JP2006223126A - Reaction vessel, method and apparatus for detecting genetic polymorphism and method and apparatus for diagnosis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a reaction vessel useful as a reagent kit suitable for automation of various measurements. <P>SOLUTION: In a preferable form, a resin plate 40 is equipped with a sample injection part 44, a PCR reaction reagent storage part 46, a typing reagent storage part 48 and a mixing part 50 and the top of each part is covered with an elastic film 42. An organism sample is injected into the sample injection part 44, a stopper 58 is removed, a proper part of a flow channel is closed, air is discharged from or sucked in the sample injection part 44 and the PCR reaction reagent storage part 46 through a nozzle so that the sample is mixed with the PCR reaction reagent and retained in a PCR reaction part 54 to carry out a PCR reaction by a fixed temperature cycle. Then, a proper part of the flow channel is closed, air is discharged from or sucked in the typing reagent storage part 48 and the mixing part 50 through a nozzle, a reaction solution is mixed with a typing reagent, then the reaction solution is sent to a probe arrangement part 30 and a fluorescence is detected by a corresponding probe to detect SNP. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention is a reaction container suitable for conducting various automatic analyzes such as genetic analysis research and clinical practice in the medical field, and using it, polymorphisms of genomic DNA of animals and plants including human beings, particularly SNP (single base) Gene polymorphism detection method for detecting polymorphism), automatic analysis device and automatic gene analysis device therefor, and diagnosis of disease incidence using the gene polymorphism detection results, types and effects and side effects of administered drugs The present invention relates to a method and an apparatus for diagnosing the relationship between and the like.

The followings have been proposed as methods or apparatuses for predicting the susceptibility of diseases using genetic polymorphism.
In order to determine whether a patient is susceptible to sepsis and / or is likely to progress rapidly to sepsis, a nucleic acid sample is taken from the patient and the pattern 2 allele in the sample, or the pattern 2 allele When a marker gene that is linkage disequilibrium is detected and a marker gene that is linkage disequilibrium with the pattern 2 allele is detected, it is determined that the patient is susceptible to sepsis (see Patent Document 1). ).

  For diagnosis of one or more single nucleotide polymorphisms in the human flt-1 gene, one or more positions of human nucleic acids: 1953, 3453, 3888 (each position in EMBL accession number X51602) ), 519, 786, 1422, 1429 (according to positions in EMBL accession number D64016, respectively), 454 (according to SEQ ID NO: 3) and 696 (according to SEQ ID NO: 5). The human constitution is determined by referring to the formality (see Patent Document 2).

Many methods have been reported for so-called typing for determining the base of an SNP site. A typical one is the following method.
In order to perform typing on hundreds of thousands of SNP sites using a relatively small amount of genomic DNA, a plurality of base sequences including at least one single nucleotide polymorphism site are used using genomic DNA and a plurality of pairs of primers. At the same time, using the plurality of amplified base sequences, the bases of single nucleotide polymorphic sites contained in the base sequences are discriminated by a typing step. As the typing process, an invader method or a Tuckman PCR method is used (see Patent Document 3).
Japanese translation of PCT publication No. 2002-533096 JP 2001-299366 A Japanese Patent Laid-Open No. 2002-300894 Japanese Patent No. 3454717 Hsu TM, Law S. M, Duan S, Neri BP, Kwok PY, "Genotyping single-nucleotide polymorphisms by the invader assay with dual-color fluorescence polarization detection", Clin. Chem., 2001 Aug; 47 (8): 1373 -7

  A first object of the present invention is to provide a reaction container that can be used as a reagent kit suitable for automating various measurements, although not limited to detection of genetic polymorphism.

  A second object of the present invention is to provide a method and apparatus for automatically detecting reactions using the reaction vessel of the present invention.

  A third object of the present invention is to provide a method for detecting a gene polymorphism using the reagent kit for detecting a gene polymorphism, particularly by using the reaction container of the present invention, and an automatic analyzer for the same. is there.

  A fourth object of the present invention is a method and apparatus for automatically performing diagnosis of disease morbidity using the genetic polymorphism detection result and diagnosis of the relationship between the type and effect of drugs to be administered and side effects. Is to provide.

  In order to achieve the first object, a reaction container of the present invention is a reaction container formed in a flat substrate and transporting a reaction solution of a sample or a sample and a reagent along a flow path, the substrate It is formed inside, connected to the reaction part through the flow path, and has a liquid reservoir part covered with a cap on the top, and the cap is made of a material that can penetrate the nozzle and restore the through hole. The liquid is transferred by discharging or sucking air into the cap through a nozzle.

  The cap is preferably an elastic deformation member, and examples of the material include a flexible material including PDMS (polydimethylsiloxane) or Si rubber.

  It is preferable that a plurality of the liquid reservoirs are provided, and the liquid is transferred by discharging or sucking air by alternately passing the nozzles through the caps.

  The flow path is preferably provided with a valve portion made of an elastically deformable member that can be closed by pressing from above, and the predetermined flow path portion is closed by pressing the valve portion, and the nozzle from the liquid reservoir portion The liquid is transferred by air discharge or suction.

  One form of the reaction container of the present invention is a reaction container in which a sample and a reagent are mixed and reacted. The sample is injected into or accommodated in a flat substrate, and a recoverable material is used above. A sample container covered with a cap having a through-hole capable of discharging or sucking air through a nozzle, and a reagent formed in the substrate is injected or accommodated therein, and the through-hole A reagent container covered with a cap, and a connection between the sample container and the first reaction part and between the reagent container and the first reaction part, and between each part And a flow path having a valve portion made of an elastically deformable member that can be closed by pressing from above.

  Although the sample can be stored in advance, this reaction container is usually used as a reagent kit, and the measurement is performed by injecting the sample into the sample storage portion each time. Therefore, it is preferable that the cap of the sample container is attached so that the sample can be opened and closed for injection.

  There is also an application in which a sample and a reagent are mixed and reacted, and then diluted and guided to a second reaction section to perform further reaction. In order to cope with such a use, this reaction vessel is formed in the substrate, accommodates a diluent, and is covered with a cap made of a material through which the nozzle can penetrate and the through hole can be restored. Diluted liquid storage section, diluted section formed in the substrate, and having an empty space covered with a cap made of a material through which the nozzle can penetrate and whose through hole can be restored, and diluted in the diluted section A second reaction section through which the reaction liquid is guided, and the flow path is connected to the dilution liquid storage section and the dilution section, and is also connected between the dilution section and the second reaction section. In addition, a valve portion made of an elastically deformable member that can be closed by pressing from above is also provided between these portions.

  One reaction using this reaction vessel is gene amplification. The reaction container of the present invention suitable for such a use is an amplification reaction section in which the reagent housed in the reagent housing section is a gene amplification reagent, and the first reaction section mixes the gene amplification reagent and the sample and performs an amplification reaction. Yes, this reaction vessel constitutes a reagent kit for the gene amplification reaction.

  The main purpose of using this reaction vessel is to detect genetic polymorphism. An example of a reaction container suitable for such a purpose is that the sample injected or stored in the sample storage unit is a biological sample, and the reagent stored in the reagent storage unit sandwiches each of a plurality of polymorphic sites. A gene amplification reagent comprising a plurality of primers to be bound, wherein the first reaction part is an amplification reaction part for mixing a gene amplification reagent and a sample to perform an amplification reaction, and is formed in the substrate, wherein the plurality of polymorphisms A typing reagent container that accommodates a typing reagent prepared in accordance with a region, and that is formed in the substrate, has a typing reagent container part that is covered with a cap made of a material through which the nozzle can penetrate and whose through hole can be restored. A mixture part consisting of an empty space covered with a cap made of a material through which the nozzle can penetrate and whose through hole can be restored, and the reaction liquid mixed in the mixing part is guided, and the plurality of polymorphic sites A probe placement section as a second reaction section, in which a plurality of probes that emit fluorescence corresponding to each of them are individually held, and the flow path is also connected to the typing reagent storage section and the mixing section In addition, the mixing portion and the probe placement portion are connected to each other, and each of the portions has a valve portion made of an elastically deformable member that can be closed by pressing from above. A reagent kit for detecting a gene polymorphism is constructed.

  Since the amplification reaction part repeats a cycle of changing the temperature, it is preferable that the thermal conductivity of the substrate is high. Therefore, it is preferable that the substrate thickness of the amplification reaction part is thinner than other parts.

  Another example of a reaction container suitable for the purpose of detecting a gene polymorphism is a typing reagent storage unit in which the liquid storage unit stores a typing reagent prepared corresponding to a plurality of polymorphic sites. The sample that has undergone the gene amplification reaction is injected into the part, and the first reaction part is guided with a reaction liquid in which the typing reagent and the sample are mixed in the liquid reservoir part, and the plurality of polymorphic sites A plurality of probes emitting fluorescence corresponding to each of the probes are individually held, and this reaction container constitutes a reagent kit for detecting a gene polymorphism.

  Yet another example of a reaction vessel suitable for the purpose of detecting a gene polymorphism is prepared in correspondence with a plurality of polymorphic sites, and a sample storage portion into which the liquid reservoir portion is injected with a sample for which a gene amplification reaction has been completed. A typing reagent storage unit that stores a typing reagent, both storage units are connected by a flow channel, and any of the storage units and the first reaction unit are connected by a flow channel. The first reaction part is a probe in which a reaction liquid in which a typing reagent and a sample are mixed is guided in the liquid reservoir part, and a plurality of probes emitting fluorescence corresponding to each of the plurality of polymorphic sites are individually held. An arrangement portion, wherein the flow path includes a valve portion made of an elastically deformable member that can be closed by pressing from above, and a predetermined flow path portion is closed by pressing the valve portion, and the liquid reservoir portion Through the nozzle Liquid can be transported to by discharging or sucking the air Te, the reaction vessel constitutes the reagent kit for detecting a genetic polymorphism.

  The reaction detection method of the present invention for achieving the second object uses a reaction container according to any one of claims 1 to 9 and discharges or sucks air into a liquid reservoir through a nozzle. Alternatively, the reaction solution of the sample and the reagent is transferred to the reaction unit along the flow path to be reacted, and the reaction result is detected.

  The detection device of the present invention that realizes the reaction detection method is equipped with the reaction container, a test reagent kit mounting part that controls each part to a predetermined temperature, a liquid feeding device that transfers the liquid, and a reaction container Fluorescence detection device for detecting fluorescence by irradiating the reaction liquid part after reaction with excitation light, temperature control of test reagent kit mounting part, liquid supply operation of liquid supply device, and control for controlling detection operation of fluorescence detection device And a section.

  An example of the liquid delivery device includes a pinch valve shaft that pushes the valve portion, and a nozzle that passes through the cap and discharges or sucks air from the cap, and the pinch valve shaft moves forward and backward each time it is pushed downward. A pressing pin is provided at the tip of a knock mechanism that is alternately fixed at a position.

  In order to achieve the third object, the gene polymorphism detection method of the present invention is characterized in that a biological sample is allowed to act on a gene amplification reaction solution containing a plurality of primers that bind to each of a plurality of polymorphic sites, Amplification step for amplification and a typing step for distinguishing the bases of the plurality of polymorphic sites by acting a typing reagent prepared corresponding to the plurality of polymorphic sites on the genomic DNA amplified in the amplification step Including.

That is, an example of a genetic polymorphism detection method uses the reaction container according to claim 10 or 11 and includes the following steps to detect a genetic polymorphism.
(A) A step of injecting a sample into the sample container,
(B) A predetermined flow path portion is closed by pressing at least one valve portion of the flow path, and a sample and a gene are amplified by discharging or sucking air to the sample storage section and the reagent storage section through a nozzle. Creating a reagent mixture and supplying it to the amplification reaction section;
(C) Gene amplification reaction of the mixed solution in the amplification reaction section,
(D) The amplification reaction part is formed by closing a predetermined flow path part by pressing at least one valve part of the flow path and discharging or sucking air to the typing reagent storage part and the mixing part via a nozzle. And (E) a step of sending the reaction solution mixed in the step (D) to the probe placement unit to detect fluorescence.

Another example of the genetic polymorphism detection method uses the reaction container according to claim 12 to detect the genetic polymorphism that includes the following steps and detects the genetic polymorphism.
(A) A step of injecting a sample into the liquid reservoir,
(B) A step of detecting the fluorescence by sending the reaction solution in which the typing reagent and the sample are mixed in the step (A) to the probe placement unit.

Still another example of the genetic polymorphism detection method uses the reaction container according to claim 13 and includes the following steps to detect the genetic polymorphism.
(A) A step of injecting a sample into the sample container,
(B) A predetermined flow path portion is closed by pressing at least one valve portion of the flow path, and a sample is typed by discharging or sucking air to the sample storage section and the typing reagent storage section through a nozzle. Mixing the reagents,
(C) A step of closing a predetermined flow path portion by pressing at least one valve portion of the flow path, and sending the reaction liquid mixed in the step (B) to the probe placement section to detect fluorescence.

Here, when the relationship between the polymorphic site and the primer is shown, in order to amplify one polymorphic site, a pair of primers that bind across the polymorphic site are required. Since there are multiple types of polymorphic sites in the target biological sample, if these polymorphic sites are located at a distance from each other, twice as many types of primers as the types of polymorphic sites are required. become. However, when two polymorphic sites are close to each other, amplification can be performed by binding a primer between each of the polymorphic sites, or between the two polymorphic sites. Amplification can also be performed by binding primers only on both sides of the sequences of the two polymorphic sites without binding. Therefore, the number of necessary primers is not necessarily twice the number of types of polymorphic sites. In the present invention, “a plurality of primers that bind each of a plurality of polymorphic sites” refers not only to a case where a pair of primers binds to a single polymorphic site but also two or more polymorphic sites. It is used to mean the type of primer necessary to amplify multiple polymorphic sites, including when binding between.
Polymorphisms include mutations, deletions, duplications, metastases and the like. A typical polymorphism is SNP.
The biological sample is blood, saliva, genomic DNA or the like.

  For the amplification step, a PCR method or the like can be used. In that case, it is preferable to carry out the PCR method under the condition that the pH at 25 ° C. is 8.5 to 9.5. In that case, the gene amplification reagent is a PCR reaction reagent.

  For SNP typing, it is essential to adjust genomic DNA at the stage of entering the amplification process, which requires labor and cost. If attention is paid only to the PCR method for amplifying DNA, a method of directly performing a PCR reaction from a sample such as blood without pretreatment has also been proposed. In the nucleic acid synthesis method for amplifying a target gene in a sample containing a gene, the gene inclusion body in the sample containing the gene or the sample containing the gene itself is added to the gene amplification reaction solution, The target gene in the sample containing the gene is amplified when the pH of the reaction solution is 8.5 to 9.5 (25 ° C.) (see Patent Document 4).

The typing system that has already been constructed requires a small amount of DNA to be initially collected in order to amplify a plurality of SNP regions to be typed by the PCR method. It is necessary to perform a pre-processing for extracting the. Therefore, it takes time and labor for the preprocessing.
Until now, no automated system has been constructed that simultaneously amplifies a plurality of SNP sites intended for typing when the direct PCR method and the typing method are combined.
In the typing process, an invader method or a Tuckman PCR method can be used. In that case, the typing reagent is an invader reagent or a Tuckman PCR reagent.

  An example of the genetic polymorphism detection apparatus of the present invention for achieving the third object is equipped with the reaction container according to claim 10 or 11, and the temperature of the amplification reaction part of the reaction container is set for gene amplification. An amplifying unit that controls the temperature of the probe, a test reagent kit mounting unit that includes a typing reaction unit that controls the temperature of the probe placement unit to a temperature at which the DNA and the probe react, and a sample storage unit and a reagent storage unit of the reaction container A liquid feeding device for transferring air between the parts and transferring the liquid from the mixing part to the probe placement part by sending air to the typing reagent storage part and the mixing part and pushing the valve part, and the probe placement Fluorescence detection device that irradiates the part with excitation light and detects fluorescence, temperature control of amplification unit and typing reaction unit, nozzle operation, liquid supply operation of liquid supply device, and detection of fluorescence detection device It is equipped with a, and a control unit for controlling the work.

  An example of the liquid feeding device includes a sample storage unit, a reagent storage unit, a typing reagent storage unit, and a mixing unit that include a nozzle that passes through a cap to feed or suck air and a pinch valve shaft that presses the valve unit. The valve shaft is provided with a pressing pin at the tip of a knock mechanism that is alternately fixed at the advance position and the retract position every time the valve shaft is pushed downward.

  Another example of the genetic polymorphism detection apparatus includes a typing reaction unit that is equipped with the reaction container according to claim 12 and that controls the temperature of the probe placement unit of the reaction container to a temperature at which the DNA and the probe are reacted. A test reagent kit mounting part, a liquid feeding device for transferring liquid to the probe placement part by discharging or sucking air to the typing reagent storage part of the reaction container via a nozzle, and an excitation light to the probe placement part And a control unit for controlling the temperature control of the typing reaction unit, the liquid supply operation of the liquid supply device, and the detection operation of the fluorescence detection device.

  Still another example of the genetic polymorphism detection apparatus is provided with the reaction container according to claim 13 and a typing reaction part for controlling the temperature of the probe placement part of the reaction container to a temperature at which the DNA and the probe are reacted. The test reagent kit mounting part provided, the nozzle that passes through the cap of the sample container and typing reagent container of the reaction container, and the pinch valve shaft that holds the valve part, and the liquid transfer and probe arrangement between the parts Liquid feeding device for transferring the liquid to the part, fluorescence detection device for detecting the fluorescence by irradiating the probe placement part with excitation light, temperature control of the typing reaction part, liquid feeding device having nozzle and pinch valve shaft And a controller for controlling the detection operation of the fluorescence detection apparatus.

  In order to achieve the fourth object, the diagnostic method of the present invention prepares a diagnostic value for a specific polymorphism or a combination of a plurality of polymorphisms as a database and detects it by the genetic polymorphism detection method of the present invention. It is a diagnostic method which reads a diagnostic value from the database based on the result of the made polymorphism.

  The diagnostic device of the present invention is detected by the genetic polymorphism detection device of the present invention, a database storing diagnostic values for a specific polymorphism or a combination of multiple polymorphisms, and the genetic polymorphism detection device. And a display device that reads and displays the diagnostic value from the database based on the polymorphic result.

FIG. 1 schematically shows the detection method of the present invention. Here, it is assumed that the PCR method is used for the amplification process and the invader method is used for the typing process.
In the PCR process, the PCR reaction reagent 4 is added to the biological sample 2 such as blood, or conversely, the biological sample 2 is added to the PCR reaction reagent 4. For example, 1 μL of the biological sample 2 is collected, and about 10 μL of the PCR reaction reagent 4 is added thereto. The PCR reaction reagent 4 is prepared in advance and includes a plurality of primers for the SNP site to be measured, and a buffer solution for adjusting pH, four types of deoxyribonucleotides, and other necessary reagents are added. The pH is adjusted to 8.5 to 9.5 when mixed with Sample 2.

  The mixture of the biological sample 2 and the PCR reaction reagent 4 is subjected to a PCR reaction according to a predetermined temperature cycle. The PCR temperature cycle includes three steps of denaturation, primer attachment (annealing), and primer extension, and DNA is amplified by repeating the cycle. As an example of each step, the denaturation step is 94 ° C. for 1 minute, the primer attachment step is 55 ° C. for 1 minute, and the primer extension is 72 ° C. for 1 minute. The biological sample may have been subjected to a genome extraction operation, but here, a sample that has not been subjected to a genome extraction operation is used. Even in the case of a biological sample that has not been subjected to genome extraction operation, DNA is released from blood cells and cells at a high temperature in the PCR temperature cycle, and the reaction proceeds when reagents necessary for the PCR reaction come into contact with the DNA.

  After the PCR reaction, the invader reagent 6 is added as a typing reagent. The invader reagent 6 includes a fluorescent (FRET) probe and a chestnut (Cleavase: structure-specific DNA degrading enzyme). A fret probe is a fluorescently labeled oligo having a sequence completely unrelated to genomic DNA, and the sequence is often common regardless of the type of SNP.

  Next, the reaction solution to which the invader reagent 6 has been added is added to the probe placement unit 8 of the typing reaction unit to cause a reaction. An invader probe and a reporter probe are individually held in each part of the probe placement unit 8 corresponding to each of the plurality of SNP parts, and the reaction solution reacts with the invader probe, and the SNP corresponding to the reporter probe is If present, it emits fluorescence.

The invader method is described in detail in paragraphs [0032] to [0034] of Patent Document 3.
Each reporter probe is prepared in two types according to the corresponding SNP base, and it can be determined whether the SNP is a homozygote or a heterozygote.

  The PCR method of the amplification step used in the present invention simultaneously amplifies a plurality of target SNP sites, and a plurality of genomes containing those SNP sites by PCR directly from a biological sample that has not been subjected to nucleic acid extraction operation. Amplify DNA. Therefore, a gene amplification reaction reagent containing a plurality of primers for these SNP sites is allowed to act on the biological sample, and a PCR reaction is caused under the condition that the pH at 25 ° C. is 8.5 to 9.5.

The PCR reaction reagent contains a pH buffer solution, salts such as MgCl ₂ and KCl, primers, deoxyribonucleotides and a thermostable synthase. In addition, substances such as surfactants and proteins can be added as necessary.

As the pH buffer solution, various pH buffer solutions can be used in addition to a combination of tris (hydroxymethyl) aminomethane and a mineral acid such as hydrochloric acid, nitric acid and sulfuric acid. The pH-adjusted buffer is preferably used at a concentration between 10 mM and 100 mM in the PCR reaction reagent.
A primer refers to an oligonucleotide that serves as a starting point for DNA synthesis by a PCR reaction. The primer may be synthesized or may be isolated from the living world.

  Synthetic enzymes are enzymes for DNA synthesis by primer addition and include chemical synthesis systems. Suitable synthases include E. coli. DNA polymerase I, E. coli Klenow fragment of DNA polymerase of E. coli, T4 DNA polymerase, Taq DNA polymerase, T. Examples include, but are not limited to, litoralis DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Hot Start Taq polymerase, KOD DNA polymerase, EX Taq DNA polymerase, and reverse transcriptase. “Thermal stability” means the property of a compound that retains its activity at elevated temperatures, preferably at 65-95 ° C.

  The invader method used in the typing step is a method of typing an SNP site by hybridizing an allele-specific oligo and a DNA containing the SNP to be typed, and the DNA containing the SNP to be typed and the SNP to be typed This method uses two types of reporter probes and one type of invader probe specific to each of the above alleles and an enzyme having a special endonuclease activity that recognizes and cleaves the DNA structure (see Patent Document 3). .)

The reaction container according to the present invention includes a liquid reservoir portion which is covered with a cap made of a material through which the nozzle can penetrate and whose through-hole can be restored, and by discharging or sucking air from the nozzle penetrating the cap. The liquid feeding efficiency is improved, and the liquid feeding mechanism of the apparatus for automatically detecting the reaction using the reaction vessel is simplified.
If the flow path is provided with a valve portion made of an elastically deformable member that can be closed by pressing from above, the liquid feeding operation can be easily controlled.

According to the gene polymorphism detection method using this reaction container, a DNA sample in which a plurality of polymorphic sites are amplified, or a biological sample, and then a plurality of target polymorphic sites are amplified simultaneously, Since the polymorphic sites are simultaneously typed, the polymorphic typing can be performed in a short time with a simple process.
Since the diagnostic method of the present invention reads the diagnostic value from the database based on the obtained polymorphic typing, it can be used in the medical field.

In the detection device of the present invention, a typing reagent, or a gene amplification reaction reagent and a typing reagent, or a diluted reagent is stored in advance, and a test reagent kit in which a probe placement unit is formed integrally is used. Multiple target polymorphic typing can be performed automatically.
In the diagnostic apparatus of the present invention, it is possible to automatically execute from polymorphic typing to display of diagnostic values based thereon.

FIG. 2 shows a first embodiment of the reaction vessel. (A) is a plan view, (B) is a perspective view, (C) is a cross-sectional view, and (D) is a cross-sectional view in a state where a nozzle penetrates.
This reaction vessel is for injecting a sample reaction solution obtained by amplifying DNA by PCR reaction and detecting SNP by invader reaction. A thin layer film 12 is joined to a substrate 10 on which fluorescence measurement can be performed. A reservoir 14 and a chamber 16 connected to the reservoir 14 through a flow path are formed on the substrate 10. The upper part of the liquid reservoir 14, the flow path and the chamber 16 is covered with the thin film 12. In order to measure fluorescence from the bottom surface side, the substrate 10 is formed of a material such as a resin having a low autofluorescence property (a property of generating less fluorescence from itself) and a light transmitting resin, for example, polycarbonate.

  The upper part of the liquid reservoir 14 is covered with a cap 18 made of a material 15 through which the nozzle can penetrate and whose through hole can be restored. The liquid reservoir 14 is a typing reagent storage unit that stores a typing reagent. The cap 18 is detachable. For example, the cap 18 can be detachable as a fitting type or a screw-in type in which only the cap 18 can be removed. In other embodiments, such a configuration can be used to make the cap removable. For example, Si rubber is fixed inside the cap 18. This may be PDMS.

At the time of sample injection, the cap 18 is removed and a sample reaction solution obtained by amplifying DNA by PCR reaction is injected into the typing reagent storage unit 14.
At the time of liquid feeding, the nozzle penetrates a part of the cap 18 and discharges or sucks air, thereby feeding or sucking the reaction liquid sealed in the housing portion into the flow path, and then sending the reaction liquid from the flow path to the above-mentioned Pull into space. When the nozzle is pulled out from the accommodating portion, the through hole is closed by elasticity.

The typing reagent container 14 and the channel are partially closed, and when the pressure inside the typing reagent container 14 exceeds a certain pressure, the blocked part opens and the typing reagent is opened. Flows out toward the flow path. As a method for blocking a part of the flow path, for example, a method using adhesion, a method using thermal welding, or the like can be employed. The pressure for opening the closed channel is, for example, 1.5 to 2.0 kg / cm ² .

  The chamber 16 is a probe arrangement part in which a plurality of probes emitting fluorescence corresponding to each of a plurality of polymorphic sites are individually held in a recess having a large area. In the chamber 16, dots indicated by reference numeral 16a indicate individual probe placement portions. A hole 20 for venting air is formed at the end of the chamber 16 on the opposite side of the typing reagent container 14.

  In this reaction container, the cap 18 of the typing reagent container 14 is removed, and a sample reaction solution in which DNA is amplified by a PCR reaction is injected by a pipette or the like. Thereafter, the cap 18 is put on again, the reaction container is mounted on the detection device, and the sample reaction solution and the typing reagent are mixed.

In the detection device, air is discharged from above the cap 18 of the dilution section through the nozzle, so that air is sent into the typing reagent storage section 14, and the reaction liquid of the sample reaction liquid and the typing reagent in the dilution liquid is chambered. If there is a predetermined SNP by reacting with the probe of the probe placement unit 16a, fluorescence is emitted from the probe. Fluorescence is detected by irradiating excitation light from the back side of the substrate 10.
The dimensions of the chamber 16 are, for example, a width of 2.5 mm, a length of 5 to 25 mm, a depth of 100 μm, and an internal volume of 1.5 to 50 μL.

FIG. 3 shows a second embodiment of the reaction vessel. The typing reagent storage unit 14, the mixing unit 28, and the probe placement unit 30 are arranged in this order in the substrate 22, and are connected to each other through a flow path. Also in this case, the typing reagent container 14 and the flow channel are partially blocked, and when the pressure inside the typing reagent container 14 exceeds a certain pressure, the blocked part is Open and the typing reagent flows out of the flow path. As a method for blocking a part of the flow path, for example, a method using adhesion, a method using thermal welding, or the like can be employed. The pressure for opening the closed channel is, for example, 1.5 to 2.0 kg / cm ² . As shown in FIG. 4 of a later embodiment, the substrate 22 is composed of a resin plate and an elastic film covering the resin plate. The mixing unit 28 is preferably configured to be a meandering flow path or to repeat a wide portion and a narrow portion of the flow path so that the stirring can be efficiently performed. The probe placement portion 30 is not particularly limited, and four rows of probe arrangement portions in which a plurality of concave portions to which one or a plurality of probes are fixed are arranged in series along the flow path are provided. A hole 32 for venting air is formed at the end of the arrangement portion 30. The probe placement unit 30 is controlled to a temperature necessary for the reaction between the DNA and the probe. A sample injection port 26 for injecting a PCR reaction solution is provided in the flow path between the typing reagent storage unit 14 and the mixing unit 28 and can be closed with a stopper except during injection.

In the reaction container of FIG. 3, after the PCR reaction liquid is injected from the sample inlet 26 and the sample inlet 26 is closed, the reaction container is mounted on the detection device. In the mounting device, the nozzle penetrates from above the typing reagent storage unit 14, air is sent through the nozzle, and the typing reagent and the PCR reaction solution are sent to the mixing unit 28 and mixed. Thereafter, the probe is sent to the probe placement unit 30, and the SNP is detected by fluorescence with a predetermined probe.
The operation of mixing the typing reagent and the PCR reaction solution by discharging or sucking air through the nozzle to the typing reagent container 14 may be performed manually outside the apparatus.

FIG. 4 shows a third embodiment of the reaction vessel. (A) is sectional drawing, (B) is a top view, (C) is sectional drawing of a PCR reaction part.
4 includes a resin plate 40 and an elastic film 42 covering the surface thereof. In the resin plate 40, a sample injection part 44, a PCR reaction reagent storage part 46, a typing reagent storage part 48, and a mixing part 50 are formed as four concave parts, and each upper part is covered with an elastic film 42. The probe placement section 30 is the same as that shown in FIG. 3, and a hole 32 for venting air is formed at the other end of the probe placement section 30. In order to measure fluorescence from the bottom surface side, the resin plate 42 is formed of a material such as low autofluorescent and light-transmitting resin, such as polycarbonate.

  A channel 54 is further formed by connecting the channel between the sample injection unit 44 and the mixing unit 50 and the channel between the PCR reaction reagent storage unit 46 and the typing reagent storage unit 48. The flow path 54 is a part that becomes a PCR reaction part.

  The mixing unit 50 and the probe placement unit 30 are also connected by a flow path. The sample injection part 44, the PCR reaction reagent storage part 46, the typing reagent storage part 48, and the mixing part 50 are each covered with a cap made of a material that can restore the through hole. The cap of the sample injection part 44 is detachable, and the cap is opened when injecting the sample.

  In order to improve the thermal conductivity, the PCR reaction part 54 has a thin wall on the lower surface side of the resin plate as shown in FIG. The thickness of the portion is, for example, 0.2 to 0.3 mm.

  The sample injection unit 44 is provided in an empty state in which a sample is not yet injected before use. The PCR reaction reagent storage unit 46 stores 10 to 20 μL of the PCR reaction reagent. The typing reagent container 48 stores about 40 μL of typing reagent. Further, in a state before use, the PCR reaction reagent storage unit 46, the typing reagent storage unit 48, and the PCR reaction unit 54 are restrained by a stopper 58 to prevent the reagent from moving. Since the upper part of the flow path is covered with the elastic film 42, the part of the flow path can be closed by pushing from above.

This reaction solution is prepared as a reagent kit for SNP detection, and a sample of the biological sample that has not been subjected to the nucleic acid extraction operation is removed as a sample by removing the cap of the sample injection unit 44. In use, the stopper 58 is removed, the appropriate part of the flow path is closed, the sample and the PCR reaction reagent are mixed by discharging or sucking air through the nozzle to the sample injection part 44 and the PCR reaction reagent storage part 46, The mixed solution is held in the PCR reaction unit 54. The PCR reaction unit 54 causes the PCR reaction to be performed at a predetermined temperature cycle. Thereafter, the appropriate portion of the flow path is closed, and the reaction solution after the PCR reaction is mixed with the typing reagent by discharging or sucking air to the typing reagent container 48 and the mixing unit 50 through the nozzle, and then mixed. The liquid is held in the mixing unit 50.
Thereafter, the reaction liquid in the mixing unit 50 is sent to the probe placement unit 30, and SNP is detected by detecting fluorescence with the corresponding probe.

Hereinafter, although the composition of each reaction reagent is shown and the present invention is described in detail, the technical scope of the present invention is not limited to these examples.
PCR is performed on 1 μL of human fresh blood using 24 μL of PCR reaction reagent. PCR reaction reagents are known, and for example, reaction reagents including primers, DNA polymerase, and TaqStart (manufactured by CLONTECH Laboratories) as described in paragraph [0046] of Patent Document 3 can be used. In addition, AmpDirect (manufactured by Shimadzu Corporation) may be mixed in the PCR reaction reagent. As the primer, for example, SNP IDs 1 to 20 described in Table 1 of Patent Document 3 and SEQ ID NOs: 1 to 40 can be used.

  An invader reagent is used as a typing reagent. As the invader reagent, an invader assay kit (manufactured by Third Wave Technology) is used. For example, a signal buffer, a fret probe, a structure-specific DNA degrading enzyme, and an allele-specific probe are prepared at concentrations as described in paragraph [0046] of Patent Document 3.

  FIG. 5 shows an embodiment of a simplified genetic polymorphism detection apparatus for detecting SNP in a biological sample using the reaction container of FIG. 4 as a reagent kit. In the apparatus, a pair of heat blocks 60 and 62 are arranged on the upper and lower sides to constitute a test reagent kit mounting portion, and five pieces of samples injected into the reaction container 41 of FIG. Installed side by side. These heat blocks 60 and 62 can move in the Y direction indicated by arrows.

  The lower heat block 60 includes an amplification unit that controls the temperature of the PCR reaction unit 54 to be a predetermined temperature cycle, and a typing reaction unit that controls the temperature of the probe placement unit 30 to a temperature at which the DNA and the probe are reacted. It has. The temperature of the amplifying unit is set so that, for example, it is changed in three stages of 94 ° C., 55 ° C., and 72 ° C. in that order, and the cycle is repeated. The temperature of the typing reaction part is set to 63 ° C., for example.

  A detector 64 for detecting fluorescence is disposed below the heater block 60, and the detector 64 moves in the direction of arrow X in the figure to detect fluorescence from the probe placement unit 30. The heater block 60 is provided with an opening for detecting fluorescence. Fluorescence detection is performed on each probe by the movement of the probe placement unit 30 in the Y direction by the test reagent kit mounting unit and the movement of the detector 64 in the X direction.

  A liquid feeding arm 66 is provided as a liquid feeding device to send air to each part of the reaction vessel or to close a part of the flow path by pushing it. The liquid feeding arm 66 includes a nozzle 67 for sending air and an actuator 68 for pushing the valve in the downward direction. The heat block 62 is embedded with a pinch valve shaft for pushing a predetermined position on the flow path of the reaction vessel 41 disposed below and a seal valve shaft for closing the air vent hole 32. Further, the nozzle 67 can discharge or suck air to the sample injection unit 44, the PCR reaction reagent storage unit 46, the typing reagent storage unit 48, and the mixing unit 50, and the pinch valve shaft presses a predetermined location. In order to be able to do so, the heat block 62 is provided with an opening at a corresponding position. The liquid feeding operation is performed by sending air to the sample injection unit 44, the PCR reaction reagent storage unit 46, the typing reagent storage unit 48, and the mixing unit 50, and pushing the pinch valve shaft.

  FIG. 6 shows the positions of the pinch valve shafts 70 a to 70 e and the seal valve shaft 70 f embedded in the upper heat block 60. As shown in (B), the pinch valve shaft is disposed at five positions 70 a to 70 e on the flow path, and the seal valve shaft is disposed at the position of the air vent hole 32.

An example of the pinch valve shafts 70a to 70e and the seal valve shaft includes a pin 72 at the tip of the knock mechanism shown in FIG. The knock mechanism portion of the pinch valve shafts 70 a to 70 e is equivalent to a Kern type knock mechanism including a cam cylinder 74, a knock bar 76 and a rotor 78. A cam groove 80 is provided in the cam cylinder 74, and the cam groove 80 meshes with the knock bar 76. Reference numeral 82 denotes a valve shaft refill, which is fitted on the distal end side of the rotor 78 and is urged upward by a spring 84. The pin 72 is attached to the tip of the valve shaft refill 82 by being biased in the tip direction by a spring 86.
As shown in FIG. 7B, the seal valve shaft is provided with a seal portion 88 for closing the air vent hole 32 at the tip of the pin 72 of the pinch valve shaft.

  FIG. 8 shows the detector 64 in detail. The detector 64 includes a laser diode (LD) and a light emitting diode (LED) 92 that emit a laser beam of 473 nm as an excitation light source, and a pair of laser beams that irradiate the laser beam on the bottom surface of the probe placement portion of the reaction vessel 41. Lenses 94 and 96 are provided. The lens 94 condenses the laser light from the laser diode 92 into parallel light, and the lens 96 is an objective lens that converges and irradiates the collimated laser light on the bottom surface of the reaction vessel 41. The objective lens 96 also functions as a lens that collects the fluorescence generated from the reaction vessel 41. A dichroic mirror 98 is provided between the pair of lenses 94 and 96, and the dichroic mirror 98 has wavelength characteristics set so as to transmit excitation light and reflect fluorescence. A dichroic mirror 100 is further arranged on the optical path of the reflected light (fluorescence) of the dichroic mirror 98. The dichroic mirror 100 has a wavelength characteristic that reflects 525 nm light and transmits 605 nm light. A lens 102 and a light detector 104 are arranged on the optical path of reflected light by the dichroic mirror 100 so as to detect fluorescence of 525 nm, and a lens so as to detect fluorescence of 605 nm on the optical path of transmitted light by the dichroic mirror 100. 106 and a photodetector 108 are arranged. By the two types of fluorescence detection by the two detectors 104 and 108, the presence or absence of the SNP corresponding to the invader probe fixed at each probe arrangement position, and whether the SNP is a homozygote or a heterozygote. Detected. As the labeling phosphor, for example, FAM, ROX, VIC, TAMRA, etc. can be used.

Next, the operation of this embodiment will be described.
The state shown in FIG. 4 shows an unused reagent kit.
(Sample injection)
First, the cap 44a of the sample injection portion 44 is opened, and a sample is injected by the pipette 45 (see FIG. 9), and then the cap is closed. The stopper 58 is removed and attached to the detection device of FIG.

(Mixing of sample and PCR reaction reagent)
In the detection apparatus, as shown in FIG. 10, the actuator 68 pushes the pinch valve shafts 70b and 70d, and closes the flow path of that portion. When the pinch valve shaft is pushed and the tip is advanced or retracted, the state is maintained until the next depression, even if the pressure by the actuator is released. Then, as shown in FIGS. 10 and 11, by discharging or sucking air to the sample injection part 44 and the PCR reaction reagent storage part 46, the liquid is passed between the two parts 44 and 46 through the PCR reaction part 54. Mix while reciprocating. The mixed solution also exists in the PCR reaction unit 54.

(PCR reaction)
As shown in FIG. 12, the flow path is closed at four places by the pinch valve shafts 70a, 70b, 70c and 70d, and the mixture of the sample and the PCR reaction reagent is confined in the PCR reaction part 54. In this state, a predetermined temperature cycle is repeated to cause the PCR reaction. The PCR temperature cycle includes three steps of denaturation, primer attachment (annealing), and primer extension, and DNA is amplified by repeating the cycle. As an example of each step, the denaturation step is 94 ° C. for 1 minute, the primer attachment step is 55 ° C. for 1 minute, and the primer extension is 72 ° C. for 1 minute. Although the sample is not subjected to the genome extraction operation, DNA is released from blood cells and cells at a high temperature in the PCR temperature cycle, and the reaction proceeds when a reagent necessary for the PCR reaction contacts the DNA.

(Mixing with typing reaction solution)
After completion of the PCR reaction, as shown in FIGS. 13 to 15, the portion of the flow path is closed by the pinch valve shafts 70 a, 70 c and 70 e, and air is discharged to the typing reagent storage unit 48 and the mixing unit 50 through the nozzles. Alternatively, the typing reagent is allowed to pass through the PCR reaction unit 54 by aspiration, and the reaction solution that has completed the PCR reaction and the typing reagent are mixed.

(Liquid feeding to the probe placement part)
As shown in FIG. 16, that portion of the flow path is closed by the pinch valve shaft 70 b, and the reaction liquid in the mixing unit 50 is sent to the probe placement unit 30 by sending air to the mixing unit 50.

(Invader reaction)
As shown in FIG. 17, the portion of the flow path is closed by the pinch valve shaft 70e, the air vent hole 32 is closed by the seal valve shaft 70f, and the invader reaction is performed while the reaction liquid is stored in the probe placement portion 30. Wake up. The invader reaction is performed with the temperature set at, for example, 63 ° C. If there is an SNP corresponding to a predetermined probe, the portion develops color, so that the detector 64 emits excitation light from the lower side of the reaction vessel to detect fluorescence.

FIG. 18 shows another embodiment of the detection device, which corresponds to nozzles 112 and 114 for discharging or sucking air to a predetermined portion of the reaction vessel on the liquid feeding arm 110 and a predetermined position of the flow path. In this example, seven pinch valve shafts 116a, 116b, 116c, 116d, 116e, 116f, and 116g are provided. In the detection device of FIG. 5, the pinch valve shaft that holds the flow path is embedded in the upper heat block 62 that constitutes the test reagent kit mounting portion. In this embodiment, the upper heat block 62 has a hole at a corresponding position. The pinch valve shaft is provided in the liquid feeding arm 110 and is operated by being pressed by another actuator different from the nozzles 112 and 114.
Next, the operation according to this embodiment will be described.

(Sample injection)
First, the cap of the sample injection portion 44 is opened, the sample is injected, and then the cap is closed. The stopper 58 is removed and attached to the detection device (see FIG. 18).

(Mixing of sample and PCR reaction reagent)
In this step, the sample injection unit 44 and the PCR reaction reagent storage unit 46 are electrically connected via the PCR reaction unit 54, and the flow path is pushed so as to close between the typing reagent storage unit 48 and the mixing unit 50. In this state, the sample and the PCR reaction reagent are mixed. For this purpose, as shown in FIG. 19, the liquid feeding arm 110 is positioned and air is sent to the PCR reaction reagent storage unit 46 by the nozzle 112 in a state where the flow paths below them are closed by the pinch valve shafts 116b and 116d. As shown in FIG. 20, the liquid feeding arm 110 is positioned, and the operation of sending air to the sample injection unit 44 by the nozzle 112 is alternately repeated with the pinch valve shafts 116d and 116f closing the flow paths below them. . As a result, the solution is mixed while reciprocating between the two parts 44 and 46 through the PCR reaction part 54. The mixed solution also exists in the PCR reaction unit 54.

(PCR reaction)
As shown in FIG. 21, the blockage of the flow path is released, and a predetermined temperature cycle is repeated in a state where the mixture of the sample and the PCR reaction reagent is present in the PCR reaction unit 54 to perform the PCR reaction.

(Mixing with typing reaction solution)
After completion of the PCR reaction, the liquid feeding arm 110 is positioned as shown in FIG. 22, the flow path below is closed by the pinch valve shafts 116 a, 116 b and 116 c, and air is discharged to the typing reagent storage unit 48 by the nozzle 112. Alternatively, as shown in FIG. 23, the suction arm 110 and the liquid feeding arm 110 are positioned, the flow paths below are closed by the pinch valve shafts 116c, 116d and 116e, and air is discharged to the mixing unit 50 by the nozzle 112 or By repeating the aspirating operation, the reaction solution that has completed the PCR reaction and the typing reagent are mixed.
Thereafter, as shown in FIG. 24, the liquid feeding arm 110 is positioned, the flow paths below are closed by the pinch valve shafts 116a, 116b, and 116c, and air is sent to the typing reagent container 48 via the nozzle 112, The mixed solution is moved to the mixing unit 50.

(Liquid feeding to the probe placement part)
As shown in FIG. 25, the liquid feeding arm 110 is positioned, the flow path below is closed by the pinch valve shaft 116g, and air is discharged or sucked into the mixing unit 50 via the nozzle 114, thereby causing the reaction in the mixing unit 50. The liquid is sent to the probe placement unit 30.

(Invader reaction)
As shown in FIG. 26, the blockage of the flow path is released, and an invader reaction is caused in a state where the reaction liquid is present in the probe placement unit 30. If there is an SNP corresponding to a predetermined probe, the portion develops color, so that the detector 64 emits excitation light from the lower side of the reaction vessel to detect fluorescence. In this example, the air vent hole 32 is not closed. In that case, in order to prevent the invader reaction from being affected by the evaporation of the reaction solution, as an example, the amount of the reaction solution that does not affect the invader reaction even if evaporation is allowed to exist in the hole 32. preferable.

  FIG. 27 shows a fourth embodiment of the reaction vessel. In this reaction container, a reaction solution in which a PCR reaction has been completed externally is used as a sample. This reaction container is provided with a sample injection part 120 and a typing reagent storage part 48 as a liquid storage part, and a probe placement part 30 is provided. The typing reagent storage unit 48 and the probe placement unit 30 are the same as those in the above embodiment.

  The sample injection unit 120 is covered with a detachable cap for injecting a reaction solution that has been subjected to the PCR reaction externally, and the cap is made of a material whose upper part can be restored. The sample injection part 120 and the typing reagent storage part 48 are connected via a flow path 122, and the sample injection part 120 and the probe placement part 30 are connected via a flow path 122. The flow paths 122 and 124 are also covered with an elastic film in the same manner as in the above embodiment, and can be closed by pressing from above.

In this reaction container, the channel 122 is pressed by the stopper 126 before use, and the space between the sample injection unit 120 and the typing reagent storage unit 48 is closed.
The operation of this reaction vessel will be described. Any of the above-described embodiments can be used as the liquid feeding arm, but here, the embodiment of FIG. 5 is used.

(Sample injection after completion of PCR reaction)
As shown in FIG. 27, the flow path 124 is closed, the cap of the sample injection unit 120 is opened, and the reaction solution that has finished the PCR reaction is injected by the pipette 45. The injection volume is about 1 μL. Thereafter, the cap of the sample injection unit 120 is closed.

(Mixing with typing reaction solution)
Next, with the stopper 126 removed and the flow path 124 closed with a pinch valve shaft, air is sent to the typing reagent container 48 through the nozzle 67 as shown in FIG. 28, and the sample is injected as shown in FIG. By repeating the operation of sending air to the unit 120 through the nozzle 67, the reaction solution having completed the PCR reaction and the typing reagent are mixed.
After that, as shown in FIG. 30, air is sent to the typing reagent container 48 through the nozzle to move the mixed solution to the sample injection unit 120.

(Liquid feeding to the probe placement part)
As shown in FIG. 31, the flow path 122 is closed with a pinch valve shaft, and the sample injection unit 120 is sent air with the nozzle 67 to send the reaction solution in the sample injection unit 120 to the probe placement unit 30.
(Invader reaction)

  As shown in FIG. 32, the portion of the flow path is closed by the pinch valve shaft 70e, the air vent hole 32 is closed by the seal valve shaft 70f, and the invader reaction is performed with the reaction liquid present in the probe placement portion 30. Wake up. If there is an SNP corresponding to a predetermined probe, the portion develops color, so that fluorescence is detected by irradiating excitation light from the lower side of the reaction vessel with a detector.

  The present invention can be used for various automatic analyzes in genetic analysis research and clinical fields. For example, it detects polymorphisms in genomic DNA of animals and plants, especially humans, especially SNPs (single nucleotide polymorphisms). In addition to diagnosing disease morbidity using the results and diagnosing the relationship between the type of drug administered and its effects and side effects, animal and plant breed determination, infectious disease diagnosis (type of infecting bacteria) It can also be used to make a determination.

It is a flowchart figure which shows the detection method of this invention roughly. It is a figure which shows the 1st Example of reaction container, (A) is a top view, (B) is a perspective view, (C) is sectional drawing, (D) is sectional drawing in the state which the nozzle has penetrated It is. It is a top view which shows the 2nd Example of reaction container. It is a figure which shows the 3rd Example of reaction container, (A) is sectional drawing, (B) is a top view, (C) is sectional drawing of the PCR reaction part. It is a schematic perspective view which shows one Example of the simple gene polymorphism detection apparatus for detecting SNP of a biological sample using the reaction container of FIG. 4 as a reagent kit. (A) is a perspective view showing the position of the pinch valve shaft and the like of the detection device, and (B) is a plan view showing the positional relationship between the pinch valve shaft and the reaction vessel. (A) is sectional drawing which shows a pinch valve axis | shaft, (B) is sectional drawing which shows the front-end | tip part of a seal valve axis | shaft. It is a schematic block diagram which shows the detector in the same detection apparatus. It is a top view which shows the sample injection | pouring operation | movement in one Example. It is a top view which shows the mixing operation | movement of the sample and PCR reaction reagent in the Example. It is a top view which shows the mixing operation | movement of the sample and PCR reaction reagent in the Example. It is a top view which shows PCR reaction operation | movement in the Example. It is a top view which shows the mixing operation | movement of the sample liquid and typing reaction liquid after PCR reaction in the Example. It is a top view which shows the mixing operation | movement of the sample liquid and typing reaction liquid after PCR reaction in the Example. It is a top view which shows the mixing operation | movement of the sample liquid and typing reaction liquid after PCR reaction in the Example. It is a top view which shows the liquid feeding operation | movement to the probe arrangement | positioning part in the Example. It is a top view which shows the invader reaction operation | movement in the Example. It is a top view which shows the sample injection | pouring operation | movement in another Example. It is a top view which shows the mixing operation | movement of the sample and PCR reaction reagent in the Example. It is a top view which shows the mixing operation | movement of the sample and PCR reaction reagent in the Example. It is a top view which shows PCR reaction operation | movement in the Example. It is a top view which shows the mixing operation | movement of the sample liquid and typing reaction liquid after PCR reaction in the Example. It is a top view which shows the mixing operation | movement of the sample liquid and typing reaction liquid after PCR reaction in the Example. It is a top view which shows the mixing operation | movement of the sample liquid and typing reaction liquid after PCR reaction in the Example. It is a top view which shows the liquid feeding operation | movement to the probe arrangement | positioning part in the Example. It is a top view which shows the invader reaction operation | movement in the Example. It is a top view which shows the sample injection | pouring operation | movement after PCR reaction in other Example. It is a top view which shows the mixing operation | movement of the sample liquid and typing reaction liquid after PCR reaction in the Example. It is a top view which shows the mixing operation | movement of the sample liquid and typing reaction liquid after PCR reaction in the Example. It is a top view which shows the mixing operation | movement of the sample liquid and typing reaction liquid after PCR reaction in the Example. It is a top view which shows the liquid feeding operation | movement to the probe arrangement | positioning part in the Example. It is a top view which shows the invader reaction operation | movement in the Example.

Explanation of symbols

2 Sample 4 PCR reaction liquid 6 Invader reagent 8 Probe placement part 14 Diluent storage part 16 Chamber 16a, 30 Probe placement part 18 Cap 26 Sample inlet 28 Mixing part 41 Reaction vessel 44, 120 Sample injection part 46 PCR reaction reagent storage part 48 Typing reagent storage unit 50 Mixing unit 54 PCR reaction unit 60, 62 Heat block 64 Detector 66, 110 Liquid feeding arm 67, 112, 114 Nozzle 68 Actuator 70a-70e, 116a-116g Pinch valve shaft 70f Seal valve shaft

Claims (30)

A reaction vessel formed in a flat substrate and transferring a reaction solution of a sample or a sample and a reagent along a flow path,
A liquid reservoir formed in the substrate, connected to the flow path, and covered with a cap on the upper side;
A reaction vessel in which a material through which a nozzle can penetrate and whose through-hole can be restored is used for the cap, and liquid is transferred by discharging or sucking air into the cap via the nozzle.   The reaction container according to claim 1, wherein the cap is an elastically deformable member.   The reaction container according to claim 2, wherein the elastically deformable member is a flexible material including PDMS or Si rubber.   The reaction container according to any one of claims 1 to 3, wherein a plurality of the liquid reservoirs are provided, and the nozzles are alternately passed through the respective caps and mixed by transferring the liquid by discharging or sucking air. The flow path includes a valve portion made of an elastically deformable member that can be closed by pressing from above, and includes a first reaction section connected via the flow path,
The reaction container according to any one of claims 1 to 4, wherein a predetermined flow path portion is closed by pressing the valve portion, and a liquid is transferred from the liquid reservoir portion by discharging or sucking air with a nozzle. A sample storage part in which a sample is injected or stored as the liquid reservoir, and a reagent storage part that stores a reagent,
The reaction vessel according to claim 5, wherein the flow path connects between the sample storage unit and the first reaction unit and between the reagent storage unit and the first reaction unit, and has a valve unit between the units.   The reaction container according to claim 6, wherein the cap of the sample storage portion is attached to be openable and closable for sample injection. The liquid reservoir further comprises a diluent storage part containing a diluent and a dilution part consisting of an empty space,
The reaction vessel further includes a second reaction unit through which the reaction solution diluted in the dilution unit is guided,
The flow path is connected to the diluent storage part and the dilution part, and is also connected between the dilution part and the second reaction part, and has a valve part between these parts. The reaction vessel as described. The reagent accommodated in the reagent accommodating portion is a gene amplification reagent,
The reaction container according to any one of claims 6 to 8, wherein the first reaction part is an amplification reaction part that mixes a gene amplification reagent and a sample and performs an amplification reaction, and the reaction container constitutes a reagent kit. . The sample injected or stored in the sample storage unit is a biological sample,
The reagent housed in the reagent container is a gene amplification reagent comprising a plurality of primers that bind across each of a plurality of polymorphic sites,
The first reaction unit is an amplification reaction unit that mixes a gene amplification reagent and a sample and performs an amplification reaction,
A typing reagent which is formed in the substrate and contains a typing reagent prepared corresponding to the plurality of polymorphic sites, and whose upper part is covered with a cap made of a material through which the nozzle can penetrate and whose through hole can be restored. A containment section;
A mixing part formed of an empty space formed in the substrate, the upper part of which is covered with a cap made of a material through which the nozzle can penetrate and whose through hole can be restored;
A probe placement section as a second reaction section, in which a reaction solution mixed in the mixing section is guided, and a plurality of probes emitting fluorescence corresponding to each of the plurality of polymorphic sites are individually held; In addition,
The flow path is connected to the typing reagent storage unit and the mixing unit, and is also connected between the mixing unit and the probe placement unit, and can be closed by pressing from above. The reaction container according to claim 6 or 7, wherein the reaction container has a valve part made of an elastically deformable member, and the reaction container constitutes a reagent kit for detecting a gene polymorphism.   The reaction container according to claim 9 or 10, wherein a substrate thickness of the amplification reaction part is thinner than other parts. The liquid reservoir is a typing reagent storage that stores typing reagents prepared corresponding to a plurality of polymorphic sites, and a sample that has been subjected to a gene amplification reaction is injected into the liquid reservoir. And
The first reaction part is a probe arrangement in which a reaction liquid in which a typing reagent and a sample are mixed is guided in the liquid reservoir, and a plurality of probes emitting fluorescence corresponding to each of the plurality of polymorphic sites are individually held. The reaction container according to claim 5, wherein the reaction container constitutes a reagent kit for detecting a genetic polymorphism. A sample storage part into which a sample that has completed the gene amplification reaction is injected as the liquid reservoir, and a typing reagent storage part that stores a typing reagent prepared corresponding to a plurality of polymorphic sites. Are connected by a flow channel, and are connected by a flow channel between any of the storage units and the first reaction unit,
The first reaction part is a probe arrangement in which a reaction liquid in which a typing reagent and a sample are mixed is guided in the liquid reservoir, and a plurality of probes emitting fluorescence corresponding to each of the plurality of polymorphic sites are individually held. Department,
The flow path includes a valve portion made of an elastically deformable member that can be closed by pressing from above, and a predetermined flow path portion is closed by pressing the valve portion, and the liquid reservoir portion is connected via a nozzle. The liquid can be transferred by discharging or sucking air,
The reaction container according to claim 5, wherein the reaction container constitutes a reagent kit for detecting a gene polymorphism.   The reaction container according to any one of claims 10 to 13, wherein the target polymorph is a single nucleotide polymorph.   The reaction container according to claim 9, wherein the sample is a biological sample that has not been subjected to a nucleic acid extraction operation.   The reaction container according to any one of claims 9, 10, 11 and 15, wherein the gene amplification reagent is a PCR reaction reagent.   The reaction container according to any one of claims 10 to 16, wherein the typing reagent is an invader reagent or a Tuckman PCR reagent.   Using the reaction vessel according to any one of claims 1 to 9, the sample or the reaction liquid of the sample and the reagent is transferred to the first reaction unit along the flow path by sending air to the liquid reservoir, and reacted. And a reaction detection method for detecting the reaction result. A genetic polymorphism detection method using the reaction container according to claim 10 or 11 and comprising the following steps to detect a genetic polymorphism.
(A) A step of injecting a sample into the sample container,
(B) A predetermined flow path portion is closed by pressing at least one valve portion of the flow path, and a sample is discharged and sucked alternately through a nozzle to the sample storage section and the reagent storage section. Creating a gene amplification reagent mixture and supplying it to the amplification reaction section;
(C) Gene amplification reaction of the mixed solution in the amplification reaction section,
(D) The amplification is achieved by closing a predetermined flow path portion by pressing at least one valve portion of the flow path, and alternately discharging or sucking air to the typing reagent storage section and the mixing section via a nozzle. A step of mixing a gene amplification reaction completion solution and a typing reagent in a reaction unit; and (E) a step of detecting the fluorescence by sending the reaction solution mixed in the step (D) to the probe placement unit. A genetic polymorphism detection method comprising detecting the genetic polymorphism using the reaction container according to claim 12 and comprising the following steps.
(A) A step of injecting a sample into the liquid reservoir,
(B) A step of detecting the fluorescence by sending the reaction solution in which the typing reagent and the sample are mixed in the step (A) to the probe placement unit. A genetic polymorphism detection method using the reaction container according to claim 13 and comprising the following steps to detect a genetic polymorphism.
(A) A step of injecting a sample into the sample container,
(B) A sample is formed by closing at least one valve part of the flow path to block a predetermined flow path, and alternately discharging or sucking air to the sample storage part and the typing reagent storage part via a nozzle. Mixing the typing reagent with
(C) A step of closing a predetermined flow path portion by pressing at least one valve portion of the flow path, and sending the reaction liquid mixed in the step (B) to the probe placement section to detect fluorescence.   The genetic polymorphism detection method according to claim 19, wherein the sample is a biological sample that has not been subjected to a nucleic acid extraction operation. Prepare a diagnostic value for a specific polymorphism or a combination of multiple polymorphisms as a database,
The diagnostic method which reads a diagnostic value from the said database based on the result of the polymorphism detected by the method in any one of Claim 19 to 22. A test reagent kit mounting part for mounting the reaction container according to any one of claims 1 to 17 and controlling each part to a predetermined temperature;
A liquid feeding device for transferring the liquid;
A fluorescence detection device for detecting fluorescence by irradiating excitation light to the reaction liquid portion after the reaction in the reaction vessel;
A control unit for controlling the temperature control of the test reagent kit mounting unit, the liquid feeding operation of the liquid feeding device, and the detection operation of the fluorescence detection device;
A detection device comprising: The liquid delivery device includes a nozzle for discharging or sucking air;
It has a pinch valve shaft that pushes the valve part,
25. The detection device according to claim 24, wherein a pressing pin is provided at a tip of a knock mechanism that is alternately fixed at a forward position and a backward position every time the pinch valve shaft is pushed downward. The test reagent kit mounting section is mounted with the reaction container according to claim 10 or 11, and an amplification section that controls the temperature of the amplification reaction section to a temperature for gene amplification, and the temperature of the probe placement section is DNA. And a typing reaction part that controls the temperature at which the probe reacts,
The liquid feeding device is configured such that air is discharged or sucked through a nozzle to the cap of the sample storage unit, reagent storage unit, typing reagent storage unit, and mixing unit, and the pinch valve shaft presses the valve unit. 26. The detection apparatus according to claim 24 or 25, wherein a genetic polymorphism is detected by transferring a liquid between and transferring the liquid from the mixing section to the probe placement section.   27. The detection apparatus according to claim 26, wherein the sample is a biological sample that has not been subjected to a nucleic acid extraction operation. The test reagent kit mounting section includes the typing reaction section that mounts the reaction container according to claim 12 and controls the temperature of the probe placement section to a temperature at which DNA and the probe are reacted.
The detection device according to claim 24 or 25, wherein the nozzle detects a gene polymorphism by transferring liquid to the probe placement unit by discharging or sucking air to a cap of the typing reagent storage unit. The test reagent kit mounting section includes the typing reaction section that mounts the reaction container according to claim 13 and controls the temperature of the probe placement section to a temperature at which DNA and the probe are reacted.
The nozzle discharges or sucks air into the cap of the sample storage unit and the typing reagent storage unit,
And the pinch valve shaft of the liquid delivery device presses the valve part,
The detection apparatus according to claim 24 or 25, wherein the genetic polymorphism is detected by transferring the liquid between the parts and transferring the liquid to the probe placement part. A detection device according to any of claims 26 to 29;
A database storing diagnostic values for a particular polymorphism or combination of polymorphisms;
A diagnostic device comprising: a display device that reads and displays a diagnostic value from the database based on a polymorphism result detected by the genetic polymorphism detection device.

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