control unit 13a on the downstream side, and the branch flow channel communicates with the pump 11 that supplies the driving liquid. The above-described reagent quantification unit is configured together with the liquid feeding control unit 13b provided in the above.

A sample extracted from blood or sputum is injected from the sample storage unit 20 of FIG. 1, and the storage unit 17b is filled with a sample in a fixed amount (2.5 μL) by the same mechanism as the reagent determination unit described above, and the subsequent flow path A fixed amount of liquid is fed to The specimen and reagent mixed solution filled in each reservoir 17 are sent to the flow channel 15e (volume: 5 μL) via the Y-shaped flow channel, and mixing and ICAN (registered trademark) reaction are performed in the flow channel 15e. Is called. Here, the liquid supply of the specimen and the reagent alternately drives each pump 11 and alternately introduces the specimen and the reagent mixed solution into the flow path 15e in a circular shape so that the specimen and the reagent are quickly diffused and mixed. Like to do.

The amplification reaction is stopped by feeding 5 μL of the reaction solution and 1 μL of the reaction stop solution stored in the stop solution storage portion 21a to the 6 μL channel 15f and mixing them. Then
A mixture of denatured solution (1 μL) housed in the denatured solution container 21b, reaction solution and stop solution (0.
5 μL) is sent to a 15 g flow channel having a volume of 1.5 μL and mixed to denature the amplified gene into a single strand. Next, the denatured treatment solution (1.5 μL) is divided into 2 minutes and sent to the detection sites 22a and 22b in which the streptazibine is adsorbed in the flow path.
-Detection site The buffer of the hybridization buffer accommodating part 21c is flowed by the single pump 11 into the flow path 22a in which the amplified gene is immobilized. Next, the probe DNA solution (2.5 μL) housed in the probe DNA housing part 21f and fluorescently labeled with FITC is fed, and the probe DNA is hybridized to the single-stranded amplified gene. Further, the washing liquid accommodated in the accommodating portions 21d and 21e and the colloidal gold solution labeled with the anti-FITC antibody are fed in the order shown in FIG. Similarly, the hybridization buffer, the washing solution, and the probe DNA solution for internal control accommodated in the accommodating portions 21c, 21d, 21g, and 21e by the single pump 11 into the channel 22b in which the amplified gene is immobilized. A solution of colloidal gold labeled with an anti-FITC antibody is fed in the order shown in FIG.

  By feeding the colloidal gold solution, the colloidal gold is bound to the immobilized amplified gene via FITC and immobilized. The presence or absence of amplification or amplification efficiency is measured by optically detecting the immobilized gold colloid.

  The flow paths 15c and 15d in FIG. 3 communicate with the positive control reaction, the detection system, and the negative control reaction and the detection system. By sending a reagent mixture to these, the above-described sample reaction and detection system are connected. In the same manner as in the above, after the amplification reaction in the flow path with the reagent, hybridization is performed in the flow path with the probe DNA housed in the probe DNA housing section, and the amplification reaction is detected based on this reaction product .

Hereinafter, the present invention will be described more specifically based on examples. The present invention is not limited in any way by such examples.
Reagents used : Streptavidin; manufactured by Nacalai Tesque.
・ Biotinylated gold colloid: Albumin-Biotin Gold Labeled 20nm (Sigma, product number A4417)
This was diluted 50 times with the following 5 × SSC and used.
・ Buffer (filter sterilized with 0.2μm filter after preparation)
5 × SSC; 750 mM sodium chloride and 75 mM trisodium citrate saline; 0.9% sodium chloride
50 mM Tris-HCl; Tris is 2-amino-2-hydroxymethyl-1,3-propandiol.

Pure water
A light-emitting diode having a detection maximum wavelength of 520 to 530 nm and a photodiode were opposed to each other, and a measurement part of the sample was placed between them, and the output of the photodiode was measured. That is, when the value of the time nothing between the light emitting diode and the photodiode I _0, the numerical value of the silicon-based non adsorbed I _b, a numerical value obtained by reacting a gold colloid was I _g, adsorption Strength is
It is calculated by log (I ₀ / (I ₀ + (I _g −I _b )).
Procedure As shown in FIG. 4, silicone rubber with a hole having a diameter of 4 mm is attached to a polystyrene sheet. Each hole is filled with 12 μL of streptavidin solution of various concentrations (9 types in the range of 10-50 μg / mL) prepared with various buffers (Tris buffer, SSC buffer, hybrid buffer, physiological saline). To do. The silicone rubber hole was covered with a silicone rubber lid and allowed to stand at room temperature for 1 hour. The streptavidin solution was extracted and the wells were washed 3 times with various buffers. Next, 2μL of biotin-labeled gold colloid in the silicone hole
Filled. The biotin-labeled gold colloid was extracted and the holes were washed three times with various buffer solutions. The silicone rubber was removed and the polystyrene sheet was dried.

Then, the optical density of the hole part and other part of a polystyrene sheet was measured, and the adsorption strength of the gold colloid part was calculated according to the above formula. The obtained results are shown in Table 1.
The optimal streptavidin concentration is 25 μg / mL. As a buffer solution to be used, physiological saline>SSC>Tris> pure water is preferable in this order.

FIG. 1 is a schematic diagram of a gene testing microreactor according to an embodiment of the present invention. The micropump 11 basically belongs to a separate device. FIG. 2 is a schematic view of a DNA testing device comprising the microreactor of FIG. 1 and the apparatus main body. FIG. 3 is a diagram showing the configuration of the reagent mixing section of the microreactor in one embodiment of the present invention. FIG. 4 shows the arrangement of the polyethylene sheet and silicone rubber used to examine the conditions for the adsorption of streptavidin onto polystyrene.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microreactor 2 Apparatus main body 11 Micropump 12 Pump connection part 13 Flow control part 15 Flow path 16 Backflow prevention part 17a Reagent storage part 17b Sample storage part 18 Reagent storage part 20 Sample storage part 21a Stop liquid storage part 21b Denatured liquid storage part 21c Hybridization buffer accommodating part 21d Washing liquid accommodating part 21e Colloidal gold accommodating part 21f Probe DNA accommodating part 21g Internal control probe DNA accommodating part 21h Positive control accommodating part 21i Negative control accommodating part 22 Detection part (streptadibin adsorption)
23 Waste liquid reservoir

Claims (9)

A microreactor having a microchannel and at least a detection portion being a transparent plastic,
In order to trap the sample gene amplified using the biotin-labeled primer in the microchannel, the biotin affinity protein is located downstream of the gene amplification reaction site in the microchannel and is detected as polystyrene as the detection site. A microreactor for genetic testing characterized by being adsorbed.   2. The microreactor for genetic testing according to claim 1, wherein the probe that hybridizes with the amplified gene is labeled with peroxidase.   The microreactor for genetic testing according to claim 2, wherein the probe is reacted with a coloring reagent. The coloring reagent may be 3,3 ′, 5,5′-tetramethylbenzidine, 3,3′-diaminobenzidine, p-phenylenediamine, 5-aminosalicylic acid, 3-amino-9-ethylcarbazole, 4 The microreactor for genetic testing according to claim 3, comprising -chloro-1-naphthol, 4-aminoantipyrine or o-dianisidine. The genetic testing microreactor;
A micropump,
A micropump and a device for controlling the temperature, and a detection device for optically detecting the gene amplification reaction, and automatically detecting the gene amplification reaction and the amplification reaction by mounting the microreactor. Characteristic DNA testing device. The micropump is
A first channel in which the channel resistance changes according to the differential pressure;
A second flow path in which a ratio of a change in flow path resistance to a change in differential pressure is smaller than the first flow path;
A pressurizing chamber connected to the first flow path and the second flow path;
6. The DNA testing device according to claim 5, wherein the DNA testing device is a piezo pump provided with an actuator for changing the pressure inside the pressurizing chamber. The detection device includes:
After the gene amplified in the microchannel is bound to the biotin-affinity protein adsorbed to the detection site of the microchannel, the hybridization product is obtained by hybridization with the probe DNA, or the microchannel The hybridization product obtained by hybridizing the amplified gene and probe DNA is bound to the biotin-affinity protein adsorbed on the detection site of the microchannel, and then the probe DNA is converted into a colored substance. The DNA test device according to claim 5, wherein the device is an optical detection device. In order to immobilize the biotin affinity protein for trapping the amplified gene in the flow path of the microreactor as the detection site of the amplified gene downstream from the gene amplification reaction site in the flow path, Is dissolved in SSC buffer or physiological saline to prepare a solution having a concentration of 10 to 35 μg / mL, and the solution is applied to the microchannel formed using polystyrene, and biotin affinity is formed on the microchannel. A method for producing a detection site of a microreactor for genetic testing, characterized by adsorbing a protein. Mixing the reaction solution containing the gene amplified at the gene amplification reaction site in the microchannel of the microreactor with the denaturation solution, and denaturing the amplified gene into a single strand;
The treatment solution obtained by denaturing the amplified gene into a single strand is sent to the detection site of the microchannel where the biotin-affinity protein is adsorbed to polystyrene, and the amplified gene is sent to the detection site. Trapping process;
Sending a probe DNA modified with peroxidase to the detection site, and hybridizing the amplified gene and the probe DNA;
Then, a solution of a coloring reagent containing a coloring substance is sent to the detection site where the amplified gene is trapped, and the coloring substance is colored by a reaction catalyzed by the peroxidase;
And a step of optically measuring the color development at the detection site.

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