JP2007244286A - Nucleic acid detection system and nucleic acid detection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid detection system for preventing that nucleic acid is contaminated in an equipment for detecting the nucleic acid; in other words to provide a system having a mechanism for preventing the transpiration of a reaction solution in a system for reacting at high temperature in the time of a nucleic acid amplification reaction. <P>SOLUTION: This nucleic acid detection system comprises a nucleic acid detection device A and a nucleic acid detection equipment B. The nucleic acid detection device A has a mechanism for preventing the transpiration of a reaction solution in a system for reacting at high temperature, and is controlled with the nucleic acid detection equipment B. Hereby, a system is provided for preventing the contamination of the nucleic acid in an equipment for detecting the nucleic acid. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nucleic acid detection system and a nucleic acid detection method.

  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 and disposable ones such as test papers. Is required.

  Furthermore, in order to easily amplify and detect nucleic acid using a technique such as a polymerase chain reaction (hereinafter referred to as “PCR”) method, the disposable device includes a mechanism for nucleic acid amplification and detection. In recent years, a device having a mechanism for achieving this has been developed. For example, a buffer solution holding unit, a reagent holding unit, a sample holding unit, etc. are provided in the vicinity of the amplification site and nucleic acid detection site provided on the same substrate, or a liquid flow that enables circulation of buffer solution, reagent, reaction solution, etc. There has been disclosed a method characterized in that a path is provided on the same substrate (Patent Documents 1 to 3). Although such a reaction base is provided with a liquid flow path, there is a problem that it takes a long time for the reaction liquid to flow through the liquid flow path. In some cases, the reaction liquid may not flow through the liquid flow path.

  On the other hand, in a device that does not have a liquid flow path, the PCR reaction system does not become a closed system, so that the aqueous reaction solution evaporates during the heat denaturation process, particularly at around 95 ° C., and the device is set. There is a problem that the equipment is contaminated.

  Such a problem seems to be solved by a lid that seals the reaction solution layer during the PCR reaction, but it is difficult to prevent evaporation when the lid is attached or detached. Since the PCR solution is very small, the problem of contamination of the equipment is serious even if transpiration occurs when the lid is attached or detached.

JP 2003-325199 A JP 2003-177114 A JP 2003-325199 A

  Therefore, it is an object of the present invention to provide a system for preventing contamination of nucleic acid to a device that detects nucleic acid. In other words, it is an object of the present invention to provide a system including a mechanism for preventing transpiration of a reaction solution in a system that reacts at a high temperature.

That is, this invention consists of the following.
1. A system for amplifying and detecting nucleic acids,
The system includes a disposable nucleic acid detection device and a nucleic acid detection apparatus,
The nucleic acid detection device comprises:
The device itself,
A slide hole for inserting slide means into the device body;
A liquid reservoir provided at a lower portion of the device body and opened with respect to the slide hole; and
Provided in the upper part of the device body facing the liquid storage part through the slide hole, an opening part communicating the upper surface of the device body and the slide hole,
A lid member for sealing the liquid reservoir;
Slide means inserted into the slide hole of the device body;
A plurality of holes provided in the sliding means and communicating the liquid storage part and the opening;
And a system for amplifying and detecting a nucleic acid comprising a nucleic acid microarray provided on the lower surface of the lid member.
2. The nucleic acid detection device
A device set mechanism for holding the nucleic acid detection device;
A heater that can be disposed at a lower portion of the liquid reservoir of the device body;
Holding means for detachably holding at least the lid member and movable up and down;
A counter heater provided in the holding means;
A system for amplifying and detecting a nucleic acid according to item 1 above, comprising liquid injection means for injecting liquid into the liquid reservoir.
3. The device itself
A first lid member for sealing the liquid reservoir;
Slide means inserted into the slide hole of the device body;
A first hole that is provided in the sliding means and communicates between the liquid storage portion and the opening;
A second hole provided in the sliding means and communicating the liquid reservoir and the opening;
A second lid member that is provided in the second hole and seals the liquid storage portion;
And a nucleic acid microarray provided on the lower surface of the second lid member,
The nucleic acid detection device comprises:
A device set mechanism for holding the nucleic acid detection device;
A heater that can be disposed at a lower portion of the liquid reservoir of the device body;
First holding means for holding at least the first lid member detachably and moving up and down;
A counter heater provided in the first holding means;
3. A system for amplifying and detecting a nucleic acid according to item 1 or 2, further comprising a liquid injection means for injecting a liquid into the liquid reservoir.
4). 4. The nucleic acid detection system according to item 3, wherein a lid holding structure for holding the second lid member is provided on the lower surface of the first lid member.
5). 5. The nucleic acid detection system according to item 3 or 4, further comprising a second holding means for holding the second lid member in the nucleic acid detection device.

  The system for amplifying and detecting a nucleic acid according to the present invention can prevent evaporation of a reaction solution in a reaction system at a high temperature in a PCR reaction, and can prevent contamination of a nucleic acid to an apparatus for detecting the nucleic acid. PCR reaction can be performed. Furthermore, it is possible to omit the time for cooling the temperature after the reaction at a high temperature, thereby enabling more rapid nucleic acid detection.

  The nucleic acid detection system of the present invention will be described below with reference to the drawings, but the present invention is not limited to these.

  The nucleic acid detection system of the present invention includes a nucleic acid detection device A (also simply referred to as device A) and a nucleic acid detection device B (also simply referred to as device B). The nucleic acid detection device A in the present invention is a disposable device and refers to a device for detecting a nucleic acid fully automatically by being set in a nucleic acid detection apparatus B described later. Further, it means a device having both a mechanism for amplifying nucleic acid and a mechanism for detecting nucleic acid.

  FIG. 1 is a view showing a nucleic acid detection device A of the present invention. The device A includes a device main body 1, a slide hole 11 for inserting the slide means 3 into the device main body 1, a liquid storage portion 12 provided at a lower portion of the device main body 1 and opened to the slide hole 11. The device main body 1 is provided at the upper portion of the liquid storage section 12 facing the slide hole through the slide hole, and the opening 13 communicating the upper surface of the device main body 1 with the slide hole 11 is sealed with the liquid storage section 12. A lid member 21, a slide means 3 fitted in the slide hole 11 of the device body 1, a first hole 31 provided in the slide means 3, which communicates the liquid storage portion 12 and the opening portion 13. A second hole 32 provided in the slide means 3 for communicating the liquid reservoir 12 and the opening 13, and a second hole 32 for sealing the liquid reservoir 12. The lid member 22, and comprises a DNA array 4 provided on the lower surface of the second cover member 22.

  The material constituting the device A can be selected from a polymer material, an inorganic material, and a metal material. However, from the viewpoint that an operator can safely handle the material without injury, the polymer material is preferable. As the polymer material, for example, general-purpose polymer materials such as polyethylene, polyethylene terephthalate, polymethyl methacrylate, polyacrylamide, polypropylene, polycarbonate, polystyrene, polytetrafluoroethylene, polyamide and epoxy resin can be used as appropriate. Although not particularly limited, polycarbonate and polypropylene are preferred from the viewpoint of easy production, low cost, and appropriate hardness that is difficult to deform.

  The above-described polymer material can be molded by a production method that can be appropriately set by those skilled in the art. For example, extrusion molding, injection molding, compression molding, vacuum forming and the like can be mentioned. Among these production methods, from the viewpoint of easy production and low production costs, extrusion molding and injection molding are preferred, but the present invention is not limited to these production methods.

  The device A of the present invention mainly has four configurations. That is, the device main body 1, the slide means 3 that can be inserted and slid into the device main body, the first lid member 21, and the second lid member 22 including the DNA array 4.

  The device main body 1 is provided with a slide hole 11 for inserting the slide means 3, a liquid storage portion 12 for storing liquid, and the device main body 1 through the slide hole 11 of the liquid storage portion 12. An opening 13 is provided in the upper part facing each other, and communicates with the upper surface of the device body and the slide hole 11.

  The slide hole 11 is not particularly limited as long as the slide means 3 to be described later can be inserted and slid. However, the slide hole 11 is designed so that a gap is reduced between the slide hole 11 and the slide means 3. Is done.

The liquid reservoir 12 is a liquid layer for mainly performing a PCR reaction. The volume is not particularly limited as long as a PCR reaction is possible, but is about 0.01 to 1.0 ml, preferably about 0.02 to 0.1 ml. The area of the opening of the liquid storage part 12, from the viewpoint of preventing the evaporation of the PCR reaction solution is preferably as small as possible, about 0.1～300Mm ^{2, preferably} about 1 to 20 mm ^2.

  Further, the opening 13 is provided in an upper portion of the device main body 1 that faces the liquid reservoir 12 through the slide hole 11. The opening 13 communicates the upper surface of the device body with the slide hole 11. This is because the slide means 3 to be described later is fitted into the slide hole 11 so that the first hole 31 and the second hole 32 provided in the slide means 3 are integrated with the liquid storage part 12 and the opening part 13. This is for forming one space. For details, refer to FIG. 1 and FIG. The inner diameter of the opening 13 is an important factor in preventing the PCR solution from evaporating. That is, at least the uppermost inner diameter is designed to be equal to or smaller than the outer diameter of the first lid member 21 described later.

  The slide means 3 can be fitted and slid into the slide hole 11 in the device body 1. The sliding means 3 includes a first hole 31 and a second hole 32. In the initial state before detecting the nucleic acid, as shown in FIGS. 1 and 2, the first hole 31 is integrated with the liquid reservoir 12 and the opening 13 to form one space. As a result, the first holding means 71 of the nucleic acid detection apparatus B, which will be described later, can move freely in the space. The inner diameters of the first hole and the second hole may be designed to be slightly larger than the outer diameters of the first lid member 21 and the second lid member 22 described later, and are not particularly limited. However, it is preferably about 0.05 to 20 mm, preferably about 1 to 5 mm larger than the outer diameter of the first lid member 21 and the second lid member 22 described later.

  The first lid member 21 and the second lid member 22 are members for sealing the liquid reservoir 12. The structure of the lid members 21 and 22 is not particularly limited as long as the liquid storage part 12 can be sealed, but the outer diameter thereof needs to be at least larger than the opening of the liquid storage part. Specifically, it is about 0.05 to 20 mm, preferably about 1 to 5 mm.

  The nucleic acid microarray 4 is provided on the lower surface of the second lid member 22. A nucleic acid microarray refers to a nucleic acid to be detected or a single-stranded nucleic acid that can be hybridized with a nucleic acid to be detected immobilized on a substrate, and is generally referred to as a DNA chip or a DNA microarray. Say. Examples of the nucleic acid include DNA, RNA, and PNA, and DNA is often used from the viewpoint of use frequency.

  The nucleic acid that can be immobilized on the substrate may be a sample nucleic acid or a nucleic acid probe, but is preferably a nucleic acid probe from the viewpoint of being able to be industrially produced in advance. A sample nucleic acid refers to a nucleic acid to be detected, and a nucleic acid probe refers to a nucleic acid that can hybridize with the sample nucleic acid. The production of the nucleic acid probe is not particularly limited, and may be, for example, synthesized DNA (oligonucleotide) or cDNA reversely transcribed from mRNA. Oligonucleotides can be synthesized using a commercially available automatic nucleic acid synthesizer or the like.

  Furthermore, if the base sequence of the nucleic acid probe is within a range capable of hybridizing with the sample nucleic acid, bases inserted, mutated and deleted from the completely complementary base sequence of the specific base sequence are used. It may be an array. 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.

  Further, the number of bases 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 in 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. is there.

  The substrate used in the nucleic acid microarray may be any material that can immobilize the nucleic acid. For example, organic materials such as polyethylene, polypropylene, polycarbonate, polymethyl methacrylate, polyamide, polystyrene, polyethylene terephthalate, and nitrocellulose. Examples include materials, inorganic materials such as glass and silica, and metal materials such as gold, silver, and trunk. Among these, 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.

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

As the shape of the substrate, a rectangular film and a substrate are preferable. In addition, as for the size of the base material, a surface to be detected needs a certain area from the viewpoint of facilitating detection. For example, if the substrate is a rectangular film and the substrate, the area of the upper surface, from the viewpoint it is easy to handle, about 40～1000Mm ^2, preferably about 60~300mm ^2.

  The base material can be manufactured according to the situation by those skilled in the art and is not particularly limited because it can be appropriately set by those skilled in the art. Examples include extrusion molding, injection molding, melt molding, and compression molding. Extrusion molding is particularly preferred from the viewpoint of production cost and ease.

  The sample nucleic acid or nucleic acid probe is immobilized on a substrate by physical or chemical treatment.

  The immobilization by physical treatment is not particularly limited as long as it is a method of spotting a sample nucleic acid or nucleic acid probe solution on a substrate. Examples of such spotting methods include an extrusion method using a dispenser and an inkjet method. From the viewpoint of production cost, the extrusion method is preferable, and from the viewpoint of immobilization accuracy, the ink jet method is preferable, and is not particularly limited.

  In addition, when immobilizing by such physical treatment, it is preferable to add a base sequence irrelevant to the nucleic acid probe because the immobilization rate on the substrate is improved by UV irradiation. Examples of the base sequence unrelated to the nucleic acid probe include polyadenine, polycytosine, polythymine and polyguanine, and polythymine is preferable from the viewpoint of the highest immobilization rate.

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

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 substrate at the end of a nucleic acid probe, or a bond capable of covalent bonding to the end of a nucleic acid probe and the substrate, respectively. Examples thereof include a method for modifying a functional group. For example:
(a) When the base material is an inorganic material such as glass or silicon, a functional group capable of a silane coupling reaction such as trimethoxysilane or triethoxysilane is modified at the end of the probe for nucleic acid detection, and the silane coupling reaction is performed. Immobilization method by:
(b) When the base material is an inorganic material such as glass or silicon, the surface of the base material is treated with silane coupling agent having an amino group such as aminoethoxysilane on the inorganic material base material. Aminated by Kepling linkage. On the other hand, the carboxylic acid is modified at the end of the nucleic acid probe. And a method of immobilizing an amide bond by an amino coupling reaction between the amino group of the substrate and the carboxylic acid of the nucleic acid probe;
Etc.

  Even if the base material is a metal material such as gold or silver, those skilled in the art can easily fix the silane coupling bond by replacing it with a metal-thiol or metal-disulfide bond. I can think of it.

  Among the above immobilization methods, with respect to immobilization of nucleic acid, from the viewpoint of easy production, a method of adding polythymine to the end of the nucleic acid probe and immobilizing by UV irradiation is preferable.

  Further, the amount of nucleic acid immobilized further increases by subjecting the base material to surface treatment such as plasma treatment. Plasma treatment means discharging the plasma in an inert gas atmosphere to irradiate the substrate surface with plasma generated by the ionizing action of the inert gas, etching the surface, improving wettability, and introducing functional groups. It refers to a process that provides an effect. Examples of the discharge include corona discharge (high pressure and low temperature plasma), arc discharge (high pressure and high temperature plasma), glow discharge (low pressure and low temperature plasma), and the like. Among these, corona discharge is preferable from the viewpoint of good surface treatment reactivity, but the present invention is not limited thereto.

  Examples of the inert gas in the plasma treatment include nitrogen gas, argon gas, oxygen gas, helium gas, neon gas, and xenon gas. Nitrogen gas, oxygen, or argon gas is preferable from the viewpoint of the highest amount of nucleic acid immobilized after plasma treatment. In particular, when nitrogen or DNA having a phosphate skeleton is fixed to a substrate, nitrogen gas generates an amine and / or amino group on the surface of the substrate, thereby improving the wettability of the surface and ion charging of the surface. Nucleic acids can be fixed more firmly, and in the case of oxygen, the surface can be easily scraped, resulting in the formation of minute protrusions. As a result, charges are concentrated, and it is preferable that DNA can be fixed easily when it is dropped.

  The second lid member 22 including the nucleic acid microarray 4 described above is provided in the second hole 32 in the slide means 3. When the sliding means 3 is slid and the second hole 32 is integrated with the liquid storage portion 12 and the opening 13 to form a space, the second lid member provided in the second hole 32 is a liquid. It functions as a lid for the reservoir 12. At this time, the first lid member 21 is held by the first holding means 71 provided in the nucleic acid detection apparatus B described later, and is arranged at the position of the opening 13 of the device body 1. That is, since the first lid member 21 is disposed in the opening portion 13 to seal the space, the liquid existing in the liquid storage portion 12 may evaporate even while the sliding means 3 is sliding. Absent. This time, the second lid member 22 that has been slid becomes a lid of the liquid storage unit 12.

  On the other hand, the nucleic acid detection apparatus B (outside view not shown) of the present invention is a device that has a mechanism for setting the nucleic acid detection device A and can be introduced and managed in a user's facility. Specifically, the device setting mechanism 5 for holding the nucleic acid detection device A, the heater 61 that can be disposed in the lower part of the liquid reservoir 12 of the device main body 1, and at least the first lid member 21 are detachable. The first holding means 71 that is held by the first holding means 71, the counter heater 62 provided in the first holding means 71, and the liquid injection means 8 that injects the liquid into the liquid storage section.

  The device setting mechanism 5 (not shown) is not particularly limited as long as the device A can be fixed to the apparatus B, but is arranged so that the opening of the liquid storage unit 12 in the device A faces upward. It is obvious that it will be done. The fixing of the device A to the device B is achieved by various mechanisms such as screwing, engagement, clamping and fitting.

  The heater 61 is for controlling the temperature of the liquid in the liquid storage part 12 of the device A, and is arranged under the liquid storage part 12 of the device A fixed by the device setting mechanism 5. The heater 61 has an effect of temperature adjustment in combination with a counter heater 62 described later. Since the liquid storage unit 12 of the present invention mainly performs a PCR reaction, the heater 61 and the counter heater 62 have a denaturation step of about 92 to 95 ° C., a rapid cooling step of about 2 to 20 ° C., and an annealing step of about 50 to 65. It is possible to apply three kinds of temperatures of about 70 to 75 ° C. in the elongation process. Of course, the set temperature can be appropriately selected by those skilled in the art according to other nucleic acid amplification reactions and the like, and is not limited only to the temperature conditions used for the PCR reaction described above.

  The apparatus B includes first holding means 71 for controlling the first lid member 21 of the device A. The first holding means 71 holds the first lid member 21 by a mechanism such as screwing, engagement, clamping, and fitting, and covers the liquid storage unit 12 of the nucleic acid detection device A. The first holding means 71 can be moved up and down, and when performing a PCR reaction or the like in the liquid storage unit 12, the liquid storage unit can be more firmly pressed by pressing the first lid member 21 downward. The system in 12 can be sealed. Furthermore, the first holding means 71 can be sealed in the above-described space by moving the first lid member 21 to the opening 13.

  The first holding means 71 is provided with a counter heater 62 corresponding to the heater 61. Thereby, the temperature of the system in the liquid reservoir 12 can be controlled in a state where the liquid reservoir 12 is sealed with the first lid member 21.

  The liquid injection means 8 is not particularly limited as long as it injects a specimen, a PCR reaction liquid, a hybridization liquid, and / or a reaction detection liquid into the liquid reservoir 12. For example, a tube and a nozzle can be used.

  Reagents such as a PCR reaction solution, a hybridization solution, and / or a reaction detection solution are hermetically held with a light-shielded film and mounted on the apparatus. Each processing step is opened by a cutter attached to the apparatus and supplied to the device through an injection means.

  The nucleic acid detection method in the nucleic acid detection system of the present invention will be described below with reference to the drawings, but the present invention is not limited to these.

  First, a sample nucleic acid is prepared. As the sample nucleic acid, genomic DNA is extracted from a biological sample of an animal according to the purpose of measurement. 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, and examples thereof include a phenol extraction method, a guanidine thiocinate extraction method, and a vanadyl ribonucleoside complex extraction method.

  Next, the nucleic acid detection device A is set in the nucleic acid detection apparatus B. The nucleic acid detection device A is fixed by the device set mechanism 5 provided in the nucleic acid detection apparatus B. Further, the first hole 31 is in a state in which a space is formed integrally with the liquid reservoir 12 and the opening 13. Then, after introducing the specimen and the PCR reaction solution into the liquid storage unit 12, the liquid storage unit 12 is sealed with the first lid member 21 held by the first holding means 71 (FIG. 1).

  Next, a PCR reaction is performed in the sealed liquid reservoir 12. The PCR method includes, for example, (I) a denaturation step in which double-stranded genomic DNA is made into a single strand by heat treatment under reaction conditions of about 92 to 95 ° C. and about 0.1 seconds to 1 minute, (II) A double-stranded portion serving as a PCR reaction starting point is prepared by binding at least two kinds of amplification primers to each of the double-stranded DNAs under reaction conditions of about 50 to 65 ° C. for about 0.1 seconds to 1 minute. Steps (I) to (III) of the annealing step and (III) strand extension step of reacting with DNA polymerase at about 70 to 75 ° C. under reaction conditions of about 0.1 second to 5 minutes, Nucleic acids can be amplified by repeating 40 times. The temperature is controlled by a heater 61 and a counter heater 62 provided in the nucleic acid detection apparatus B. The amount of the liquid in the PCR reaction is not particularly limited because it depends on the size of the liquid reservoir 12, but is, for example, about 10 to 1,000 μl, preferably 20 to 100 μl.

  Here, the primer pair used in the PCR method has a base sequence capable of hybridizing with the extracted genomic DNA and capable of amplifying a nucleic acid containing the specific base sequence, and the degree of polymerization thereof is about What is necessary is just about 15-40 bases. The primer can be synthesized by an automatic synthesizer or the like, similar to the nucleic acid probe.

In order to facilitate detection of the nucleic acid during amplification of the target nucleic acid, labeling is preferred. The labeling method includes an RI method and a non-RI method, but it is preferable to use the non-RI method because of easy handling. Non-RI methods include fluorescent labeling and enzyme labeling. Any fluorescent label can be used as long as it can bind to the base moiety of the nucleic acid, and cyanine dyes (for example, Cy Dye ^(TM) series Cy3, Cy5 etc.), rhodamine 6G reagent, N-acetoxy-N Examples include ₂ -acetylaminofluorene (AAF), AAIF (iodine derivative of AAF), and the like. Examples of enzyme labels include alkaline phosphatase and peroxidase.

  After the PCR reaction, the first holding means 71 and the first lid member 21 move upward. Thereby, the space united by the first hole 31, the liquid reservoir 12, and the opening 13 is sealed by the first lid member 21 (FIG. 2).

  Next, the sliding means 3 slides so that the second hole 32 is disposed at the position of the first hole 31. As a result, a space in which the second hole 32, the liquid reservoir 12, and the opening 13 are integrated is formed. At this time, the second lid member 32 provided in the second hole 32 becomes a lid of the liquid storage unit 12 and is sealed (FIG. 3). With these mechanisms, the next hybridization reaction can be started without waiting until the temperature of the PCR reaction solution in the liquid reservoir 12 reaches room temperature, and the time required for nucleic acid detection can be shortened. Moreover, since the PCR reaction solution does not evaporate in the nucleic acid detection device B, the device B is not contaminated. In this case, an alkaline solution may be added, or the nucleic acid amplified by heat shock may be denatured and single-stranded, or only a single-stranded nucleic acid is specifically amplified by performing an asymmetric PCR reaction. Also good.

  The control of the second lid member 22 is performed by the second holding means 72 that holds the second lid member 22 provided in the apparatus B (FIG. 4). Further, as another example of the control of the second lid member 22, the first lid member 21 is connected to the second lid member 22 by connecting the first lid member 21 held by the first holding unit 71 to the first holding unit. You may control by 71. At this time, the first lid member 21 and the second lid member 22 are connected by providing a mechanism for connecting the second lid member 22 to the lower surface of the first lid member 21.

  The hybridizing solution is preheated to about 40 to 50 degrees with a heater. The second lid means 22 is injected through the liquid injection means 8 with the PCR reservoir 12 sealed (FIG. 5). Thereafter, the second lid member 22 is opened to mix the amplified sample nucleic acid present in the PCR reservoir 12 and the hybridizing solution. That is, the nucleic acid microarray 4 disposed on the lower surface of the second lid member 22 is immersed in the hybridization reaction solution. At this time, it is preferable to stir by operating the second holding means 72 up and down to allow the reaction to proceed more rapidly (FIG. 6).

  The volume of the hybridization solution with respect to the PCR reaction solution may be such that the PCR reaction solution does not inhibit the hybridization reaction. For example, when the volume of the PCR reaction solution is 100%, it is about 100 to 1,000,000%, preferably about 1,000 to 100,000%. That is, the PCR reaction solution is diluted with the hybridization reaction solution. At this time, the space formed by integrating the liquid storage unit 12, the opening 13 and the second hole 32 becomes one reaction liquid tank, so that the liquid storage unit 12 is injected by excessively injecting the hybridizing solution. There is no problem even if it overflows from.

  The temperature condition for the hybridization reaction depends on the thermal denaturation temperature of the DNA probe immobilized on the substrate. For example, when the heat denaturation temperature of the DNA probe is about 55.0 to 75.0 ° C, it is generally about 61.5 to 62.5 ° C. In this case, unreacted sample nucleic acid and / or nucleic acid probe is preferably removed by a washing step.

  The detection method can be appropriately set according to the label. For example, in the case of RI labeling, it can be detected by autoradiography. In the case of a fluorescent label, it can be detected by irradiating light having an excitation wavelength suitable for each fluorescent label and measuring the fluorescence intensity. In the case of enzyme labeling, measurement can be performed by adding a substrate for each enzyme. When alkaline phosphatase is used, for example, p-nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) are added as substrates, and the degree of color development is increased. It can be measured visually or with a CCD camera or densitometer.

  The detection method based on the combination of the alkaline phosphatase label and the substrate NBT / BCIP can be suitably used for clinical diagnosis because the measurement result can be visually observed.

  In the present invention, analysis can also be suitably achieved by processing and outputting a digital image obtained by processing each data.

  The nucleic acid detection system of the present invention includes a nucleic acid detection device A and a nucleic acid detection apparatus B. The nucleic acid detection device A is provided with a mechanism for preventing evaporation of the reaction solution in a system that performs the reaction at a high temperature, and the device A is controlled by the nucleic acid detection device B. Thus, it is possible to provide a system for preventing contamination of nucleic acid to a device that detects nucleic acid. In particular, it eliminates the time for cooling the temperature after the reaction at a high temperature such as a PCR reaction, and enables more rapid detection of nucleic acids.

In the nucleic acid detection device A, it is a figure which shows the state which sealed the liquid storage part 12. FIG. In the nucleic acid detection device A, the first holding means 71 and the first lid member 21 are moved upward, so that the space integrated with the first hole 31, the liquid reservoir 12 and the opening 13 is the first. It is a figure which shows the state sealed by the 1 cover member. In the nucleic acid detection device A, the slide means 3 is slid so that the second hole 32 is disposed at the position of the first hole 31, and the second hole 32, the liquid reservoir 12, and the opening 13 are formed. FIG. 4 is a diagram showing a state in which a unitary space is formed and the second lid member 22 provided in the second hole 32 becomes a lid of the liquid storage unit 12 and is sealed. In the nucleic acid detection device A, it is a figure which shows the state which seals the liquid storage part 12 in 2nd hole 32, and performs PCR reaction. The second lid member 22 is controlled by the second holding means 72 provided in the apparatus B. In the nucleic acid detection device A, it is a diagram showing a state in which the hybridization liquid is injected through the liquid injection means in a state where the liquid storage section 12 is sealed by the second lid means 22. In the nucleic acid detection device A, it is a figure which shows the state stirred by operating the 2nd holding means 72 up and down.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Device main body 11 Slide hole 12 Liquid storage part 13 Opening part 21 1st cover member 22 2nd cover member 3 Slide means 31 1st hole 32 2nd hole 4 Nucleic acid microarray 5 Device set mechanism 61 Heater 62 Counter heater 71 First holding means 72 Second holding means 8 Injection means

Claims (5)

A system for amplifying and detecting nucleic acids,
The system includes a disposable nucleic acid detection device and a nucleic acid detection apparatus,
The nucleic acid detection device comprises:
The device itself,
A slide hole for inserting slide means into the device body;
A liquid reservoir provided at a lower portion of the device body and opened with respect to the slide hole; and
Provided in the upper part of the device body facing the liquid storage part through the slide hole, an opening part communicating the upper surface of the device body and the slide hole,
A lid member for sealing the liquid reservoir;
Slide means inserted into the slide hole of the device body;
A plurality of holes provided in the sliding means and communicating the liquid storage part and the opening;
And a system for amplifying and detecting a nucleic acid comprising a nucleic acid microarray provided on the lower surface of the lid member. The nucleic acid detection device
A device set mechanism for holding the nucleic acid detection device;
A heater that can be disposed at a lower portion of the liquid reservoir of the device body;
Holding means for detachably holding at least the lid member and movable up and down;
A counter heater provided in the holding means;
The system for amplifying / detecting a nucleic acid according to claim 1, further comprising liquid injection means for injecting the liquid into the liquid reservoir. The device itself
A first lid member for sealing the liquid reservoir;
Slide means inserted into the slide hole of the device body;
A first hole that is provided in the sliding means and communicates between the liquid storage portion and the opening;
A second hole provided in the sliding means and communicating the liquid reservoir and the opening;
A second lid member that is provided in the second hole and seals the liquid storage portion;
And a nucleic acid microarray provided on the lower surface of the second lid member,
The nucleic acid detection device comprises:
A device set mechanism for holding the nucleic acid detection device;
A heater that can be disposed at a lower portion of the liquid reservoir of the device body;
First holding means for holding at least the first lid member detachably and moving up and down;
A counter heater provided in the first holding means;
The system for amplifying / detecting a nucleic acid according to claim 1 or 2, further comprising a liquid injection means for injecting a liquid into the liquid storage section. The nucleic acid detection system according to claim 3, further comprising a lid holding structure that holds the second lid member on a lower surface of the first lid member. The nucleic acid detection system according to claim 3 or 4, further comprising a second holding means for holding the second lid member in the nucleic acid detection device.

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