JP2014081329A - Nucleic acid analysis cartridge - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a nucleic acid analysis cartridge made of an organic material, encapsulating a plurality of reagents to be used in electrophoresis including formamide, the cartridge capable of reducing pollutant contamination, having high storage stability and inexpensive, and a packing kit for the cartridge.SOLUTION: A nucleic acid analysis cartridge has at least one vent hole communicating to a space making contact with a reagent. The nucleic acid analysis cartridge is packaged in a packing bag having low vapor permeability in a decompressed state. Through packaging in a decompressed state, vapor concentration in the packing bag can be reduced, and through using the packing bag having low vapor permeability, vapor permeation from atmosphere inside the packing bag at transport or storage can be reduced. Through the vapor concentration reduction, formamide hydrolysis at storage can be reduced, DNA fragment separability at electrophoresis can be prevented from decreasing, and storage stability can be increased.

Description

  The present invention relates to a nucleic acid analysis cartridge, for example, a packing kit for packing it and an analysis apparatus using the same.

  Applications using nucleic acid analysis have been put to practical use in fields such as forensic science, personalized medicine, and bioterrorism.

  In the field of forensic science, STR (Short Tandem Repeat) analysis has been put into practical use. STR analysis analyzes a repetitive base sequence of a certain region in the genome. Using the fact that the length of the STR base sequence is individual-specific, DNA identification such as individual identification and parent-child identification is performed.

  Patent Document 1 discloses a process for analyzing 13 types of areas designated by the US FBI at a time. The analysis process consists of DNA sample collection, DNA amplification, DNA fragment separation, and DNA fragment detection processes. In the DNA amplification process, multiplex PCR (Polymerase Chain Reaction) amplification is performed on one measurement DNA sample using 13 types of primer sets. During DNA amplification, the DNA fragment that is the amplification product is labeled. In the DNA fragment separation process, labeled DNA fragments are separated by electrophoresis. In the DNA fragment detection process, the electrophoresis pattern of the obtained separated DNA fragment is detected and analyzed.

  Patent Document 2 discloses a method using an acrylamide gel in a DNA fragment separation process. The method involves mixing 95% formamide with the DNA fragment solution, which is an amplification product, then denaturing the mixed solution at 90 ° C. for 2 minutes, loading the denatured DNA fragment solution onto an acrylamide gel, and performing electrophoresis. Features.

  Non-Patent Document 1 discloses a method of using a capillary for the separation process. Similar to the acrylamide gel described above, after mixing formamide with the DNA fragment solution that is the amplification product, the mixed solution is denatured at 95 ° C. for 3 minutes.

  The existing STR analysis described above has problems that analysis time is long and analysis cost is high. Analysis time is up to 3 to 4 hours for DNA sample collection and DNA quantification, up to 3 hours and 30 minutes for DNA amplification, and up to 45 minutes for DNA fragment separation and detection. Reference 2). In addition, a plurality of specialized devices and engineers with specialized knowledge are required, and there is a problem that the analysis system cannot be generalized.

  Therefore, it is effective to use microfluidics technology as a method of reducing analysis time and analysis cost. The microfluidics technology is a technology for handling a trace amount liquid using a microchip or the like. Moreover, handling time and cost are reduced by greatly reducing the amount of liquid to be handled.

  Patent Document 3 discloses an automated system that collects a DNA sample using microfluidics technology. That is, a swab obtained by collecting an intraoral sample or an intravaginal sample is inserted into a container, and a plurality of reagents in the reagent container are sequentially transferred to the swab container via a microfluidic channel to extract a DNA sample from the measurement sample. After the obtained DNA sample is amplified, the DNA fragment is separated, indicating that STR analysis is possible.

  Non-Patent Document 2 discloses an automated system that performs STR analysis using microfluidics technology. This analysis system uses two types of microdevices and one type of microchip. The microdevice is for collecting a DNA sample and for amplifying the DNA, and separates DNA fragments with one type of microchip. The analysis time is within 3 hours.

  Patent Document 4 discloses an automated system for performing another form of STR analysis. This system includes a module for collecting a DNA sample, a module for amplifying DNA, a module for cleaning up amplification products, and a capillary electrophoresis module. A disposable cartridge is used for the analysis. The cartridge is composed of a chamber, a connecting pipe, and a microchip having a fine flow path and a fluid disk valve. After inserting the swab having the measurement sample attached to the chamber in the cartridge, the reagent solution in the reagent cartridge is transferred to the chamber via the connecting tube, and the measurement sample is dissolved in the chamber. A reagent solution in which a measurement sample is dissolved is transferred into a chamber with magnetic fine particles, and DNA dissolved in the reagent solution is attached to the magnetic fine particles. The magnetic fine particles having DNA attached are moved to a plurality of chambers through a fluid disk valve, and a plurality of reagents are sequentially supplied to the plurality of chambers to amplify DNA and clean up amplification products. Finally, the cleaned up amplification product is introduced into a capillary electrophoresis module and subjected to STR analysis.

US Pat. No. 6,531,282 Japanese Patent Application Publication Special Table 2001/511018 Specification US Patent Application Publication No. US2010 / 0285578 US Patent Application Publication No. US2011 / 0005932 Specification

J Forensic Sci 1998; 43: 164-170 Analytica Chimica Acta 2011; 687: 150-158

  In general, the DNA amplified in the previous measurement to be measured and cells derived from the worker are floating in the ambient air around the analysis system. In order to prevent false detection by these contaminants, contamination inside the cartridge It is necessary to prevent material contamination. In order to prevent contamination, it is desirable to have a system that reduces the amount of substances brought into the cartridge as much as possible. In this respect, it is desirable that all the reagents necessary for the analysis are built in the cartridge. Reagents necessary for STR analysis include DNA amplification enzymes, primers, deoxynucleosides, size markers, denaturing formamide, and the like. Among these, formamide needs to reduce contact with the atmosphere as much as possible during storage. This is because formamide absorbs water vapor in the atmosphere and hydrolyzes to produce ammonia and formic acid, thereby reducing the ability to separate DNA fragments during electrophoresis.

Furthermore, cost reduction is required for the cartridge. As a method for reducing the cost, it is desirable to use an organic material such as plastic for the cartridge material and the packing material.
However, in general, since the organic material transmits water vapor, when the organic material is used in a cartridge or a cartridge packaging kit, a packaging package is required in which the reagent contained in the cartridge minimizes contact with the atmosphere.

  Accordingly, the present invention provides a cartridge and a cartridge packaging kit that reduce contamination contamination, have high storage stability, and are low-cost in a cartridge made of an organic material in which a plurality of reagents used in electrophoresis containing formamide are enclosed. The purpose is to do.

  As a result of diligent research, the inventor of the present invention has a cartridge and a cartridge in which a plurality of reagents used in electrophoresis containing formamide are enclosed, and the contamination of contaminants is reduced, storage stability is high, and cost is low. A packaging kit was realized.

That is, the features of the main nucleic acid analysis cartridge of the present invention are as follows.
In a kit packed with a nucleic acid analysis cartridge containing multiple reagents used in electrophoresis containing formamide, the nucleic acid analysis cartridge having at least one vent that conducts to the space in contact with the reagent is decompressed in a packing bag with low water vapor permeability. A nucleic acid analysis cartridge packaging kit is provided that is packaged.

  The reduced pressure package reduces the water vapor concentration in the packaging bag, and the use of the packaging bag with low water vapor permeability reduces the permeation of water vapor from the atmosphere into the packaging bag during transportation and storage. The vent efficiently discharges water vapor in the space in contact with the reagent into the decompression package, and reduces the water vapor concentration in the space. The reduction of water vapor concentration reduces formamide hydrolysis during storage, prevents a decrease in DNA fragment separation ability during electrophoresis, and enhances storage stability.

  According to the present invention, there are provided a cartridge and a cartridge packaging kit which are reduced in contamination contamination, high in storage stability and low in cost in a cartridge made of an organic material in which a plurality of reagents used in electrophoresis containing formamide are enclosed.

It is a conceptual diagram for demonstrating an example of the whole structure of the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating the structure of the nucleic acid analysis cartridge of this invention, a packaging kit, and a packaging kit preparation process. It is a conceptual diagram for demonstrating another structure of the nucleic acid analysis cartridge and packaging kit of this invention, and a packaging kit preparation process. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention. It is a conceptual diagram for demonstrating an example of a structure of the nucleic acid analyzer for operating and detecting the nucleic acid analysis cartridge of this invention.

Hereinafter, the novel features and effects of the present invention will be described with reference to the drawings.
Here, in order to have a complete understanding of the present invention, specific embodiments will be described in detail, but the present invention is not limited to the contents described herein. In addition, the embodiments can be appropriately combined, and the present specification also discloses the combination form.

An example of the nucleic acid analysis cartridge of the present invention will be described with reference to FIG. 1 and FIG.
FIG. 1 is a plan view showing the overall outline of the nucleic acid analysis cartridge 1. The nucleic acid analysis cartridge 1 shown in FIG. 1 is a configuration example that can simultaneously analyze a total of 8 samples including a control sample.
The nucleic acid analysis cartridge 1 includes a reagent enclosure chamber (2, 3) in which a reagent is enclosed and holds the reagent therein, a branch chamber 4 for branching the reagent solution, a mixing chamber 6 for mixing the reagent solution, and heating to be heated. It has the flow path 13 which connects a room (7,10) and these rooms. Here, as the reagent enclosure chamber, there are a Master mix enclosure chamber 2, a Primer mix enclosure chamber 3, an electrophoresis marker enclosure chamber 8, a formamide enclosure chamber 9, and a sample solution enclosure chamber 12. The heating room includes a DNA amplification heating room 7 for performing DNA amplification and a DNA denaturation heating room 10.
Moreover, there are a measurement sample insertion room 5 into which a measurement sample is inserted and a capillary insertion room 11 into which a capillary is inserted as a room other than the reagent enclosure room, the branch room, the mixing room, and the heating room. In this figure, only one end of the capillary is shown, but the capillary is provided extending in the depth direction with respect to the figure.

  The reagent solution basically flows from the left side to the right side of the plan view shown in FIG. The reagent in the Master mix enclosure 2 and the Primer mix enclosure 3 flows through the flow path, for example, to the branch chamber 4 located at the lower side of the figure, and further flows through the measurement sample 5 arranged at the lowermost stage. The mixture chamber 6, the DNA amplification heating chamber 7, the electrophoresis marker enclosure chamber 8, the formamide enclosure chamber 9, and the DNA denaturation heating chamber 10 are arranged in this order in the lower stage and reach the capillary insertion chamber 11. In the present embodiment, a configuration example in which 8 samples can be analyzed simultaneously has been described, and the case of the sample flowing in the lowermost stage in the figure has been described. However, the flow of the reagent is the same for the other samples.

  However, between some rooms, the reagent solution that has once advanced is returned to the original room, or is reciprocated to advance to the previous room, thereby efficiently mixing the reagent solution. As a combination of a room and a room for performing such reciprocating liquid feeding, a sample solution enclosing room 12 and a measurement sample insertion room 5, a DNA amplification heating room 7 and a mixing room 6, a formamide enclosing room 9 and a mixing room 6 are listed. It is done.

FIG. 2 shows a schematic cross-sectional view of the nucleic acid analysis cartridge 1. This sectional view is in line with ^the cutting line A-A of FIG. The nucleic acid analysis cartridge 1 includes a membrane 15, a cartridge body 14, an upper lid 16, and a film 17 in order from the bottom. As a manufacturing method, after the cartridge body 14 and the upper lid 16 are joined, the cartridge body 14 and the membrane 15 are joined or bonded. Thereafter, the reagent is sealed in the sealing chamber through the opening at the top of the sealing chamber 9, and then the film 17 is bonded to complete.

  The nucleic acid analysis cartridge 1 is used by being installed in a cartridge holder of a nucleic acid analyzer. A mechanism for changing the membrane is provided in the cartridge holder, and the reagent solution is moved from room to room by changing the membrane. Further, by providing the cartridge holder with a heating mechanism, the reagent solution in the cartridge is heated to perform DNA amplification or DNA denaturation. The reagent feeding method will be described later with reference to FIGS.

The outline | summary of the nucleic acid analysis cartridge packing kit of this invention and a packing kit preparation process is demonstrated using FIG. 3 (a) is a sectional view of a nucleic acid analysis cartridge 1, in particular, is obtained by emphasizing the cross-sectional portion along the ^cutting line A-A shown in the Figure 1. FIG. 3D shows a state where the nucleic acid analysis cartridge 1 shown in FIG. 3A is packed with the nucleic acid analysis cartridge packing kit of the present invention.

  3A to 3D will be described in order. First, the rubber plug 18 of the nucleic acid analysis cartridge 1 in which the formamide 24 is enclosed is removed and a vent 21 is provided (FIG. 3A). After the nucleic acid analysis cartridge 1 with the rubber plug removed is packed in the packing bag 22 (FIG. 3B), the packing bag 22 is decompressed, and the nucleic acid analysis cartridge 1 and the packing bag 22 are discharged from the packing bag discharge port 23. The air containing water vapor is discharged (FIG. 3C). Thereafter, the packing bag discharge port is welded, the inside of the packing bag is sealed, and the nucleic acid analysis cartridge packing kit of the present invention is completed (FIG. 3 (d)).

  The reduced pressure package as shown in FIG. 3C reduces the water vapor concentration in the packing bag 22 and uses the packing bag 22 having low water vapor permeability to transfer the air from the atmosphere into the packing bag 22 during transportation and storage. Reduce water vapor transmission. The vent 21 and the air hole 20 reduce the water vapor concentration in the space in contact with the reagent by efficiently discharging the water vapor in the space in contact with the formamide 24 in the formamide enclosure 9 in the reduced pressure package. The reduction in water vapor concentration reduces formamide hydrolysis during transportation and storage, prevents a decrease in DNA fragment separation during electrophoresis, and enhances storage stability.

  Next, the further form of the nucleic acid analysis cartridge packing kit of this invention is demonstrated using FIG. The nucleic acid analysis cartridge of FIG. 4 has a valve structure with a rubber plug 18 and a vent 21. As shown in FIG. 4C, when the rubber plug 18 descends to the wide vent 21 in the drawing at the time of depressurization, a gas such as air is discharged from the other narrow vent 21 and the pressure is reduced. The rubber plug 18 closes both the wide vent 21 and the narrow vent 21 to seal the nucleic acid analysis cartridge (FIG. 4D).

  The vent 21 of the nucleic acid analysis cartridge shown in FIG. 3 is sealed with the packing bag 22, whereas the vent 21 of the nucleic acid analysis cartridge shown in FIG. 4 is doubled by the rubber plug 18 and the packing bag 22. Since it is sealed, water vapor permeation from outside air to the space in the formamide enclosure 9 can be more reliably reduced. Therefore, higher storage stability can be obtained.

  In the nucleic acid analysis cartridge packing kit of the present invention, the space surrounded by the reagent, the cartridge material, and the film material is selected from nitrogen, argon, and helium before packing (FIGS. 3A and 4A). At least one kind of gas may be substituted. Since the water vapor concentration in the cartridge internal space before packing can be reduced, the water vapor concentration in the cartridge internal space after the decompression package can also be reduced, and hydrolysis of formamide can be prevented.

  The material used for the packaging bag having a low water vapor permeability used in the present invention is not particularly limited, but a plastic material having a small specific gravity and a small bending strength is desirable. Examples of such plastic materials include polypropylene, polyethylene, polyacetal, polybutylene terephthalate, polyethylene terephthalate, acrylonitrile butadiene styrene, polystyrene, acrylic, polycarbonate, modified polyphenylene ether, polyvinyl acetate, and copolymers thereof. A plurality of types of sheets made of these materials may be stacked to form one packing material. Regarding the packaging material in which a plurality of types of sheets are stacked, it is desirable to use a plastic material having a low softening point as the plastic material of the innermost layer of the packaging bag because the packaging bag can be easily welded at a low temperature in a short time. Examples of such plastic materials include polypropylene, polyethylene, polyvinyl acetate, acrylic, and copolymers thereof. Moreover, in order to reduce water vapor permeability, a layer made of a metal such as aluminum or an inorganic material such as silica may be introduced into the packaging material in order to reduce the water vapor permeability.

  Moreover, there is no restriction | limiting in particular about the vacuum degree in the packaging bag at the time of a pressure reduction package, However, About 0.01-0.9 atm is desirable. If it is less than 0.01 atm, the effect of discharging water vapor in the packaging bag at the time of packaging is high, but the deformation amount of the nucleic acid analysis cartridge after decompression packaging becomes large. On the other hand, when it is larger than 0.9 atm, the water vapor discharge effect is reduced. For this reason, a desirable vacuum range is determined.

Moreover, there is no restriction | limiting in particular about the water vapor permeability of a packaging bag or a film material, However, It is desirable that it is 100.0 g / m < ² > or less in 24 hours under 1 atmosphere. A water vapor permeability of a resin film such as a general polyacetal, ABS resin, polymethyl methacrylate, polycarbonate, or polystyrene is about 50.0 g / m ² . When formamide contained in a glass container having an open mouth is packed in a film material using these resins and kept for one year in an environment of a temperature of 25 ° C. and a humidity of 60%, hydrolysis of formamide occurs. On the other hand, by using a film material of 50.0 g / m ² or less, water vapor mixed from the atmosphere can be reduced, and decomposition of formamide can be reduced. For these reasons, the desirable water vapor permeability range described above is determined.

  The material used for the cartridge body 14 and the upper lid 16 is preferably a thermoplastic plastic material that is inexpensive and can be molded and is suitable for mass production. Examples of such plastic materials include polypropylene, polyethylene, polyacetal, polybutylene terephthalate, polyethylene terephthalate, acrylonitrile butadiene styrene, polystyrene, acrylic, polycarbonate, modified polyphenylene ether, polyvinyl acetate, cycloolefin, and copolymers thereof. In addition, a composite material in which a thermosetting plastic material, an inorganic material, a metal material, or the like is finely dispersed in the thermoplastic plastic material can also be used.

  The material used for the membrane 15 is preferably an elastic body suitable for a cartridge holder mechanism described later. Such elastic bodies include isoprene rubber, styrene rubber, butyl rubber, butadiene rubber, ethylene / propylene rubber, nitrile rubber, chloroprene rubber, hyperon rubber, urethane rubber, polysulfide rubber, silicone rubber, fluorine rubber, acrylic rubber, ethylene Examples thereof include vinyl acetate rubber, hydrin rubber, and copolymers thereof.

  Moreover, there is no restriction | limiting in particular as a material used for the film 17, However, The plastic material with small specific gravity and small bending strength is desirable like the material used for a packaging bag. Examples of such plastic materials include polypropylene, polyethylene, polyacetal, polybutylene terephthalate, polyethylene terephthalate, acrylonitrile butadiene styrene, polystyrene, acrylic, polycarbonate, modified polyphenylene ether, polyvinyl acetate, and copolymers thereof. A plurality of types of sheets made of these materials may be stacked to form a single film. Moreover, about the film which laminated | stacked multiple types of sheet | seat, in order to reduce water vapor permeability, you may introduce | transduce into the inside the layer which consists of inorganic materials, such as metals, such as aluminum, and a silica.

  There are no particular restrictions on the bonding or joining of the cartridge body 14 and the membrane 15, the cartridge body 14 and the upper lid 16, and the upper lid 16 and the film 17 for assembling the nucleic acid analysis cartridge. Adhesion using an adhesive or double-sided tape is also possible. Bonding such as thermal welding, ultrasonic welding, and laser welding, and chemical bonding using a silane coupling material or a solvent can be used.

  FIG. 5 shows a further example of the nucleic acid analysis cartridge 1 of the present invention. The difference from the cartridge for nucleic acid analysis shown in FIG. 2 is that the formamide enclosed chamber 9 is divided into a chamber in which formamide 24 is held and another chamber in the upper part. The orifice 33 is provided. Since an orifice having a small space cross-sectional area exists between the two chambers, it is possible to prevent the formamide 24 from being scattered through the air holes 20 to the adjacent mixing chamber 6 or the like. Therefore, since scattering can be prevented, the volume of the reagent to be initially sealed can be reduced, and the analysis cost can be reduced.

  An example of a process for producing the nucleic acid analysis cartridge packaging kit of the present invention will be described with reference to FIGS. In this embodiment, the cartridge main body 14 and the upper lid 16 shown in FIG. 2 are produced using an injection molding process. Here, each material is made of polypropylene.

Next, after the cartridge main body 14 and the upper lid 16 are bonded using ultrasonic welding, the cartridge main body 14 and the membrane 15 are bonded together using a double-sided tape (in this embodiment, manufactured by Nitto Denko Corporation). For the membrane 15, a silicon rubber sheet (product name: T_30, manufactured by Inoac Corporation) having a thickness of 0.2 mmt is used. STR analysis kit (product name: AmpFlSTR Identifiler Direct PCR Amplification Kit, manufactured by Life Technologies), cell lysate (product name: Prep-n-Go ^TM Buffer, manufactured by Life Technologies), formamide (product name: Hi-) Di Formamide (manufactured by Life Technologies) and electrophoresis marker (product name: GeneScan 500LIZ, manufactured by Life Technologies) are enclosed. Thereafter, a film 17 (product name: plate seal MS-30020, manufactured by Sumitomo Akita Bake Co., Ltd.) is bonded onto the upper lid 16. Thereafter, as shown in FIG. 3, the nucleic acid analysis cartridge 1 is packed in a packing bag 22 (product name: aluminum lami zip, manufactured by As One Co., Ltd.), and then a ventilated sealer (product surface: FCB-200, manufactured by Fuji Impulse Sales Co., Ltd.). To prepare a nucleic acid analysis cartridge packing kit of the present invention.

An outline of a nucleic acid analyzer for performing STR analysis and detecting a nucleic acid analysis cartridge will be described with reference to FIG.
The nucleic acid analyzer includes a cartridge holder 26, a liquid feeding / temperature adjustment unit 27, a liquid feeding pump 28, an electrophoresis capillary 29, a laser unit 30, a detection unit 31, and a control substrate unit 32.

  The cartridge holder 26 holds the nucleic acid analysis cartridge 1. Further, in order to transfer the reagent solution in the cartridge, it has a mechanism for changing the membrane 15 under the cartridge and a mechanism for adjusting the temperature of the cartridge. The liquid feeding / temperature adjustment unit 27 controls the membrane fluctuation mechanism and temperature adjustment mechanism of the cartridge holder 26 and the liquid feeding pump 28.

  The electrophoresis capillary 29 is connected to the nucleic acid analysis cartridge 1, sucks the DNA fragment amplified and fluorescently labeled in the cartridge, and then separates the DNA fragment under a high voltage. The laser unit 30 excites fluorescent molecules in the electrophoresis capillary 29 by irradiating the electrophoresis capillary 29 with a laser. The detection unit 31 detects the excited fluorescent molecule.

  The configuration of a pneumatic control system that varies the membrane 15 (see FIG. 2) in the lower part of the cartridge, which is built in the liquid feeding / temperature control unit 27 (see FIG. 6), will be described with reference to FIG. A liquid feed pump 28 serving as a pneumatic drive source performs air suction and discharge. The discharged air passes through the pipe, passes through the filter 34 and the air pressure adjusting valve 35, and is connected to the IN side of the three-way valve manifold 36. Three-way valves 37 are mounted in series on the three-way valve manifold 36 and are connected to each other through the same flow path. Pipes are connected from the three-way valves 37, respectively, and each is connected to the cartridge holder 26 (see FIG. 6) through the speed controller 38. The three-way valve manifold 36 also has an exhaust OUT-side flow path that is open to the atmosphere. A silencer 39 is attached to the outlet of the OUT side flow path.

  As the air discharged from the liquid feed pump 28 passes through the filter 34, dust and dust contained in the air are removed. This prevents foreign matter from entering the three-way valve 37 and the speed controller 38. Further, the air pressure applied to the cartridge holder 26 can be adjusted to an appropriate pressure by the air pressure adjusting valve 35. By mounting the three-way valve 37 on the three-way valve manifold 36, the connection of the pipes can be integrated into one place. Even if the number of the three-way valves 37 is increased, only one pipe connection is required, so that it can be more compactly accommodated. By connecting a speed controller 38 to each pipe connected to the three-way valve 37, the flow rate of air pressure can be controlled. This time, it is important to manage the flow rate because the liquid is sent by air pressure. In addition, since a sound is generated when the pipe with increased pressure is released to the atmosphere, a silencer 39 is provided at the OUT side outlet to reduce the sound.

  FIG. 8 is a diagram showing the direction control of the three-way valve 37 configured in the pneumatic control system. In the current piping, the three-way valve 37 switches the pneumatic flow path 40 connected from the IN side to the cartridge holder 26 (see FIG. 6) side and the pneumatic flow path 41 connected from the cartridge holder 26 side to the OUT side. The three-way valve 37 is normally closed. In the normal state, the pneumatic flow path 40 is closed, and the pneumatic flow path 41 is connected. At this time, air coming from the IN side is connected to the three-way valve manifold 36, but since the pneumatic flow path 40 is closed, no air pressure is applied to the cartridge holder 26 side. However, since the pneumatic flow path 41 is open, the flow path on the cartridge holder 26 side and the OUT side is open to the atmosphere. When the three-way valve 37 is energized, the pneumatic channel 40 is opened and the pneumatic channel 41 is closed. At this time, the air coming from the IN side is connected to the three-way valve manifold 36, and since the pneumatic flow path 40 is open, the air can be sent to the cartridge holder 26 side. Further, since the air pressure channel 41 is closed, it is possible to apply air pressure to the cartridge holder 26 side. Since each pipe is connected to the cartridge holder 26 side via the three-way valve 37, air pressure can be applied to an arbitrary flow path.

  FIG. 9 shows a liquid feeding mechanism due to membrane fluctuation in the cartridge holder 26 (see FIG. 6). In the cartridge holder main body 50, when the nucleic acid analysis cartridge 1 is set, a pin 51 and a pin 52 that are driven by a pneumatic control system to act as a valve are incorporated. A packing 53 and a packing 55 are attached to the pin 51 and the pin 52, respectively. A packing 54 and a packing 56 are built in the tip of the pin 51 and the pin 52 on the cartridge holder main body 50 side. The cartridge holder body 50 is provided with a sealing protrusion 57 that can crush the membrane 15 when the nucleic acid analysis cartridge 1 is set and block the periphery of the flow path 13 of the nucleic acid analysis cartridge 1. The cartridge holder body 50 is formed with pneumatic ports 58, 59, 60, 61, 62 connected to the pneumatic control system. Since each pneumatic port is connected to the three-way valve 37 of the pneumatic control system, each can be controlled separately.

  The cartridge holder body 50 is preferably an acrylic resin. As the number of liquid feeding sites of the nucleic acid analysis cartridge 1 increases, the air flow path of the cartridge holder main body 50 becomes more complicated. Acrylic can be joined and bonded, so it can handle complicated flow paths. Since the number of pins 51 and pins 52 increases as the number of liquid feeding points increases, it is desirable to mold with a rigid resin such as PPS resin. However, care should be taken when making by molding because air may leak from the parting line. The packing 53, packing 54, packing 55, and packing 56 are pneumatic reciprocating packings, and vacuum grease is also applied to the sliding portions. This reduces the sliding resistance when the pins 51 and 52 are driven.

  FIG. 10 is a view when the nucleic acid analysis cartridge 1 (see FIG. 1) is set in the cartridge holder 26 (see FIG. 6). When set, the membrane 15 is crushed by the sealing projection 57 in the cartridge holder main body 50 and the periphery of the flow path 13 is closed. The cartridge holder 26 has a pneumatic port 60 for pushing the pin 51, a pneumatic port 59 for returning the pin 51 to its original position, a pneumatic port 62 for pushing the pin 52, and a pin 52 for returning the pin 52 to its original position. There is a pneumatic port 61, and a pipe from the pneumatic control system is connected to each port. Thus, air pressure is supplied to each port by the air pressure control system, and each pin is driven like an air cylinder. A pneumatic port 58 for pressing the membrane 15 against the flow path 13 is also formed. However, the air pressure is not given to the cartridge holder 26 only by connecting the piping of the air pressure control system to each port. By controlling the direction of the three-way valve 37 described above, all the ports of the cartridge holder 26 are released to the atmosphere in the normal state.

7 and FIGS. 11 to 20 show the flow of the liquid feeding in the nucleic acid analysis cartridge 1 of the present invention.
As preparation before liquid feeding, first, the liquid feeding pump 28 is driven before connecting the cartridge holder 26 and the pneumatic control system. Since the three-way valve 37 is normally closed, the pressure between the liquid feed pump 28 and the three-way valve 37 increases. In this state, the pressure is adjusted to an appropriate pressure by the pressure adjustment valve 35. Thereafter, each three-way valve 37 is energized, the pneumatic flow path 40 is opened, and the pneumatic flow path 41 is closed. Then, since air is sent to the pipe connected to the cartridge holder 26, the flow rate of each pipe connected to the cartridge holder 26 is adjusted by the speed controller 38 in that state. After adjusting the air pressure and flow rate, the pneumatic control system is connected to the cartridge holder 26, and the nucleic acid analysis cartridge 1 is set in the cartridge holder 26.

  Hereinafter, the pneumatic control method and the movement of the pin and fluid accompanying therewith will be described. First, the three-way valve 37 of the pneumatic port 59 and the three-way valve 37 of the pneumatic port 61 are opened. Thereby, the pin 51 and the pin 52 are lowered as shown in FIG. This state is the initial position of the pin.

  Next, the three-way valve 37 of the pneumatic port 60 is opened, and the three-way valve 37 of the pneumatic port 59 is closed. As a result, the air pressure accumulated on the air pressure port 59 side is released to the atmosphere, and air pressure is applied from the air pressure port 60 side, so that the pin 51 is pressed against the nucleic acid analysis cartridge 1 by air pressure as shown in FIG. The pin 51 pushes up the plug 19 that closes the formamide enclosure 9 through the membrane 15. Then, the stopper 19 that closed the formamide enclosure 9 is opened. The plug 19 that has been opened once is kept open from now on by keeping it from moving from the pushed-up position. However, since the pin 51 is pressed between the formamide enclosure 9 and the flow path 13, the space between the formamide enclosure 9 and the flow path 13 remains closed.

  Next, the three-way valve 37 of the pneumatic port 58 is opened. Then, as shown in FIG. 13, the membrane 15 is pushed by air pressure and is brought into close contact with the flow path 13. As a result, the air originally in the flow path 13 can be pushed out to the mixing chamber 6. Since the inside of the nucleic acid analysis cartridge 1 is hermetically sealed, the pressure inside the nucleic acid analysis cartridge 1 increases during this time. Since there is an air hole 20 passing through the upper part of the chamber between the formamide enclosure 9 and the mixing chamber 6, the pressure in each chamber is the same.

  Next, the three-way valve 37 of the pneumatic port 62 is opened, and the three-way valve 37 of the pneumatic port 61 is closed. As a result, the air pressure accumulated on the air pressure port 61 side is released to the atmosphere, and air pressure is applied from the air pressure port 62 side, so that the pin 52 is pressed against the nucleic acid cartridge 1 with air pressure as shown in FIG. Since the pin 52 is pressed between the mixing chamber 6 and the flow path 13 through the membrane 15, the space between the mixing chamber 6 and the flow path 13 is blocked.

  Next, the three-way valve 37 of the pneumatic port 60 is closed, and the three-way valve 37 of the pneumatic port 59 is opened. As a result, the air pressure accumulated on the air pressure port 60 side is released to the atmosphere, and air pressure is applied from the air pressure port 59 side, so that the pin 51 returns to the original position as shown in FIG. Since the air pressure is still applied from the air pressure port 58, the membrane 15 remains pressed against the flow path 13.

  Next, the three-way valve 37 of the pneumatic port 58 is closed. As a result, the air pressure accumulated on the air pressure port 58 side is released to the atmosphere, and the membrane 15 pressed against the flow path 13 returns to its original position by its own elastic force and the pressure inside the nucleic acid cartridge 1 as shown in FIG. . At this time, since the mixing chamber 6 and the flow path 13 remain blocked by the pins 52, the reagent flows into the flow path 13 from the formamide enclosure 9 and the air in the mixing room 6 moves to the formamide enclosure 9 through the air holes 20. .

  Next, the three-way valve 37 of the pneumatic port 60 is opened, and the three-way valve 37 of the pneumatic port 59 is closed. As a result, the air pressure accumulated on the air pressure port 59 side is released to the atmosphere, and air pressure is applied from the air pressure port 60 side, so that the pin 51 is pressed against the nucleic acid cartridge 1 again as shown in FIG. At this time, the pin 51 again closes the space between the formamide enclosure 9 and the flow path 13, but the reagent remains in the flow path 13.

  Next, the three-way valve 37 of the pneumatic port 62 is closed, and the three-way valve 37 of the pneumatic port 61 is opened. Accordingly, the air pressure accumulated on the air pressure port 62 side is released to the atmosphere, and air pressure is applied from the air pressure port 61 side, so that the pin 52 returns to the original position as shown in FIG.

  Next, the three-way valve 37 of the pneumatic port 58 is opened again. As a result, as shown in FIG. 19, the membrane 15 is pushed by air pressure and is in close contact with the flow path 13. At that time, since the space between the formamide enclosure chamber 9 and the flow path 13 remains blocked by the pin 51, the reagent accumulated in the flow path 13 flows into the mixing chamber 6. As a result, the reagent is mixed into the sealed sample.

  Next, the three-way valve 37 of the pneumatic port 61 is closed again, and the three-way valve 37 of the pneumatic port 62 is opened. As a result, the air pressure accumulated on the air pressure port 61 side is released to the atmosphere, and air pressure is applied from the air pressure port 62 side, so that the pin 52 is pressed against the nucleic acid analysis cartridge 1 as shown in FIG. At this time, the space between the mixing chamber 6 and the flow path 13 is blocked by the pin 52.

  By repeating this operation with FIGS. 14 to 20, the reagent sealed in the formamide sealed chamber 9 can be sent to the mixing chamber 6. As a result, the liquid can be fed in the sealed nucleic acid analysis cartridge 1 without being in contact with the fluid. By repeating this operation many times, all the reagents in the room can be fed, regardless of whether it is a small amount of reagent or a reagent with a large capacity. However, after purification or reaction, there are cases where it is desired to send only a certain volume, not all reagents in the room. In that case, by managing the number of times this operation is repeated, it is possible to feed only a prescribed volume.

  By providing this structure for each flow path between the rooms and performing the same operation, it becomes possible to send various reagents at an arbitrary timing. Further, when performing purification, reaction, and stirring, the chambers can be arbitrarily sealed, so that the control of the fluid can be stabilized.

  There are many types of reagents necessary for pretreatment in gene analysis. On the other hand, by adopting this system, the drive source can deal with a large number of reagents while only the liquid feed pump 28 configured in the pneumatic control system is left. Further, even if the nucleic acid analysis cartridge 1 is added on the apparatus, the connection of the three-way valve 37 and the pipe can be increased in this system without increasing the drive source. For this reason, it can be said that it is a versatile system. Furthermore, the cost of the apparatus can be reduced and the apparatus can be downsized.

  A method for performing STR analysis using the nucleic acid analysis cartridge packaging kit of the present invention and the nucleic acid analyzer of Example 2 will be described.

The oral cells of the subject are collected using a swab (product name: 4N6 ^™ FLOQ Swabs (registered trademark), manufactured by COPAN). After removing the nucleic acid analysis cartridge from the packing bag, after inserting a swab with oral cells attached to the measurement sample insertion chamber 5 on the nucleic acid analysis cartridge, and sealing the measurement sample insertion chamber 5 with the rubber plug 18, The nucleic acid analysis cartridge 1 is attached to the cartridge holder 26. Thereafter, the nucleic acid analyzer automatically operates to perform STR analysis including (1) DNA sample collection, (2) DNA amplification, (3) DNA denaturation, and (4) DNA fragment separation and detection. The operating conditions of the apparatus are as follows.
O Device operating conditions (1) DNA sample collection: Oral cells are immersed in Prep-n-Go ^™ Buffer (registered trademark) for 5 minutes.
(2) DNA amplification: 95 ° C./10 min hold → temperature cycle (number of cycles: 25 times, 1 cycle condition: 94 ° C./20 sec → 59 ° C./2 min → 72 ° C./1 min) → 60 ° C./25 min Holding → 4 ° C./25 minutes holding (3) DNA denaturation: after mixing with formamide, 95 ° C./3 minutes holding (4) DNA fragment separation and detection: injection 1.2 kV / 15 sec

1 ... Nucleic acid analysis cartridge,
2 ... Master Mix enclosure room,
3 ... Primer Mix enclosure room,
4 ... Branch room,
5 ... Measurement sample insertion room,
6 ... mixing room,
7 ... DNA amplification heating room,
8 ... Room containing electrophoresis marker,
9 ... Formamide enclosed room,
10 ... DNA denaturation heating room,
11 ... Capillary insertion room,
12 ... Sample solution enclosure room,
13 ... channel,
14 ... cartridge body,
15 ... Membrane,
16 ... upper lid,
17 ... Film,
18 ... rubber stopper,
19 ... stopper,
20 ... Air hole,
21 ... Vents,
22 ... Packing bag,
23 ... Packing bag outlet,
24 ... formamide,
25 ... valve structure,
26 ... cartridge holder,
27 ... Liquid feeding / temperature control unit,
28 ... liquid pump,
29 ... electrophoresis capillary,
30 ... Laser unit,
31 ... Detection unit,
32 ... Control board unit,
33: Orifice,
34 ... filter,
35 ... Air pressure adjusting valve,
36 ... Three-way valve manifold,
37 ... Three-way valve,
38 ... Speed controller,
39 ... Silencer,
40 ... pneumatic flow path 50 ... cartridge holder body,
51,52 ... pin,
53, 54, 55, 56 ... packing,
57 ... projection for sealing,
58, 59, 60, 61, 62 ... pneumatic ports.

Claims (7)

In a nucleic acid analysis cartridge containing a plurality of reagents used in electrophoresis containing formamide,
A reagent enclosure that has a first opening open to the atmosphere and encloses the reagent;
A reagent mixing part having a second opening connected to the flow path for passing the reagent from the reagent sealing part and opened to the atmosphere;
An air hole enabling air movement between the reagent mixing part and the reagent sealing part,
A region including at least the first and second openings is covered with a packaging bag made of a material having low permeability to water vapor contained in the atmosphere, and the inside of the packaging bag is lower than the atmospheric pressure of the atmosphere. A nucleic acid analysis cartridge which is decompressed. The region including the reagent enclosing part and the reagent mixing part is composed of a cartridge material and a film material,
The film material is made of a material having low water vapor permeability,
The nucleic acid analysis cartridge according to claim 1, wherein the film material is provided so as to cover at least the first opening.   The nucleic acid analysis cartridge according to claim 1, wherein the second opening has a valve structure. The reagent enclosure includes a first enclosure chamber in which the reagent is enclosed and a second enclosure chamber having the first opening and connected to the air hole,
The first enclosure chamber and the second enclosure chamber are stacked via an orifice, and the orifice has a cross-sectional area smaller than a cross-sectional area of a surface facing the first enclosure chamber and the second enclosure chamber. The cartridge for nucleic acid analysis according to claim 1, comprising:   In the region including the reagent enclosing part and the reagent mixing part, the atmosphere in the space surrounded by the reagent, the cartridge material, and the film material is replaced with at least one gas selected from nitrogen, argon, and helium. The nucleic acid analysis cartridge according to claim 2, wherein the cartridge is a nucleic acid analysis cartridge.   Each of the cartridge material and the packing bag is either an organic material, a mixed material composed of an organic material and an inorganic material, a mixed material composed of an organic material and a metal material, or a mixed material composed of an organic material, an inorganic material and a metal material. The nucleic acid analysis cartridge according to any one of claims 2 to 5, wherein the cartridge is made of any of the above materials. The nucleic acid analysis cartridge according to any one of claims 2 to 6, wherein the water vapor permeability of the packing bag or the film material is 100.0 g / m ² or less at 24 hours under 1 atmosphere.

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