JP4527582B2 - Reaction vessel processing equipment - Google Patents

Description

  In the present invention, polymorphisms of genomic DNA of animals and plants including human beings, particularly SNP (especially using a reaction vessel suitable for conducting various automatic analyzes such as chemical reactions, medical studies, for example, genetic analysis research and clinical practice. The present invention relates to a reaction vessel processing apparatus for detecting a single nucleotide polymorphism). By using the detected gene polymorphism detection result, it is possible to diagnose the disease morbidity and the relationship between the kind and effect of the administered drug and side effects.

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 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 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 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 2001-299366 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

  The present inventors have proposed a reaction vessel suitable for automating the measurement of a chemical reaction and the detection of a gene polymorphism for the purpose of automatically detecting the measurement of the chemical reaction and the gene polymorphism.

  The reaction vessel includes at least one reaction unit that is formed on a flat substrate and causes a sample to react. The reaction vessel is used for measuring various reactions including chemical reactions and biochemical reactions.

  A reaction vessel processing apparatus that uses such a reaction vessel to measure chemical reactions and detect gene polymorphisms has also been proposed. The reaction container processing apparatus includes a reaction container mounting unit for mounting the reaction container, a dispensing unit that includes a mechanism for suction and discharge by a nozzle, and transfers and dispenses liquid, and a dispensing operation of the dispensing unit And at least a control unit for controlling the above.

In the reaction vessel processing apparatus as described above, it is necessary to correctly transfer the liquid with a nozzle.
An object of the present invention is to make it possible to accurately perform operations such as dispensing of a reaction solution using a nozzle in the reaction vessel processing apparatus as described above.

The reaction container processing apparatus of the present invention includes a reaction container mounting part for mounting a reaction container in which at least one reaction part for causing a sample to react on a flat substrate is mounted. Further, as shown in FIG. And a dispensing portion 112 for transferring the liquid in the reaction vessel by moving the nozzle 28 for discharge in the X and Y directions in the plane and the Z direction in the height direction, and the position of the tip of the nozzle 28 in the X direction. , A nozzle tip position sensor unit 116 having a detector that detects in at least one of the Y direction and the Z direction, a nozzle tip position detection operation by the nozzle tip position sensor unit 116, and a detection by the nozzle tip position sensor unit 116 And a control unit 118 that controls the dispensing operation of the dispensing unit 112 based on the nozzle tip position information.
The reaction container may further include a non-volatile liquid storage portion that is formed as a concave portion on the same substrate, stores a non-volatile liquid having a specific gravity lower than that of the reaction solution, and is sealed with a film.
The reaction container further includes at least one reagent container formed as a recess on the same substrate, containing a reagent used for the sample reaction, and sealed with a film to constitute a sample reaction reagent kit. It can also be.

  When this reaction container processing apparatus is used as a genetic polymorphism detection apparatus, the reaction container attached to the reaction container attachment part includes at least a typing reagent storage part as a reagent storage part, and a plurality of polymorphic sites as reaction parts The reaction container for diagnosing a gene polymorphism including a plurality of probe placement portions individually holding fluorescent probes corresponding to each of the above. As shown in FIG. 1, the reaction vessel processing apparatus controls the temperature of the probe placement unit to a temperature at which the reaction solution of the sample genomic DNA and the typing reagent reacts with the probe of the probe placement unit. It includes at least a reaction temperature control unit 110, and further includes a fluorescence detection unit 64 that detects fluorescence by irradiating each probe placement unit with excitation light, and a control unit 118 controls temperature and fluorescence of the typing reaction temperature control unit 110. The genetic polymorphism is detected by controlling the detection operation of the detection unit 64.

When the invader reaction is used as the typing reaction, the typing reaction temperature control unit 110 serves as a temperature control unit for the invader reaction.
In addition, when a gene amplification reaction is also performed in the reaction vessel, the reaction vessel processing apparatus also includes an amplification reaction temperature control unit 120 for the gene amplification reaction. 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. In this case, the control unit 118 also performs temperature control of the amplification reaction temperature control unit 120.

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.
The detector of the nozzle tip position sensor unit 116 is preferably a non-contact type detector. An example of a non-contact type detector is a photo sensor.

An example of the nozzle 28 has a tip detachably attached to the tip. In that case, the nozzle tip position detected by the nozzle tip position sensor unit is the tip position of the chip mounted on the nozzle.
As such a chip, a disposable chip, a disposable chip holding a filter inside, a replaceable probe, or the like can be used.
The control unit 118 can repeat a plurality of times in the direction in which the detection operation of the nozzle tip position by the nozzle tip position sensor unit is detected, and use the average value as nozzle tip position information in that direction.

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.
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 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.
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. 12 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. 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 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 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). .)

The reaction container processing apparatus of the present invention is provided with a nozzle tip position sensor unit, and detects the position of the tip of the nozzle of the dispensing unit in at least one of the X direction, the Y direction, and the Z direction, and controls the operation. Since the control unit for controlling the nozzle tip position by the nozzle tip position sensor unit and the function for controlling the dispensing operation of the dispensing unit based on the nozzle tip position information detected by the nozzle tip position sensor unit are provided. The dispensing operation by the nozzle can be performed accurately.
When a non-contact type detector is used as the detector of the nozzle tip position sensor unit, the nozzle does not come into contact with the detector and the position does not shift.

When using a nozzle with a removable tip attached to the tip as a nozzle, warpage that occurs when the tip itself is molded, misalignment when attached to the tip of the nozzle, the reaction container seals the reagent container with a film When the film is inserted, the tip position of the tip may be shifted by several hundred μm or more due to a tip position shift that occurs when the film is penetrated by the tip end. Therefore, when a disposable tip is attached to the tip of the nozzle, detection of the tip tip position is important for dispensing the liquid into the small area by the nozzle, and the present invention exerts a remarkable effect . be able to.
If the nozzle tip position detection operation by the nozzle tip position sensor unit is repeated a plurality of times and the average value is used, more accurate nozzle tip position information can be obtained.

FIG. 2 is a first example of a reaction vessel used in the reaction vessel treatment apparatus of the present invention. (A) is a front view, (B) is a plan view.
A reagent container 14 and a non-volatile liquid container 16 are formed as recesses on the same side of the flat substrate 10. A reaction portion 18 is also formed on the same side of the substrate 10. The reagent container 14 and the non-volatile liquid container 16 are sealed with a film 20, and when the reagent and the non-volatile liquid are sucked with a nozzle and transferred to another place, the film 20 is removed and the nozzle 20 Inhalation is performed, or the film 20 is made to be piercable by the nozzle, and the nozzle is pierced and sucked by the nozzle.
The surface of the substrate 10 is covered with a peelable sealing material 22 having a size covering the reagent storage unit 14, the non-volatile liquid storage unit 16, and the reaction unit 18 from the film 20.
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.
Mineral oil is used as the non-volatile liquid, and hereinafter, the non-volatile liquid container is referred to as a mineral oil container.

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.
On the same side of the flat substrate 10, a sample injection part 12, a typing reagent storage part 14, and a mineral oil storage part 16 are formed as recesses. A plurality of probe placement portions 18 are also formed on the same side of the substrate 10.

  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 nozzle.

  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 10 is covered from above the film 20 with a peelable sealing material 22 of a size that covers the sample injection part 12, the typing reagent storage part 14, the mineral oil storage part 16 and the probe placement part 18. The sealing material 22 is also an aluminum foil, a laminated film of aluminum and resin, etc., 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 with an adhesive or the like.

  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 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.

The usage method of the reaction container of this Example is shown.
As shown in FIG. 3, the seal material 22 is peeled off during use. 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 is attached to the detection device.

  In the detection apparatus, as shown in FIG. 4, the nozzle 28 penetrates the film 20 and is inserted into the typing reagent container 14 to suck the typing reagent, and the typing reagent is transferred to the sample injection unit 12 by the nozzle 28. The In the sample injection unit 12, the sample reaction solution and the typing reagent are mixed by repeating the suction and discharge by the nozzle 28.

Thereafter, the reaction solution of the sample reaction solution and the typing reagent is dispensed to each probe placement unit 18 by the nozzle 28. Mineral oil is dispensed from each mineral oil container 16 to each probe placement unit 18 by a nozzle 28. 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 10.

FIG. 5 shows a second embodiment of the reaction vessel. (A) is a front view, (B) is a plan view, and (C) is an enlarged cross-sectional view at the position XX of (B).
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.

  On the same side of the flat substrate 10a, 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. 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 10a.

  The gene amplification reagent storage unit 30 is also formed as a recess in the substrate 10a, 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 nozzle. 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 for mixing the reaction solution that has been subjected to 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 10a 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. 6 shows an enlarged cross section of the amplification reaction section 32. 6 is a cross-sectional view taken along the line YY in FIG. As shown in FIG. 6, the liquid dispensing ports 34 a and 34 b of the amplification reaction section 32 have openings 36 a and 36 b having shapes corresponding to the tip shape of the nozzle 28, so that the PDMS ( (Polydimethylsiloxane) and silicone rubber.

In order that the amplification reaction part 32 may improve thermal conductivity, the lower surface side of the substrate 10a of that part is thin as shown in FIG. 5C 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 10a 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 10a 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 10 is 1 to 2 mm.

The usage method of the reaction container of this Example is shown.
As shown in FIG. 7, the seal 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.

  In the detection apparatus, as shown in FIG. 8, the nozzle 28 penetrates the film 20 and is inserted into the gene amplification reagent storage unit 30 to suck in the PCR reaction reagent. 2 to 20 μL. In the sample injection unit 12, the suction and discharge by the nozzle 28 are repeated, whereby the sample reaction solution and the PCR reaction reagent are mixed to form a PCR reaction solution.

  Next, as shown in FIG. 6A, the PCR reaction solution is injected into the amplification reaction unit 32 through the nozzle 28. That is, in order to prevent the PCR reaction solution 38 from evaporating during the reaction in the amplification reaction unit 32, the nozzle 28 is inserted into one port 34a of the amplification reaction unit 32 and the PCR reaction solution 38 is injected. Mineral oil 40 is injected into the ports 34 a and 34 b by the nozzle 28, and the surface of the PCR reaction solution 38 at the ports 34 a and 34 b is covered with the mineral oil 40.

After completion of the PCR reaction, the PCR reaction solution is recovered by the nozzle 28. At this time, in order to facilitate recovery, as shown in FIG. 6B, mineral oil is supplied from one port 34a of the amplification reaction unit 32. 40 is injected. After completion of the reaction, the PCR reaction solution 38a is pushed to the other port 34b. Therefore, the nozzle 28 is inserted, and the PCR reaction solution 38 a is sucked into the nozzle 28. The ports 34a and 34b have openings 36a and 36b formed in accordance with the shape of the nozzle 28, and are made of an elastic material, so that the nozzle 28 is in close contact with the ports 34a and 34b to prevent liquid leakage, and PCR. The reaction liquid injection and recovery operations are easy.
The PCR reaction solution 38a after the reaction collected from the amplification reaction unit 32 by the nozzle 28 is transferred to the PCR completion solution injection unit 31 and injected.

  Next, the nozzle 28 penetrates the film 20 and is inserted into the typing reagent container 14 to suck the typing reagent, and the typing reagent is transferred to the PCR end solution injection unit 31 by the nozzle 28 and injected. In the PCR end liquid injection unit 31, the PCR reaction liquid and the typing reagent are mixed by repeating the suction and discharge by the nozzle 28.

Thereafter, the reaction solution of the PCR reaction solution and the typing reagent is dispensed by the nozzle 28 to each probe placement unit 18 in an amount of 0.5 to 4 μL. Mineral oil is dispensed from each mineral oil container 16 to each probe placement unit 18 by a nozzle 28. 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. 9 shows an embodiment in which the present invention is applied to a simple reaction container processing apparatus for detecting SNP of a biological sample using the above reaction container 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 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 opens when the liquid is transferred, sucked, or discharged by the nozzle 28.

  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 amplification reaction temperature control unit and the typing reaction temperature control unit are denoted by reference numerals 120 and 110 in FIG. 1, respectively. 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.

A fluorescence detection unit 64 that performs fluorescence detection is disposed below the heater block 60, and the fluorescence detection unit 64 detects the fluorescence from the probe placement unit 18 by moving in the arrow X direction in the figure. The heater block 60 is provided with an opening for detecting fluorescence. 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.
In order to perform transfer, suction, and discharge of the liquid by the nozzle 28, a liquid supply arm 66 that moves in the X direction, the Y direction, and the Z direction is provided as a dispensing unit, and the liquid supply arm 66 includes the nozzle 28. Yes. A disposable tip 70 is detachably attached to the tip of the nozzle 28. The dispensing part is indicated by reference numeral 112 in FIG.

FIG. 10 shows the tip accommodating portion 132 and the nozzle tip position sensor portion 116 provided within the movement range of the nozzle 28 by the liquid feeding arm 66.
The tip 70 has a hollow conical shape, and the tip receiving portion 132 holds the plurality of tips 70 in a state of being fitted into the holes with the opening of the cone facing up. When the tip 70 is attached to the nozzle 28, the tip 28 is attached to one tip 70 and the tip of the nozzle 28 is pushed into the opening of the tip 70, so that the tip 70 is attached to the tip of the nozzle 28.
After the chip is used, the nozzle 28 moves to the chip disposal place, and the chip 70 is removed from the nozzle 28 and discarded.

The nozzle tip position sensor unit 116 includes a detector that constitutes the nozzle tip position sensor unit 116 in order to detect the tip position of the tip 70 attached to the tip of the nozzle 28 in three directions, the X direction, the Y direction, and the Z direction. Photosensors 134, 136, and 140 are provided. The photosensors 134 and 136 are arranged on the horizontal plane of the substrate 130 having a horizontal plane, the photosensor 134 is arranged in a direction for detecting the X direction position of the tip of the chip 70, and the photosensor 136 sets the Y direction position of the tip of the chip 70. It is arranged in the direction to detect. The photo sensor 140 is attached to the lower side of the substrate 130 and is arranged in a direction for detecting the position of the tip of the chip 70 in the Z direction. A hole 138 is formed at the position of the substrate 130 above the photosensor 140 so that the nozzle 28 to which the chip 70 is attached can be lowered toward the photosensor 140.
Here, the chip accommodating portion 132 and the nozzle tip position sensor portion 116 are integrally configured on the same substrate 130, but may be configured separately.

Returning to FIG. 9, 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 near them. The control unit 118 includes a CPU and holds a program for operation. The control unit 118 controls the temperature of the typing reaction temperature control unit 110 and the amplification reaction temperature control unit 120 realized by the heat blocks 60 and 62, the detection operation of the fluorescence detection unit 64, and the position of the nozzle 28 tip position by the nozzle tip position sensor unit 116. The detection operation and the dispensing operation of the liquid feeding arm 66 of the dispensing unit 112 based on the nozzle tip position information detected by the nozzle tip position sensor unit 116 are controlled.
When the reaction vessel 41 having no amplification reaction unit such as the reaction vessel of FIG. 2 is used, an amplification reaction temperature control unit for controlling the temperature of the amplification reaction unit is not necessary, and the control unit 118 is also amplified. It is not necessary to provide a function for controlling the temperature of the reaction temperature controller.

  FIG. 11 shows the fluorescence detection unit 64 in detail. The fluorescence detection unit 64 includes a laser diode (LD) or a light emitting diode (LED) 92 that emits a laser beam of 473 nm as an excitation light source, and a pair of the laser beam is focused and irradiated on the bottom surface of the probe placement unit 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. The presence or absence of a SNP corresponding to an invader probe fixed at each probe placement position and whether the SNP is a homozygote or a heterozygote by two types of fluorescence detection by the two photodetectors 104 and 108. Is detected. As the labeling phosphor, for example, FAM, ROX, VIC, TAMRA, Redmond Red, or the like can be used.

  The detector 64 in FIG. 11 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 (single nucleotide polymorphisms) can be detected, and the results are used to diagnose disease morbidity, diagnose the relationship between the type and effect of drugs, and side effects, as well as animal and plant varieties. It can also be used for determination, infectious disease diagnosis (type determination of infecting bacteria), and the like.

1 is a block diagram schematically illustrating the present invention. It is a figure which shows the 1st example of reaction container, (A) is a front view, (B) is a top view. It is a figure which shows the first half part of the process of the SNP detection method using the reaction container, (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, (A) is a front view, (B) is a top view. It is a figure which shows the 2nd example of reaction container, (A) is a front view, (B) is a top view, (C) is sectional drawing in the XX line position of (B). It is a figure which shows the amplification reaction part in the same reaction container as a sectional view in the YY line position of Drawing 5 (B), (A) is in the state where the reaction liquid was poured, and (B) collects the reaction liquid. It is in a state. It is a figure which shows the first half part of the process of the SNP detection method using the reaction container, (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, (A) is a front view, (B) is a top view. It is a schematic perspective view which shows one Example of the reaction container processing apparatus of this invention. It is a schematic perspective view which shows the chip | tip accommodating part and nozzle tip position sensor part in the Example. It is a schematic block diagram which shows the fluorescence detection part 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 10, 10a Substrate 12 Sample injection part 14 Typing reagent storage part 16 Mineral oil storage part 18 Probe placement part 20 Film 22 Sealing material 28 Nozzle 30 Gene amplification reagent storage part 31 PCR End solution injection unit 32 Amplification reaction unit 34a, 34b Amplification reaction unit port 36a, 36b Port opening 41 Reaction vessel 60, 62 Heat block 64 Fluorescence detection unit 66 Liquid feeding arm 70 Chip 110 Typing reaction temperature control unit 112 Dispensing unit 112
116 Nozzle tip position sensor unit 118 Control unit 134, 136, 140 Photo sensor

Claims (7)

A reaction vessel mounting portion for mounting a reaction vessel formed with at least one reaction portion for causing a sample to react on a flat substrate;
A dispensing unit for transferring the liquid in the reaction vessel by moving a nozzle for suction and discharge in the X and Y directions in the plane and the Z direction in the height direction;
A nozzle tip position sensor unit comprising a detector that detects the position of the tip of the nozzle in at least one of the X direction, the Y direction, and the Z direction;
A control unit that controls a nozzle tip position detection operation by the nozzle tip position sensor unit and a dispensing operation of the dispensing unit based on nozzle tip position information detected by the nozzle tip position sensor unit; A reaction vessel treatment device comprising :
The nozzle has a tip detachably attached to the tip, and the nozzle tip position detected by the nozzle tip position sensor unit is the tip position of the tip attached to the nozzle,
The reaction container further includes at least one reagent storage portion formed as a recess in the substrate, containing a reagent used for the reaction of the sample, and sealed with a film that can be penetrated at the tip of the chip. A reagent kit for
When the controller sucks the reagent in the reagent container with the nozzle, the tip penetrates the film and inserts the chip tip into the reagent container, and before dispensing the sucked reagent A reaction vessel processing apparatus for detecting the tip position of the tip . The reaction vessel processing apparatus according to claim 1, wherein the reaction vessel further includes a non-volatile liquid storage portion that is formed as a recess in the substrate and contains a non-volatile liquid having a specific gravity lower than that of the reaction solution and is sealed with a film. . The reaction container includes at least a typing reagent storage unit as the reagent storage unit, and includes a plurality of probe placement units that individually hold fluorescent probes corresponding to each of a plurality of polymorphic sites as the reaction unit. A reaction vessel for genetic polymorphism diagnosis,
The reaction vessel processing apparatus controls the temperature of the probe placement unit as the reaction temperature control unit to control the reaction solution of the genomic DNA of the sample and the typing reagent to a temperature at which the reaction solution of the probe placement unit reacts with the probe of the probe placement unit. And
A fluorescence detection unit for detecting fluorescence by irradiating each probe placement unit with excitation light;
The control unit may reactor apparatus according to claim 1 or 2 for detecting a gene polymorphism by also controls the detection operation of the temperature control of the typing reaction temperature control unit fluorescence detector. Reactor apparatus according to any one of the detectors of the nozzle tip position sensor unit of claims 1, which is a non-contact type detector 3. The reaction container processing apparatus according to any one of claims 1 to 4, wherein the chip is a disposable chip or a disposable chip holding a filter therein. The reaction vessel processing apparatus according to claim 1, wherein the tip is a replaceable probe. Wherein the control unit repeats a plurality of times about the direction of detecting the detection operation of the nozzle tip position by the nozzle tip position sensor unit, according to the average value in any one of claims 1 to 6 that the nozzle tip position information in that direction Reaction vessel processing device.

Images