JP2007178328A - Reaction container kit and reaction container processing apparatus - Google Patents

Abstract

A reaction container kit suitable for automating various measurements is provided.
A reaction container kit is attached to a reaction container 10 having a reagent container 14 and a non-volatile liquid container 16 formed as recesses on the same side of a flat plate-like substrate 9 and the tip of a dispensing probe. Dispensing tips 70a and 70b for sucking and discharging the liquid are provided. Dispensing tips 70a and 70b are detachably held by a holding member 42 attached to the end of the substrate 9 constituting the reaction vessel 10 via a movable member.
[Selection] Figure 2

Description

  The present invention is a reaction vessel kit suitable for conducting various automatic analyzes such as chemical reactions and on-site, for example, genetic analysis research and clinical use, and polymorphisms of genomic DNA of animals and plants including humans, For example, the present invention relates to a reaction vessel processing apparatus for detecting SNP (single nucleotide polymorphism) 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, 1470 (according to positions in EMBL accession number D64016, respectively), 454 (according to SEQ ID No. 3) and 696 (according to SEQ ID No. 5), and the multiple in the flt-1 gene The human constitution is determined by referring to the formality (see Patent Document 2).

Many methods have been reported for so-called typing for discriminating 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 nucleic acid, a plurality of base sequences including at least one single nucleotide polymorphism site can be simultaneously obtained using a nucleic acid and a plurality of pairs of primers. Using a plurality of amplified base sequences, the bases of single nucleotide polymorphic sites contained in the base sequences are discriminated by a typing process. 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 2001-709366 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

  Attach a reaction vessel for analysis such as chemical reaction and genetic analysis to the reaction processing device, and dispense the reagent with the dispensing probe when dispensing the reagent to the sample in the reaction vessel. However, a disposable dispensing tip is attached to the tip of the dispensing probe to suck and discharge the reagent. At this time, if the reaction container is disposable, the dispensing tip attached to the dispensing probe is generally replaced with the reaction container as being disposable. At that time, at least the number of reaction vessels to be analyzed must be set for the dispensing tips. If the number of dispensing tips is small or if you forget to set them, the dispensing tip, which should be disposable, will be used repeatedly in multiple samples, and the analysis accuracy will decrease. In particular, when it is included in the PCR process, the contamination is amplified and the influence on the test result is increased.

Therefore, it is desirable that as many dispensing tips as the number of reaction vessels be brought into the reaction vessel processing apparatus.
The first object of the present invention is to provide a reaction vessel kit suitable for automating measurement of chemical reaction and detection of gene polymorphism.

  The second object of the present invention is to provide an apparatus for automating chemical reaction measurement and gene polymorphism detection using the reaction container kit of the present invention.

  The reaction container kit according to the present invention includes a reaction container in which at least one reaction part for causing a sample to react on one surface of a flat substrate is formed, and the reaction container mounted on a dispensing probe of a dispensing mechanism. A dispensing tip for dispensing a sample and / or a reagent into the reaction section, a holding member for detachably holding the dispensing tip, and the dispensing tip held by the holding member are provided in the reaction container. A reaction container arranged along the flat surface of the substrate and a packaging member for packaging a set of dispensing tips are provided. The term “sample and / or reagent” is used to include both “sample and reagent” and “sample or reagent”.

In the state where the reaction container and the dispensing tip are packaged, the holding member holding the dispensing tip may be integrated with the reaction container or may be a separate body.
When the holding member and the reaction vessel are integrated, the holding member is a movable member so that the held dispensing tip can be displaced between the state along the flat surface of the substrate of the reaction vessel and the rising state. It is preferable that it is attached to the reaction vessel via.

  When the holding member is attached to the reaction vessel, the attachment position is not particularly limited, but one form is that the holding member is attached to the tip of the reaction vessel. In that case, the holding member is bent so that the dispensing tip comes to the reaction container side in the packaged state.

  The other form of the attachment position where the holding member is attached to the reaction vessel is one in which the holding member is attached to an intermediate portion of the reaction vessel. In that case, in the packaged state, the dispensing tip is bent so as to come to the side where the bulge is small on the flat plate surface of the substrate on the side to be bent.

It is preferable that the used dispensing tip is again held by the holding member and discarded together with the reaction container. At this time, at least the tip of the dispensing tip when the holding member holds the dispensing tip so that the sample or reagent attached to the tip of the dispensing tip does not adhere to other objects or scatter. It is preferable that a storage part that covers the liquid is provided, and an absorption material that absorbs the liquid is provided inside the storage part. Examples of such an absorbent include filter paper.
To facilitate the work of taking out the reaction container from the packaging member and extending the folded holding member so that the dispensing tip rises with respect to the flat surface of the reaction container substrate, It is preferable that a grip portion is formed.

The reaction container in the reaction container kit of the present invention may further include a nonvolatile liquid container that is formed as a recess in the same substrate, contains a nonvolatile liquid having a specific gravity lower than that of the reaction liquid, and is sealed with a film. Good.
Further, the reaction container is formed as a recess in the same substrate, and further comprises at least one reagent container that contains a reagent used for the sample reaction and is sealed with a film to constitute a sample reaction reagent kit. May be.

  The reagent or non-volatile liquid sealed with a film can be obtained by inserting a dispensing tip attached to the tip of a dispensing probe through the film in the reaction container processing apparatus of the present invention, or the film. By inserting a dispensing tip after peeling off, it can be sucked into the dispensing tip and transferred to another location such as a reaction part.

  This reaction vessel is used for measuring various reactions including chemical reactions and biochemical reactions. One use of the reaction container as a reaction reagent kit is to detect genetic polymorphism. A first form in the case of a reaction vessel for detecting a gene polymorphism is a reaction vessel for detecting a gene polymorphism by injecting a biological sample that has undergone a gene amplification reaction into this reaction vessel. The reaction container according to the first aspect includes a typing reagent storage unit that stores typing reagents prepared corresponding to a plurality of polymorphic sites as a reagent storage unit, and each of the plurality of polymorphic sites as a reaction unit. Correspondingly, a genetic polymorphism diagnostic reagent kit is configured including a plurality of probe arrangement parts individually holding fluorescent probes.

In the second embodiment when a reaction container for detecting a gene polymorphism is used, a plurality of primers that bind to the reaction reagent kit of the first embodiment with a plurality of polymorphic sites sandwiched as reagent storage units are provided. The apparatus further includes a gene amplification reagent container containing the gene amplification reagent, and further includes, as a reaction part, an amplification reaction part that performs a gene amplification reaction on the mixture of the gene amplification reagent and the sample.
In such a case, if two dispensing tips are provided for one reaction vessel as a combination of the reaction vessel and the dispensing tip included in the reaction vessel kit, one of them handles the amplification reaction solution before the gene amplification reaction. The other can be used to handle the amplification reaction completion solution after completion of the gene amplification reaction.
A plurality of sets of reaction containers and dispensing tips may be accommodated and packaged in the packaging member.

The target of polymorphism detection according to the present invention is not particularly limited, and examples include cDNA, genes of various organisms including humans, genes of various microorganisms including viruses and bacteria, and the like. Therefore, a nucleic acid having a nucleic acid sequence can be used as a template nucleic acid for nucleic acid amplification. Moreover, it does not ask | require whether it is a gene of DNA or a gene of RNA as a gene used as the object of these polymorphism detection.
A sample containing these genes is not particularly limited. That is, polymorphism detection can be performed on biological samples, biological samples, extracted nucleic acid samples, and the like. Here, a biological sample or a biological sample is a sample in a form in which a nucleic acid inclusion body (for example, a membrane structure containing nucleic acid inside, such as cells, fungi, bacteria, and viruses) is maintained without performing a nucleic acid extraction operation. is there. Examples of biological samples include organs, tissues, body fluids, excreta and the like. Body fluids include blood samples, spinal fluid, saliva, milk and the like. Blood samples include whole blood, plasma, serum and the like. Excrements include urine and feces. A biological sample is a nucleic acid inclusion body recovered from a biological sample. The method for recovering the nucleic acid inclusions is not particularly limited, and examples thereof include centrifugation and ultracentrifugation, methods using a coprecipitation agent such as polyethylene glycol, and an adsorption carrier. The extracted nucleic acid sample is a sample subjected to nucleic acid extraction operation, and may be a sample obtained by further purifying nucleic acid after extraction. Examples of the extraction method include a method using an enzyme, a surfactant, a chaotropic agent, and the like. Examples of the purification method include a method using phenol / chloroform, an ion exchange resin, a glass filter, glass beads, magnetic beads, a reagent having a protein aggregation action, and the like.
The polymorphisms that can be detected in the present invention are not particularly limited, and include both SNPs and polymorphisms that span multiple nucleotide sequences. In the present invention, the polymorphism further includes a mutation. Specifically, examples of the polymorphism that can be detected in the present invention include SNP, insertion polymorphism, deletion polymorphism, and repeat sequence polymorphism.
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.
An example of a 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 nucleic acids, 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, the gene inclusion in the sample containing the gene, that is, the biological sample, or the sample containing the gene itself, that is, the biological sample is added to the gene amplification reaction solution. Then, the target gene in the sample containing the gene is amplified when the pH of the reaction solution after addition is 8.5 to 9.5 (25 ° C.) (see Patent Document 4).

  The typing system that has already been constructed uses a PCR method to amplify a plurality of SNP regions to be typed by the PCR method. However, the amount of nucleic acid to be collected at first can be reduced. 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.
The invader method or the Tuckman PCR method can be used for the typing process. In that case, the typing reagent is an invader reagent or a Tuckman PCR reagent.

FIG. 15 schematically shows a detection method when a genetic polymorphism is detected using the reaction container of the present invention as a genetic polymorphism diagnostic reagent kit. 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.

The PCR reaction reagent 4 is prepared in advance and includes a plurality of primers for the SNP site to be measured, pH buffer for adjusting the pH, four types of deoxyribonucleotides, and thermostable synthesis. Necessary reagents such as enzymes and salts such as MgCl ₂ and KCl are added. In addition, substances such as surfactants and proteins can be added as necessary. The PCR method of the amplification step that may be used in the present invention is to simultaneously amplify a plurality of target SNP sites. The biological sample may be subjected to a nucleic acid extraction operation or may not be subjected to a nucleic acid extraction operation. Here, the biological sample that has not been subjected to the nucleic acid extraction operation is the biological sample, the biological sample, and the biological sample or biological body in which the membrane structure of the nucleic acid inclusion body is destroyed by an operation such as heat treatment or freezing treatment. Includes derived samples. When a plurality of genomic DNAs containing those SNP sites are directly amplified by PCR from a biological sample that has not been subjected to nucleic acid extraction operation, a gene amplification reaction reagent containing a plurality of primers for those SNP sites is used. The PCR reaction is allowed to occur under conditions such that when mixed with sample 2, the pH at 25 ° C. is 8.5-9.5.

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.

  In the PCR step, a PCR reaction is performed on the mixture of the biological sample 2 and the PCR reaction reagent 4 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 nucleic acid extraction operation, but here, a biological sample that has not been subjected to a nucleic acid extraction operation is used. Even in the case of a biological sample that has not been subjected to nucleic acid extraction operation, the nucleic acid 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 nucleic acid.

  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 is added is added to the plurality of probe placement units 8 to cause a reaction. In each probe placement section 8, an invader probe and a reporter probe are individually held corresponding to each of the plurality of SNP sites, and the reaction solution reacts with the invader probe, and the SNP corresponding to the reporter probe exists. Emits fluorescence.

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

  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. The DNA containing the SNP to be typed and the SNP to be typed Using two types of reporter probes and one type of invader probe specific to each of the alleles and an enzyme having a special endonuclease activity that recognizes and cleaves the DNA structure (see Patent Document 3). .)

  It is preferable that at least the non-volatile liquid dispensed in the reaction part is a recess capable of holding the non-volatile liquid.

A sample injecting portion for injecting a sample may be further provided as a recess in the same substrate.
At least the reaction part is preferably covered with a peelable sealing material before use.
The typing reagent is an invader reagent or a Taqman PCR reagent.

  In order to achieve the second object, the reaction container processing apparatus of the present invention comprises a reaction container mounting part for mounting the reaction container of the reaction container kit of the present invention, and a dispensing tip of the reaction container kit of the present invention at the tip part. It is equipped with a dispenser that transports and dispenses liquid with a mechanism for performing suction and discharge by the attached dispensing probe, and a controller that controls the dispensing operation of the dispenser. is there. As shown in FIG. 1, an example of a preferable form of such a reaction vessel processing apparatus is a dispensing unit 112 including two dispensing probes 28 a and 28 b each having a dispensing tip 70 a and 70 b attached to the tip. The control part 118 which controls the dispensing operation | movement of the dispensing part 112 is provided. In order to prevent contamination between the liquid before the reaction treatment and the liquid after the completion of the reaction treatment when the reaction vessel treatment apparatus shown here performs a reaction treatment such as gene amplification reaction in the reaction vessel, for example, A dispensing probe 28a for handling the liquid before the reaction process and a dispensing probe 28b for handling the liquid after the completion of the reaction process are provided. In such a reaction vessel processing apparatus, a reaction vessel kit including two dispensing tips 70a and 70b is used. However, the reaction vessel processing apparatus of the present invention may be provided with only one dispensing probe. In this case, a reaction container kit provided with only one dispensing tip may be used.

In a preferred form of the reaction vessel processing apparatus, the reaction vessel of the reaction vessel kit of the present invention, a reaction vessel mounting portion for mounting the substrate in a state where the flat surface of the substrate is in the horizontal direction and the dispensing tip is in the vertical direction, A dispensing unit that has a mechanism for suction and discharge by a dispensing probe and that transfers and dispenses liquid, and a control unit that at least controls the dispensing operation of the dispensing unit, and further, the control unit reacts With the proximal end side of the dispensing tip held by the container holding member facing up, the dispensing probe is lowered toward the dispensing tip, inserted into the dispensing tip, and lifted to raise the dispensing tip. The operation of detaching the tip from the reaction vessel and attaching it to the tip of the dispensing probe is also controlled.
The reaction vessel processing apparatus further includes reaction temperature control units 110 and 120 for controlling the temperature of the reaction unit, and the control unit 116 can also control the temperature of the reaction temperature control unit.

When this reaction container processing apparatus is used as a genetic polymorphism detection apparatus, the first embodiment further includes a typing reagent storage section that stores a typing reagent as a reaction container, and a plurality of polymorphic sites as reaction sections. A genetic polymorphism diagnosis reaction vessel provided with a plurality of probe placement sections each holding a fluorescent probe corresponding to each is used. As shown in FIG. 1, the reaction container is provided with a typing reaction temperature control unit 110 that controls the temperature of the probe placement unit as a reaction temperature control unit to a temperature at which the reaction liquid of the sample and the typing reagent reacts with the probe. The processing apparatus further includes a fluorescence detection unit 64 that detects fluorescence by irradiating each probe placement unit with excitation light. The control unit 118 also controls the temperature control of the typing reaction temperature control unit 110 and the detection operation of the fluorescence detection unit 64.
When the invader reaction is used as the typing reaction, the typing reaction temperature control unit 110 serves as a temperature adjusting unit for the invader reaction.

  A second mode in which this reaction vessel processing apparatus is used as a gene polymorphism detection apparatus is a gene container containing a gene amplification reagent containing a plurality of primers that bind to each of a plurality of polymorphic sites as a reaction container. And a reaction vessel for diagnosing a gene polymorphism that further includes an amplification reaction unit that performs a gene amplification reaction on a mixed solution of a gene amplification reagent and a sample as a reaction unit. As shown in FIG. 1, an amplification reaction temperature control unit that controls the temperature of the amplification reaction unit as a reaction temperature control unit to a temperature for gene amplification that amplifies DNA in the reaction solution of the sample and the gene amplification reagent. The control unit 118 also controls the temperature of the amplification reaction temperature control unit 120.

When a PCR reaction is used as the gene amplification reaction, the amplification reaction temperature control unit 120 is a temperature control unit for a temperature cycle for the PCR reaction.
A personal computer (PC) 122 may be connected to the control unit 118 in order to operate the control unit 118 from the outside or display the inspection result.

  An example of a dispensing probe is one in which a disposable tip is detachably attached to the tip. When the liquid container of the reaction vessel is sealed with a film and is attached to the reaction vessel processing apparatus in a state of being sealed with the film, the liquid is sucked through the film of the reaction vessel with the chip. Will be performed.

Since the reaction container kit of the present invention includes a reaction container and a dispensing tip, as many dispensing tips as the number of reaction vessels are always taken into the reaction container processing apparatus, and contamination due to a shortage of dispensing tips. The fear of disappears.
By the way, since the dispensing tip generally has a capacity larger than the inhalation amount of the reagent or the like to be dispensed, its length becomes long. Therefore, if the long dispensing tip is held by the holding member so that the base end side of the dispensing tip is up when the surface of the reaction vessel where the reaction portion is formed is up, The container kit becomes large, and a large area is required to store the reaction container kit, which causes a problem that the transportation cost and storage location of the reaction container kit, which is a consumable item, increase.

  Therefore, in the reaction container kit of the present invention, the reaction container and the dispensing chip are packaged together by the packaging member in a state where the dispensing chip held by the holding member is disposed along the flat plate surface of the substrate of the reaction container. Therefore, the size of the reaction container kit itself can be made compact.

  In the reaction container kit of the present invention, the holding member is attached to the reaction container via the movable member so that the held dispensing tip can be displaced between the state along the flat surface of the substrate of the reaction container and the standing state. By doing so, it is easy to take out the reaction container from the packaging member and attach it to the processing apparatus so that the dispensing tip stands up with respect to the flat surface of the substrate of the reaction container. This operation can be performed manually or automatically. In this reaction vessel kit, the dispensing probe is lowered from above the dispensing tip so that the dispensing tip rises perpendicularly to the flat surface of the reaction vessel substrate in the reaction vessel treatment apparatus when in use. By inserting into the dispensing tip, the dispensing tip can be attached to the tip of the dispensing probe.

  If the holding member is attached to the reaction container, the holding member is attached to the tip of the reaction container, and in the packaged state, the dispensing tip is folded so that it comes to the reaction container side. The size of the reaction vessel kit in the state of being made can be made compact.

  Further, even when the holding member is attached to the middle portion of the reaction vessel, it is folded so that the dispensing tip comes to the side where the bulge is small on the flat plate surface of the substrate on the side where the dispensing tip is folded. If it makes it, the thickness of the reaction container kit in the packaged state can be made small.

When the holding member of the reaction container holds the dispensing tip, it is provided with a storage portion that covers at least the tip of the dispensing tip, and an absorbent that absorbs the liquid is provided inside thereof. It is possible to prevent the liquid remaining on the dispensing tip from being scattered or adhering to other members, and contamination can be avoided.
If the grip portion is formed on the tip side of the substrate or the holding member, manual operation for raising the dispensing tip is facilitated.

If a non-volatile liquid having a specific gravity lower than that of the reaction liquid is accommodated on the same substrate provided with the reaction part, the reaction liquid is covered with the non-volatile liquid in the reaction part, so that the reaction liquid is reacted with the reaction part. It is possible to avoid a situation in which the reaction solution evaporates even when heated at.
Furthermore, the one provided with a reagent container becomes a reagent kit for sample reaction, and the trouble of separately arranging the reagents is eliminated.

  The first form of using this reaction container as a genetic polymorphism diagnostic reagent kit is integrally provided with a typing reagent storage unit, a non-volatile liquid storage unit, and a probe placement unit, so that a plurality of polymorphic sites are amplified. These polymorphic sites can be simultaneously typed for the DNA sample thus obtained, and polymorphic typing can be performed in a short time by a simple process.

  In addition, the second mode in which this reaction container is used as a genetic polymorphism diagnostic reagent kit further includes a gene amplification reagent storage unit and an amplification reaction unit, so that a plurality of target multiple samples can be obtained from a biological sample. After simultaneously amplifying the mold sites, the polymorphic sites can be typed at the same time, and polymorphic typing can be performed in a short time with a simple process.

If the reaction part is a recess and can hold the non-volatile liquid, the evaporation of the reaction liquid in the reaction part can be more effectively prevented.
If the reaction part is covered with a peelable sealing material, it can be covered with the sealing material before use, and the sealing material can be peeled off during use, thereby preventing the adhesion of dust and dirt before use.

  In a reaction vessel equipped with an amplification reaction section, the liquid dispensing port of the amplification reaction section can be in close contact with the tip of the dispensing tip by opening the shape corresponding to the tip shape of the dispensing tip attached to the dispensing probe. If the elastic material is used, the operation of injecting the mixed solution into the amplification reaction unit and the recovery of the reaction solution from the amplification reaction unit can be easily performed.

  In the reaction container kit of the present invention, two dispensing chips are provided, one for handling an amplification reaction solution before a gene amplification reaction and the other for handling an amplification reaction end solution after the completion of a gene amplification reaction. By doing so, the amplification reaction solution before the gene amplification reaction and the amplification reaction end solution after the completion of the gene amplification reaction are not mixed through the dispensing chip, so that the occurrence of contamination can be prevented. .

In the reaction container processing apparatus of the present invention, since the liquid is transferred by the dispensing probe, the dispensing operation can be performed with a simple mechanism.
The reaction vessel processing device is mounted on the reaction vessel mounting portion with the reaction tip mounted with the dispensing tip in a state where the respective base ends are up, and the dispensing probe is lowered toward the dispensing tip to bring the tip If the dispensing tip is attached to the part, the mechanism for attaching the dispensing tip is simple.

  As the non-volatile liquid having a specific gravity lower than that of the reaction liquid, mineral oil (mineral oil), vegetable oil, animal oil, silicone oil, diphenyl ether, or the like can be used. Mineral oil is a liquid hydrocarbon mixture obtained by distillation from petrolatum and is also called liquid paraffin, liquid petrolatum, white oil, etc., and also includes low specific gravity light oil. Examples of animal oils include cod liver oil, halibut oil, herring oil, orange luffy oil, and shark liver oil. As the vegetable oil, canola oil, tonsil oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil, and the like can be used.

  First, examples of the reaction container kit will be described. As shown in FIG. 1, the reaction container kit described below has two dispensing tips for use in a reaction container processing apparatus having two dispensing probes for transferring and dispensing liquids. It is equipped with. In addition, the reaction container kit of the present invention is not limited to the one in this example, and it may be provided with only one dispensing tip.

FIG. 2 is a first embodiment of a reaction container kit. (A) is the front view at the time of mounting | wearing with the reaction container processing apparatus, (B) is a top view, (C) is the front view in the state where it was packed at the time of unused.
This reaction container kit includes a reaction container 10 in which a reagent container 14 and a nonvolatile liquid container 16 are formed as recesses on the same side of a flat plate-like substrate 9. Mineral oil is used as the non-volatile liquid, and hereinafter, the non-volatile liquid container is referred to as a mineral oil container. A reaction part 18 is also formed on the same side of the substrate 9. The reagent container 14 and the mineral oil container 16 are sealed with a film 20, and when the reagent and mineral oil are sucked with a dispensing probe and transferred to another place, the film 20 is removed and dispensed. Inhalation is performed with a probe, or the film 20 is made penetrable with a dispensing probe, and the dispensing probe is penetrated to inhale with the dispensing probe. Such a film 20 is, for example, an aluminum foil, a laminated film of aluminum and a resin film such as a PET (polyethylene terephthalate) film, and is attached by fusion or adhesion so as not to be easily peeled off.

  The surface of the reaction vessel 10 is covered from above the film 20 with a peelable sealing material 22 having a size covering the reagent storage unit 14, the mineral oil storage unit 16 and the reaction unit 18. The sealing material 22 is also an aluminum foil, a laminated film of aluminum and resin, or the like, but the bonding strength is weaker than that of the film 20 and is bonded to such an extent that it can be peeled off by fusion or adhesive.

  An example of a specific use of this reaction vessel is a reagent kit for diagnosing gene polymorphism that injects a sample reaction solution obtained by amplifying DNA by PCR reaction and detects SNP by invader reaction. With reference to FIG. 2, the Example as the reagent kit for a genetic polymorphism diagnosis is demonstrated in detail.

  A sample injection part 12, a typing reagent storage part 14, and a mineral oil storage part 16 are formed as recesses on the same side of the flat substrate 9. A plurality of probe placement portions 18 are also formed on the same side of the substrate 9 as reaction portions.

  The sample injection unit 12 is for injecting a biological sample reaction solution obtained by amplifying DNA by a PCR reaction, but is provided in an empty state where a sample is not yet injected before use. The typing reagent storage unit 14 stores 10 to 300 μL of typing reagents prepared corresponding to a plurality of polymorphic sites, and the mineral oil storage unit 16 stores 20 to 300 μL of mineral oil for preventing evaporation of the reaction solution. The typing reagent container 14 and the mineral oil container 16 are sealed with a film 20 that can be penetrated by a dispensing probe.

  Each probe placement unit 18 individually holds a fluorescent probe corresponding to each of a plurality of polymorphic sites, and holds the mineral oil when the mineral oil from the mineral oil storage unit 16 is dispensed. It is a recess that can be made. The size of the concave portion of each probe placement portion 18 is, for example, a circle having a diameter of 100 μm to 2 mm and a depth of 50 μm to 1.5 mm.

  The surface of the substrate 9 is covered from above the film 20 with a peelable sealing material 22 having a size covering the sample injection part 12, the typing reagent storage part 14, the mineral oil storage part 16 and the probe placement part 18.

  In order to measure fluorescence from the bottom surface side, the substrate 9 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 thickness of the substrate 10 is 0.3 to 4 mm, preferably 1 to 2 mm. From the viewpoint of low autofluorescence, the substrate 10 is preferably as thin as possible.

  This reaction container kit includes dispensing tips 70a and 70b that are attached to the tip of a dispensing probe to suck and discharge liquid. Dispensing tips 70a, 70b are detachably held by a flat holding member 42. In the holding member 42, circular openings 42a and 42b having a diameter smaller than the maximum diameter of the cross-section of the dispensing tips 70a and 70b are formed in order to detachably hold the dispensing tips 70a and 70b. Dispensing tips 70a and 70b are inserted into openings 42a and 42b.

  The holding member 42 is attached to the distal end portion of the reaction vessel 10. The holding member 42 is moved through a movable member (not shown) so that the held dispensing tips 70a and 70b can be displaced between a state along the flat surface of the substrate 9 of the reaction vessel 10 and a standing state. Attached to the substrate 9 of the reaction vessel 10.

  The movable member between the substrate 9 and the holding member 42 may be integrally molded with the same resin together with the substrate 9 and the holding member 42, and the movable member portion may be thinned to be bent or assembled. A hinge may be used.

In the packaged state before use, the reaction container kit is folded so that the dispensing tips 70a and 70b held by the holding member 42 are on the reaction container 10 side as shown in FIG. 2C. The reaction vessel 10 and the dispensing tips 42 a and 42 b are both housed and packaged in the packaging member 74 in a state where the dispensing tips 42 a and 42 b are arranged along the flat plate surface of the substrate 9 of the reaction vessel 10. . In such a state, the thickness of the reaction container kit can be reduced, which is convenient for storing and transporting the reaction container kit.
The packaging member 74 is made of a resin or a film in which a resin is coated with aluminum.

When the reaction container kit is used, as shown in FIG. 2A, the reaction container kit is taken out from the packaging member 74, the holding member 42 is in a direction parallel to the flat surface of the substrate 9 of the reaction container 10, and the holding member The holding member 42 is extended so that the dispensing tips 70 a and 70 b held by the plate 42 are in a direction perpendicular to the flat plate surface of the substrate 9. In this state, when the reaction container 10 is mounted on the reaction container processing apparatus, the dispensing probe 28a is lowered from above, inserted into the dispensing tip 70a, and lifted upward so that the dispensing tip 70a opens the opening of the holding member 42. It is pulled out from the portion 42a and attached to the tip of the dispensing probe 28a. Similarly, the dispensing probe 28b is lowered from above, inserted into the dispensing tip 70b, and pulled upward to pull out the dispensing tip 70b from the opening 42b and be attached to the tip of the dispensing probe 28b.
The tips of the dispensing tips 70 a and 70 b are thin so that the film of the reagent holding part of the reaction vessel 10 can be broken through.

  The holding member 42 includes storage portions 43a and 43b that cover at least the tip portions of the dispensing tips 70a and 70b when holding the dispensing tips 70a and 70b. Absorbing materials 44a and 44b such as filter paper are provided in the storage portions 43a and 43b in order to absorb the liquid adhering to the used dispensing tips 70a and 70b. Thereby, the waste liquid adhering to the dispensing tips 70a and 70b can be absorbed by the absorbents 44a and 44b by returning the dispensing tips 70a or 70b into the storage portions 43a and 43b after use. By discarding the used dispensing tips 70a and 70b in the state where they are returned to the storage portions 43a and 43b, it is possible to prevent liquid spillage of the waste liquid and to avoid contamination.

  In the reaction container kit shown in FIG. 2, the holding member 42 is provided at the end of the reaction container 10, but the holding member 42 may be provided at a position different from the end of the reaction container 10. In FIG. 3, an opening 9 a into which the holding member 42 can be fitted is provided in the intermediate part of the substrate 9, and a part of the peripheral part of the holding member 42 is attached to a part of the opening 9 a so as to be bent. ing. The holding member 42 is bent so as to come to the side where the bulge is small on the flat plate surface of the substrate on which the dispensing tips 70 a and 70 b are bent, in this example, the back surface side of the probe placement portion 18.

In a packaged state before use, as shown in FIG. 3C, the dispensing tips 70a and 70b are bent so that they are in a direction parallel to the flat plate surface of the substrate on the back surface side of the probe placement portion 18. And packaged.
Also in this reaction container kit, the holding member 42 is attached to the substrate 9 via a movable member, and when used, as shown in FIG. 3A, the dispensing tips 70a and 70b are placed on the flat surface of the substrate 9. Are set up in a vertical direction and attached to the reaction vessel processing apparatus.

A plurality of raised portions 10a may be formed at the end of the reaction vessel 10 of the reaction vessel kit shown in FIG. By forming the grip portion 10a, manual operation when starting up the dispensing tips 70a and 70b is facilitated.
Also in the reaction container shown in FIG. 2, such a gripping portion 10 a may be provided at the end of the holding member 42.

  Moreover, as a reaction container kit of this invention, the reaction container 10 and the holding member may not be integrated. For example, as shown in FIG. 4A, in the state of being accommodated in the packaging member 74, the packaging tips 74a and 70b are held in a direction parallel to the flat plate surface of the substrate of the reaction vessel 10 in the packaging member 74. The reaction vessel 10 and the dispensing tips 70a and 70b are enclosed. Also in this case, the dispensing tips 70a and 70b are enclosed in the packaging member 74 in a state where the dispensing tips 70a and 70b are held by the holding member 72 provided with the storage portions 71a and 71b that at least cover the tips of the dispensing tips 70a and 70b. Is preferred. And it is preferable that absorption material 73a, 73b which absorbs the liquid adhering to dispensing tip 70a, 70b is provided in storage part 71a, 71b.

  As shown in FIG. 4B, the holding member 72 can be installed in a reaction vessel processing apparatus that handles the reaction vessel 10, and the operator places the holding member 72 at a predetermined position of the reaction vessel processing apparatus. Install. The used dispensing tips 70a and 70b can be automatically returned to the holding member 72 and discarded in the reaction vessel processing apparatus. At that time, liquid spillage of the waste liquid adhering to the used dispensing tips 70a and 70b can be prevented.

Next, the method of using the reaction vessel of the embodiment of FIG. 2 will be described, but the same applies to the reaction vessels of other embodiments.
The chip holding member 42 of the reaction container kit is packaged in the state shown in FIG. 2C before the reaction container 10 is attached to the processing apparatus. After the reaction vessel 10 is taken out from the packaging member 74, the sealing material 22 is peeled off as shown in FIG. The film 20 that seals the typing reagent container 14 and the mineral oil container 16 remains without being peeled off.

2 to 20 μL of a sample reaction solution 24 in which DNA is amplified by a PCR reaction is injected into the sample injection unit 12 by a pipette 26 or the like. Thereafter, the reaction container kit is extended by the operator's hand or automatically so that the tip holding member 42 is stretched so that the base ends of the dispensing tips 70a and 70b are up, and the dispensing tips 70a and 70b. Is raised vertically to the flat plate surface of the substrate of the reaction vessel 10 (state of FIG. 2A).
In the reaction container processing apparatus, the dispensing tip 70a is attached to the distal end portion of the dispensing probe 28a, and the dispensing tip 70b is attached to the distal end portion of the dispensing probe 28b.

  As shown in FIG. 6, a dispensing tip 70a attached to the distal end portion of the dispensing probe 28a passes through the film 20 and is inserted into the typing reagent container 14, and the typing reagent is inhaled. The sample is transferred to the sample injection unit 12 by the injection probe 28a. In the sample injection part 12, the sample reaction solution and the typing reagent are mixed by repeating suction and discharge by the dispensing tip 70a attached to the tip of the dispensing probe 28a.

Thereafter, the reaction solution of the sample reaction solution and the typing reagent is dispensed to each probe placement portion 18 by the dispensing tip 70b attached to the distal end portion of the dispensing probe 28b. Mineral oil is dispensed from the mineral oil storage portion 16 to each probe placement portion 18 by a dispensing tip 70b attached to the tip of the dispensing probe 28b. The dispensing of the mineral oil to the probe placement unit 18 may be before the reaction solution is dispensed to the probe placement unit 18. In each probe placement unit 18, 0.5-10 μL of mineral oil is dispensed, and the mineral oil covers the surface of the reaction solution, and the typing reaction time is accompanied by heating in the typing reaction temperature control unit of the detection device. Prevent evaporation of the reaction solution inside.
In each probe placement unit 18, if the reaction solution reacts with the probe and there is a predetermined SNP, fluorescence is emitted from the probe. Fluorescence is detected by irradiating excitation light from the back side of the substrate 9.

FIG. 7 shows still another embodiment of the reaction container kit. (A) is a front view at the time of use, (B) is a plan view, (C) is an enlarged cross-sectional view at the XX line position of (B), and (D) is a front view of a packaged state before use. It is.
In this reaction vessel, a biological sample that has not been subjected to nucleic acid extraction operation is injected as a sample, and both amplification of DNA by PCR reaction and SNP detection by invader reaction are performed. However, a biological sample that has not been subjected to nucleic acid extraction may be injected.

  The same sample injection part 12, typing reagent storage part 14, mineral oil storage part 16, and a plurality of probe placement parts 18 as in the embodiment of FIG. 2 are formed on the same side of the flat substrate 76 of this reaction container. . In this reaction container, a gene amplification reagent storage unit 30, a PCR end solution injection unit 31, and an amplification reaction unit 32 are further formed on the same side of the substrate 76.

  Similarly to the reaction container kit of FIG. 2, the reaction container kit of this embodiment also includes a holding member 42 that holds the dispensing tips 70a and 70b in a detachable manner. 2, the holding member 42 is attached to the end portion of the substrate 76 via a movable member (not shown). Before use, as shown in FIG. 7D, the dispensing tips 70a, 70b are folded so as to be parallel to the flat plate surface of the substrate 76, and when used, as shown in FIG. 7A. In addition, the dispensing tips 70 a and 70 b are raised so as to be in a direction perpendicular to the flat plate surface of the substrate 76.

  The gene amplification reagent storage unit 30 is also formed as a recess in the substrate 76, and stores a gene amplification reagent including a plurality of primers that are bonded with each of a plurality of polymorphic sites interposed therebetween. The gene amplification reagent container 30 is sealed together with the typing reagent container 14 and the mineral oil container 16 by a film 20 that can be penetrated by a dispensing probe. The gene amplification reagent storage unit 30 stores 2 to 300 μL of PCR reaction reagent. Similar to the embodiment of FIG. 2, 10 to 300 μL of typing reagent is stored in the typing reagent storage unit 14, and 20 to 300 μL of mineral oil is stored in the mineral oil storage unit 16.

The PCR end solution injection unit 31 is used to mix the reaction solution that has completed the PCR reaction in the amplification reaction unit 32 and the typing reagent. The PCR end solution injection unit 31 is formed as a recess in the substrate 76 and is provided in an empty state before use. The
The amplification reaction unit 32 performs a gene amplification reaction on the mixture of the PCR reaction reagent and the sample.

  FIG. 9 shows an enlarged cross section of the amplification reaction section 32. FIG. 9 is a cross-sectional view taken along the line YY in FIG. As shown in FIG. 9, the liquid dispensing ports 34a and 34b of the amplification reaction section 32 have openings 36a and 36b having shapes corresponding to the tip shapes of the dispensing probes 28a and 28b. It is made of an elastic material such as PDMS (polydimethylsiloxane) or silicone rubber so as to be in close contact with the tips of the dispensing tips 70a and 70b attached to the tips.

In order that the amplification reaction part 32 may improve thermal conductivity, the lower surface side of the substrate 76 of that part is thin as shown in FIG. 7C and FIG. The thickness of the portion is, for example, 0.2 to 0.3 mm.
In this embodiment, the sample injection unit 12 is provided with a biological sample that has not been subjected to the nucleic acid extraction operation, but is provided in an empty state in which the sample is not yet injected before use.

As in the embodiment of FIG. 2, the typing reagent storage unit 14 stores typing reagents prepared corresponding to a plurality of polymorphic sites, and the mineral oil storage unit 16 prevents mineral oil from evaporating. Is housed.
Similarly to the embodiment of FIG. 2, each probe placement unit 18 individually holds a probe that emits fluorescence corresponding to each of a plurality of polymorphic sites, and the mineral oil from the mineral oil storage unit 16 is dispensed. It is a recess that can hold the mineral oil.

The surface of the substrate 76 is formed on the film 20 from the sample injection unit 12, the PCR end solution injection unit 31, the typing reagent storage unit 14, the mineral oil storage unit 16, the gene amplification reagent storage unit 30, the amplification reaction unit 32, and the probe placement unit. 18 is covered with a peelable sealing material 22 having a size covering 18. The material of the film 20 and the sealing material 22 and the method of attaching them are the same as in the embodiment of FIG.
The substrate 76 is also made of a material such as a low autofluorescent and light-transmitting resin, such as polycarbonate, in order to measure fluorescence from the bottom side. The thickness of the substrate 76 is 1 to 2 mm.

  FIG. 8 shows a reaction container kit of another embodiment in which the holding members 42 of the dispensing tips 70 a and 70 b are attached to the intermediate portion of the substrate 76. An opening 76a into which the holding member 42 can be fitted is formed between the probe placement portion 18 and the amplification reaction portion 32, and the holding member 42 is bent inside the opening 76a via a movable member (not shown). It is attached as possible. In a packaged state before use, as shown in FIG. 8C, the dispensing tips 70 a and 70 b are in a horizontal direction with respect to the flat plate surface of the substrate 76 on the back surface side of the probe placement portion 18. It is folded and stored in the packaging member 74, and when used, as shown in FIG. 8A, the dispensing tips 70a and 70b are raised so as to be perpendicular to the flat surface of the substrate 76. .

The usage method of the reaction container kit shown in FIG. 7 is shown.
As shown in FIG. 10, the sealing material 22 is peeled off during use. The film 20 sealing the typing reagent container 14, the mineral oil container 16, and the gene amplification reagent container 30 remains as it is without being peeled off.
0.5 to 2 μL of sample 25 is injected into the sample injection unit 12 by a pipette 26 or the like. In the embodiment of FIG. 2, the sample to be injected is a sample reaction solution in which DNA is amplified by a PCR reaction externally, but the sample injected in this embodiment is a biological sample that has not been subjected to nucleic acid extraction operation, for example, blood It is. The sample may be a biological sample subjected to a nucleic acid extraction operation. After sample injection, the reaction vessel is attached to the detection device. When the reaction container is mounted on the detection device, the dispensing tips 70 a and 70 b are started up manually or automatically so as to be in a direction perpendicular to the flat plate surface of the substrate 76.

  In the reaction vessel processing apparatus, as shown in FIG. 11, a dispensing tip 70a attached to the tip of the dispensing probe 28a penetrates the film 20 and is inserted into the gene amplification reagent storage unit 30 so that the PCR reaction reagent is added. Inhaled and transferred to the sample injection part 12 by 5 to 20 μL. In the sample injection unit 12, the sample reaction solution and the PCR reaction reagent are mixed to become a PCR reaction solution by repeating suction and discharge by the dispensing tip 70a attached to the tip of the dispensing probe 28a.

  Next, as shown in FIG. 9A, the PCR reaction solution is injected into the amplification reaction section 32 by the dispensing tip 70a attached to the tip of the dispensing probe 28a. At this time, both ends of the PCR reaction solution 38 are closed with mineral oil 40 to prevent the PCR reaction solution 38 from evaporating during the reaction in the amplification reaction section 32. Moreover, it is preferable that the injected PCR reaction solution 38 is placed only in the gene amplification reaction temperature control region of the amplification unit, and the control device causes the PCR reaction solution in the dispensing device to be in such a state. The dispensing amount of 38 and mineral oil 40 is controlled.

  As one method for dispensing the PCR reaction solution 38 into the amplification reaction section 32 in such a state, a dispensing tip 70a attached to the tip of the dispensing probe 28a is inserted into one port 34a of the amplification reaction section 32. Then, the PCR reaction solution 38 is injected, and then the mineral oil 40 is injected into the ports 34a and 34b by the dispensing tip 70a attached to the tip of the dispensing probe 28a.

  In another method of dispensing the PCR reaction solution 38 to the amplification reaction unit 32, the mineral oil 40, the PCR reaction solution 38, and the mineral oil 40 are sucked in this order by the dispensing tip 70a attached to the tip of the dispensing probe 28a. In the dispensing tip 70a, the upper layer is made of mineral oil 39, the intermediate layer is made of the PCR reaction liquid 38, and the lower layer is made of three layers of mineral oil. Thereafter, the three layers of liquid are injected into the amplification reaction section 32 as they are.

After completion of the PCR reaction, the PCR reaction end solution 38a is recovered from the port 34a or 34a by the dispensing tip 70b attached to the tip of the dispensing probe 28b. At this time, it is recovered in the three layers of mineral oil 40 / PCR reaction end solution 38a / mineral oil 40 or in two layers of mineral oil 40 / PCR reaction end solution 38a. Here, in order to facilitate recovery, mineral oil 40 may be injected from one port 34a of the amplification reaction section 32 as shown in FIG. 9B. After completion of the reaction, the PCR reaction completion solution 38a is pushed to the other port 34b. Therefore, the dispensing tip 70b is inserted into the port 34b, and the PCR reaction end solution 38a is sucked into the dispensing tip 70b. When collecting the PCR reaction end solution 38a, three layers of mineral oil 40 / PCR reaction end solution 38a / mineral oil 40 may be inhaled into the dispensing tip 70b, or the mineral oil 40 / PCR reaction end solution 38a Two layers may be inhaled. The ports 34a and 34b have openings 36a and 36b formed in accordance with the shapes of the dispensing tips 70a and 70b, and are formed of an elastic material, so that the dispensing tips 70a and 70b are in close contact with the ports 34a and 34b. Thus, the liquid leakage is prevented, and the PCR reaction liquid injection and recovery operations are easy.
Here, the liquid injected from the port 34a is not limited to the mineral oil 40 but may be other liquids. Preferably, the specific gravity is the same as or lower than that of the reaction liquid.

The PCR reaction end solution 38a after completion of the reaction collected from the amplification reaction unit 32 by the dispensing tip 70b attached to the tip of the dispensing probe 28b is transferred to the PCR end solution injection unit 31 and injected.
The ports 34a and 34b are formed in accordance with the shape of the dispensing tips 70a and 70b attached to the distal ends of the dispensing probes 28a and 28b and the openings 36a and 36b are formed of an elastic material. Dispensing tips 70a and 70b attached to the distal ends of the dispensing probes 28a and 28b are in close contact with the ports 34a and 34b to prevent liquid leakage, and the PCR reaction solution injection and recovery operations are easy.

  Next, the dispensing tip 70b attached to the distal end portion of the dispensing probe 28b penetrates the film 20 and is inserted into the typing reagent storage portion 14, and the typing reagent is inhaled. The typing reagent is the distal end of the dispensing probe 28b. It is transferred to the PCR end liquid injection unit 31 and injected by the dispensing tip 70b attached to the unit. In the PCR end solution injection unit 31, the PCR reaction solution and the typing reagent are mixed by repeating suction and discharge by the dispensing tip 70b attached to the tip of the dispensing probe 28b.

Thereafter, the reaction solution of the PCR reaction solution and the typing reagent is dispensed in an amount of 0.5 to 4 μL to each probe placement portion 18 by the dispensing tip 70b attached to the tip of the dispensing probe 28b. Mineral oil is dispensed from the mineral oil storage portion 16 to each probe placement portion 18 by a dispensing tip 70b attached to the tip of the dispensing probe 28b. The dispensing of the mineral oil to the probe placement unit 18 may be before the reaction solution is dispensed to the probe placement unit 18. In each probe arrangement unit 18, mineral oil covers the surface of the reaction solution, and prevents evaporation of the reaction solution during the typing reaction time accompanied by heating in the typing reaction temperature control unit of the detection device.
In each probe placement unit 18, if the reaction solution reacts with the probe and there is a predetermined SNP, fluorescence is emitted from the probe. Fluorescence is detected by irradiating excitation light from the back side of the substrate 10.

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 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. 12A shows an embodiment of a simplified reaction container processing apparatus for detecting SNP of a biological sample using the reaction container kit described with reference to FIG. The reaction container processing apparatus of this embodiment includes a dispensing probe 28a that handles an amplification reaction solution before a gene amplification reaction, and a dispensing probe 28b that handles an amplification reaction end solution after the completion of the gene amplification reaction. A dispensing tip 70a is attached to the distal end portion of the dispensing probe 28a, and a dispensing tip 70b is attached to the distal end portion of the dispensing probe 28b.

In the apparatus, a pair of heat blocks 60 and 62 are arranged on the upper and lower sides to constitute a reaction vessel mounting portion, and five pieces of samples injected into the reaction vessel 41 of the present invention are placed on the lower heat block 60 in parallel. Installed side by side. These heat blocks 60 and 62 can move in the Y direction indicated by arrows.
The upper heat block 62 is provided with a window that can be opened and closed so that the lid is opened when the dispensing probes 28a and 28b transfer, suck, and discharge the liquid.

  The lower heat block 60 is an amplification reaction temperature control unit that controls the temperature of the amplification reaction unit 32 to be a predetermined temperature cycle, and typing that controls the temperature of the probe placement unit 18 to a temperature at which DNA and the probe are reacted. And a reaction temperature control unit. The temperature of the amplification reaction temperature control 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 temperature control unit is set to 63 ° C., for example.

As shown in FIG. 13, the upper heat block 62 constituting the typing reaction temperature controller has an opening 150 only at a position corresponding to the probe placement portion, and the lower heat block 60 constitutes the typing reaction temperature controller. This portion also has an opening 152 only at a position corresponding to the probe placement portion. A typing reaction temperature control unit cover 154 is put on the heat block 62, and the opening 156 is also opened only at the position of the opening 150 of the heat block 62. The reaction solution is dispensed to the probe placement portion of the reaction vessel 41 by the nozzle 28 through the openings 150 and 156.
When using a reaction vessel 41 that does not include an amplification reaction section as in the embodiment of FIG. 2, an amplification reaction temperature control section that controls the temperature of the amplification reaction section is unnecessary.

  A fluorescence detection unit 64 that performs fluorescence detection is disposed below the heater block 60, and the fluorescence detection unit 64 irradiates the probe arrangement unit with excitation light from the lower surface side of the reaction vessel 41 through the opening 152 of the heater block 60. Then, the fluorescence from the probe placement portion is detected through the opening 152 of the heater block 60 on the lower surface side of the reaction vessel 41. The fluorescence detection unit 64 moves in the direction of the arrow X in FIG. Fluorescence detection is performed on each probe by moving the probe placement unit 18 in the Y direction by the reaction vessel mounting unit and moving the fluorescence detection unit 64 in the X direction.

  Returning to FIG. 12A, in order to transfer, inhale, and discharge liquid by the dispensing probes 28a and 28b, a liquid feeding arm 66 is provided as a dispensing unit. Two dispensing probes 28a and 28b are provided. Disposable dispensing tips 70a and 70b are detachably attached to the tips of the dispensing probes 28a and 28b.

  In order to control the operations of the heat blocks 60 and 62, the fluorescence detection unit 64, and the liquid feeding arm 66, a control unit 118 is disposed in the vicinity thereof. The control unit 118 includes a CPU and holds a program for operation. The control unit 118 operates to attach the dispensing tip 70a to the distal end of the dispensing probe 28a and attach the dispensing tip 70b to the distal end of the dispensing probe 28b, the typing reaction unit 110 realized by the heat blocks 60 and 62, and the amplification. The temperature control of the unit 120, the detection operation of the fluorescence detection unit 64, and the dispensing operation of the liquid feeding arm 66 of the dispensing unit 112 are controlled.

  When the reaction vessel 41 having no gene amplification reaction unit such as the reaction vessel of FIG. 2 is used, an amplification unit for controlling the temperature of the gene amplification reaction unit is not necessary, and the control unit 118 is also an amplification unit. It is not necessary to provide a function for temperature control.

  In the reaction container processing apparatus of this embodiment, two dispensing probes 28a and 28b are attached to one liquid feeding arm 66. However, as shown in FIG. -1, 66-2 may be provided, and the two dispensing probes 28a, 28b may be attached to separate liquid feeding arms 66-1, 66-2.

  FIG. 14 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 placement position, and whether the SNP is a homozygote or a heterozygote. Detected. As the labeling phosphor, for example, FAM, ROX, VIC, TAMRA, Redmond Red, or the like can be used.

  The detector 64 in FIG. 14 is configured to be excited with excitation light from one light source and measure fluorescence of two wavelengths, but the detector 64 can be excited with different excitation wavelengths for fluorescence measurement of two wavelengths. As described above, two light sources may be used.

  In addition to measurement of various chemical reactions, the present invention can be used for various automatic analyzes in, for example, genetic analysis research and clinical fields. For example, polymorphisms of genomic DNA of animals and plants including humans, In particular, SNPs can be detected, and the results are used to diagnose disease prevalence, diagnose the relationship between the type of drug administered, effects and side effects, animal and plant breeding, and infection diagnosis It can also be used for (type determination of infecting bacteria).

1 is a block diagram schematically illustrating the present invention. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure which shows 1st Example of reaction container kit, (A) is a front view at the time of mounting | wearing with a processing apparatus, (B) is a top view, (C) is a front cross section of the packaged state before use. FIG. It is a figure which shows 2nd Example of reaction container kit, (A) is a front view at the time of use, (B) is a top view, (C) is front sectional drawing of the packaged state before use. It is a figure which shows the 3rd Example of the reaction container kit, (A) is front sectional drawing of the packaged state before use, (B) is a front view at the time of use. It is a figure which shows the first half part of the process of the SNP detection method using the reaction container kit of the Example of FIG. 2, (A) is a front view, (B) is a top view. It is a figure which shows the latter half part of the process of the SNP detection method using the reaction container kit of the Example, (A) is a front view, (B) is a top view. It is a figure which shows the 4th Example of reaction container kit, (A) is a front view at the time of use, (B) is a top view, (C) is an expanded sectional view in the XX line position of (B). , (D) is a front sectional view of the packaged state before use. It is a figure which shows 5th Example of reaction container, (A) is a front view at the time of mounting | wearing with a processing apparatus, (B) is a top view, (C) is a front sectional view of the packaged state before use. is there. FIG. 8 is a diagram showing an amplification reaction part in the embodiment of FIG. 7 as an enlarged cross-sectional view at the YY line position in FIG. 7B, where (A) is a state in which a reaction solution is injected, and (B) is a reaction It is in a state for recovering the liquid. It is a figure which shows the first half part of the process of the SNP detection method using the reaction container of the Example of FIG. 7, (A) is a front view, (B) is a top view. It is a figure which shows the latter half part of the process of the SNP detection method using the reaction container of the Example, (A) is a front view, (B) is a top view. BRIEF DESCRIPTION OF THE DRAWINGS It is a schematic perspective view which shows the Example of the simple reaction container processing apparatus for detecting SNP of a biological sample using the reaction container of this invention as a reagent kit, (A) is the case where there is one liquid feeding arm, B) shows a case where there are two liquid feeding arms. It is sectional drawing which shows the typing reaction temperature control part in the Example. It is a schematic block diagram which shows the detector in the Example. FIG. 2 is a flow chart diagram schematically illustrating a SNP detection method to which the present invention may relate.

Explanation of symbols

2 Sample 4 PCR reaction reagent 6 Invader reagent 8 Probe placement part 9 Substrate 10 Reaction vessel 12 Sample injection part 14 Typing reagent storage part 16 Mineral oil storage part 18 Probe placement part 20 Film 22 Sealing material 28a, 28b Dispensing probe 30 Gene amplification Reagent storage unit 31 PCR end solution injection unit 32 Amplification reaction unit 34a, 34b Amplification reaction unit port 36a, 36b Port opening 42, 72 Holding member 43a, 43b Storage unit 44a, 44b Absorbing material 60, 62 Heat block 64 Detector 66a, 66b Liquid feeding arm 70a, 70b Dispensing tip

Claims (14)

A reaction vessel in which at least one reaction portion for causing a sample to react on one surface of a flat substrate is formed;
A dispensing tip attached to a dispensing probe of a dispensing mechanism for dispensing a sample and / or reagent to the reaction part of the reaction vessel;
A holding member for detachably holding the dispensing tip;
A reaction vessel kit comprising: a reaction vessel in which the dispensing tips held by the holding member are arranged along a flat plate surface of the substrate of the reaction vessel; and a packaging member for packaging the set of dispensing tips. .   The holding member is attached to the reaction vessel via a movable member so that the held dispensing tip can be displaced between a state along the flat surface of the substrate of the reaction vessel and a standing state. The reaction container kit according to 1.   The reaction container kit according to claim 2, wherein the holding member is attached to a distal end portion of the reaction container and is bent so that a dispensing tip comes to the reaction container side in a packaged state.   The reaction according to claim 2, wherein the holding member is attached to an intermediate portion of the reaction container, and is folded so as to come to a side where the bulge is small on the flat plate surface of the substrate on which the dispensing tip is folded in a packaged state. Container kit.   The holding member includes a storage portion that covers at least a tip portion of the dispensing tip when the dispensing tip is held, and includes an absorbent that absorbs the liquid in the storage portion. 5. The reaction container kit according to any one of 4 above.   The reaction container kit according to any one of claims 1 to 5, wherein a grip portion is formed on a distal end side of the substrate or the holding member.   The said reaction container is provided with the non-volatile liquid storage part which was formed as a recessed part in the said board | substrate, accommodated the non-volatile liquid whose specific gravity is lower than a reaction liquid, and was sealed with the film. The reaction container kit described.   The reaction container further includes at least one reagent container formed as a recess in the substrate, containing a reagent used for the sample reaction, and sealed with a film to constitute a sample reaction reagent kit. The reaction container kit according to claim 7. Including a typing reagent container containing a typing reagent prepared corresponding to a plurality of polymorphic sites as the reagent container;
Including a plurality of probe placement parts individually holding probes that emit fluorescence corresponding to each of the plurality of polymorphic sites as the reaction part,
The reaction container kit according to claim 8 constituting a genetic polymorphism diagnostic reagent kit. Further comprising a gene amplification reagent containing part containing a gene amplification reagent containing a plurality of primers that bind across each of the plurality of polymorphic sites as the reagent containing part,
The reaction container kit according to claim 9, further comprising an amplification reaction part for causing a gene amplification reaction to be performed on the mixture of the gene amplification reagent and the sample as the reaction part.   The reaction container kit according to any one of claims 1 to 10, wherein the reaction container and the dispensing chip are provided with two dispensing chips for each reaction container.   The reaction container kit according to claim 1, wherein a plurality of sets of the reaction container and the dispensing tip are accommodated and packaged in the packaging member. For suction and discharge by a reaction container mounting portion for mounting the reaction container of the reaction container kit according to any one of claims 1 to 12, and a dispensing probe having a dispensing tip of the reaction container kit mounted at a tip portion A dispensing unit that transports and dispenses liquid with the mechanism of
And a control unit that controls at least the dispensing operation of the dispensing unit. A reaction vessel mounting for mounting the reaction vessel of the reaction vessel kit according to any one of claims 2 to 12 in a state where a flat plate surface of the substrate is in a horizontal direction and a dispensing tip held in the reaction vessel is in a vertical direction. A reaction part comprising: a dispensing part, a dispensing part that includes a mechanism for suction and discharge by a dispensing probe and that transfers and dispenses liquid; and a control part that at least controls the dispensing operation of the dispensing part A container processing device,
The control unit lowers the dispensing probe toward the dispensing tip, inserts it into the dispensing tip, and lifts the dispensing tip from the holding member to attach it to the tip of the dispensing probe. A reaction vessel processing device that also controls operations.

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