JP4759299B2 - 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, the Invader (registered trademark) method or the Tuckman (registered trademark) 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 is provided with at least one reaction part for causing a sample to react on a flat substrate. This reaction vessel is used for measuring various reactions including chemical reactions and biochemical reactions.

  There has also been proposed a reaction vessel processing apparatus that detects a reaction in a reaction section using such a reaction vessel. The reaction container processing apparatus includes at least a fluorescence detection unit that detects fluorescence by irradiating the reaction unit with excitation light.

When detecting the reaction in the reaction part, some reactions require a long time for the reaction to proceed until sufficient detection sensitivity is obtained.
For example, when a genetic polymorphism is to be diagnosed, a reaction container for a genetic polymorphism diagnosis in which the reaction part is a plurality of probe placement parts individually holding fluorescent probes corresponding to each of a plurality of polymorphic sites. It is necessary to dispense the reaction solution of the genomic DNA of the sample and the typing reagent into the probe placement portion and maintain the temperature at a predetermined temperature to cause the typing reaction. However, the typing reaction takes time. When a device equipped with an LED (light emitting diode) is used as the fluorescence detection device and the fluorescence is detected by continuously emitting the LED as usual, 20 to 40 until a state where sufficient fluorescence intensity can be obtained is obtained. It takes a minute. As a result, the entire measurement time is long.

Although it is conceivable to increase the fluorescence intensity by increasing the concentration of the fluorescent label in the reaction section, the fluorescent reagent used for the fluorescent label is expensive, which leads to high costs.
An object of the present invention is to make it possible to detect fluorescence from a reaction part of a reaction vessel with high sensitivity.

The reaction vessel processing apparatus of the present invention includes a reaction vessel mounting portion for mounting a reaction vessel having a flat substrate with at least one reaction portion for causing a reaction to occur in a sample. Further, as shown in FIG. At least a fluorescence detection unit 64 for detecting fluorescence by irradiating excitation light, and a control unit 118 for controlling at least the detection operation of the fluorescence detection unit 64. The fluorescence detection unit 64 uses a light emitting element as an excitation light source. It is characterized in that the fluorescence is detected while the light emitting element blinks and lights up so that the light emission intensity is higher than the continuous light emission.
As the light emitting element, it is appropriate to use an LED or an LD (laser diode).

  The reaction vessel may be further provided with a non-volatile liquid storage portion that is formed as a recess in 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. In addition, the reaction container may further include at least one reagent storage unit that is formed as a recess in the same substrate, stores a reagent used for sample reaction, and is sealed with a film. Both the nonvolatile liquid container and the reagent container can be provided on the same substrate. In the case where a non-volatile liquid storage unit and a reagent storage unit are provided, the reaction vessel processing apparatus includes a mechanism for suction and discharge by a nozzle 28 to transfer and separate liquid as shown in FIG. A dispensing unit 112 for dispensing is provided, and the control unit 118 also controls the dispensing operation of the dispensing unit 112.

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 the reaction part has a plurality of polymorphic sites. A reaction vessel for diagnosing a gene polymorphism having a plurality of probe arrangement portions individually holding fluorescent probes corresponding to each of the above. In this case, 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. A temperature control unit 110 is further provided, and the control unit 118 also performs temperature control of the typing reaction temperature control unit 110.
When an Invader (registered trademark) reaction is used as a typing reaction, the typing reaction temperature control unit 110 is a temperature control unit for the Invader (registered trademark) reaction.

  When a gene amplification reaction is also performed in the reaction vessel, the reaction vessel contains a gene amplification reagent containing portion containing a gene amplification reagent containing a plurality of primers that bind to each of a plurality of polymorphic sites, and a gene This is a genetic polymorphism diagnostic reaction vessel further comprising an amplification reaction section for performing a gene amplification reaction on the mixture of the amplification reagent and the sample. In this case, as shown in FIG. 1, this reaction vessel processing apparatus controls the temperature of the amplification reaction section to a temperature for gene amplification that amplifies DNA in the reaction solution of the sample and the gene amplification reagent. A temperature control unit 120 is further provided, and the control unit 118 also performs temperature control 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.

  The excitation light irradiation part of the optical unit is preferably provided with light emitting elements having different emission wavelengths as the excitation light source. As the light emitting element, LD (laser diode) or LED (light emitting diode) can be used, and LED is preferable in terms of cost. A high-brightness LED is particularly preferable.

When a light-emitting element is used as the excitation light source, the light-emitting elements emit light at different timings, and fluorescence detection from each optical unit can be distinguished by performing fluorescence detection with a photodetector at the light emission timing.
When detecting two types of fluorescence, two optical units are provided, the light emitting elements of each optical unit emit light alternately, and the fluorescence received by each optical unit is emitted while the light emitting element of the optical unit emits light. Can be detected.

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 (registered trademark) method and the Tuckman (registered trademark) PCR method can be used for the typing process. In that case, the typing reagent is an Invader (registered trademark) reagent or a Tuckman (registered trademark) PCR reagent.

FIG. 13 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 step and the Invader (registered trademark) method is used for the typing step.
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 completion of the PCR reaction, Invader (registered trademark) reagent 6 is added as a typing reagent. The Invader (Registered Trademark) 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 (registered trademark) reagent 6 is added is added to the plurality of probe placement units 8 to cause the reaction. Each probe placement unit 8 individually holds an Invader (registered trademark) probe and a reporter probe corresponding to each of the plurality of SNP sites. The reaction solution reacts with the Invader (registered trademark) probe, and the reporter. If a SNP corresponding to the probe exists, it emits fluorescence.

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

The Invader (registered trademark) method used in the typing step is a method of typing an SNP site by hybridizing an allele-specific oligo and a DNA containing the SNP to be typed, and a DNA containing the SNP to be typed, Two types of reporter probes and one type of Invader (registered trademark) specific to each allele of the SNP to be typed and an enzyme having a special endonuclease activity for recognizing and cleaving the DNA structure are used. It is a method (refer patent document 3).

In the reaction container processing apparatus according to the present invention, the fluorescence detection unit includes a light emitting element as an excitation light source, and the light emission element detects the fluorescence while blinking and lighting so that the emission intensity is higher than continuous light emission. Therefore, it is possible to perform a measurement of a reaction that has to wait for a long time until a sufficient fluorescence intensity is obtained.
Further, even if an expensive element having a large light emission intensity is not used as the light emitting element, the light emission intensity is increased by blinking and lighting instead of continuous light emission, so that the reaction vessel processing apparatus can be realized in a small size and at a low cost. .

Since it is not necessary to use many expensive fluorescent reagents, the reaction vessel can be provided at low cost when the reaction part of the reaction vessel is fluorescently labeled.
Since a light emitting element is used as an excitation light source, it can contribute to size reduction of the reaction vessel processing apparatus, and if an LED is used as the light emitting element, it can contribute to further cost reduction.

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.
On the same side of the flat substrate 10, a reagent container 14 and a nonvolatile liquid container 16 containing a nonvolatile liquid having a specific gravity lower than that of the reaction liquid are formed as recesses.

  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.

In the embodiment, 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 portion 18 is also formed on the same side of the substrate 10.
The reagent storage unit 14 and the mineral oil storage unit 16 are sealed with a film 20, and when the reagent and mineral oil are sucked by the nozzle and transferred to another place, the film 20 is removed and the nozzle is sucked by the nozzle. Alternatively, the film 20 can be penetrated by a nozzle, and the nozzle is penetrated and sucked by the nozzle. 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 substrate 10 is covered 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 from the film 20.

An example of a specific use of this reaction vessel is a polymorphism diagnostic reagent kit for injecting a sample reaction solution obtained by amplifying DNA by PCR reaction and detecting SNP by Invader (registered trademark) reaction. is there. 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 or a laminated film of aluminum and a resin such as a PET film. However, the sticking strength is weaker than that of the film 20 and is attached 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 by the nozzle 28 to each probe placement unit 18 by 0.5 to 4 μL. Mineral oil is dispensed into each probe placement portion 18 by 0.5 to 10 μL from the mineral oil storage portion 16 by the 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 DNA amplification by PCR reaction and SNP detection by Invader (registered trademark) reaction are both performed. However, a biological sample subjected to nucleic acid extraction operation 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 by 0.5 to 4 μL. Mineral oil is dispensed into each probe placement portion 18 by 0.5 to 10 μL from the mineral oil storage portion 16 by the 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.

Invader (registered trademark) reagent is used as a typing reagent. As the Invader (registered trademark) reagent, an Invader (registered trademark) 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.
A pair of heat blocks 60 and 62 are arranged vertically as heaters in the apparatus. The reaction vessel mounting portion is configured by forming a guide portion on the lower heat block 60 where the reaction vessel 41 shown in FIG. 2 or 5 slides and is positioned at a predetermined position. In the reaction vessel mounting portion, five pieces of samples injected into the reaction vessel 41 are arranged in parallel. These heat blocks 60 and 62 can move in the Y direction indicated by arrows.

  The lower heat block 60 constitutes an amplification reaction temperature control unit that controls the temperature of the gene amplification reaction unit 32 to be a predetermined temperature cycle. In addition, a typing reaction temperature control unit that controls the temperature of the probe placement unit 18 to a temperature at which the DNA and the probe are reacted by both the heat blocks 60 and 62 is provided. 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.

  As shown in FIG. 10, the upper heat block 62 constituting the typing reaction temperature control unit has an opening 150 only at a position corresponding to the probe placement unit, and the lower heat block 60 constitutes the typing reaction temperature control unit. 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 can be dispensed to the probe placement portion of the reaction vessel 41 by the nozzle 28 through the openings 150 and 156.

  Returning to FIG. 9, in order to transfer, suck, and discharge 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. The arm 66 includes a nozzle 28. The dispensing part is indicated by reference numeral 112 in FIG.

  FIG. 11 shows the tip of the nozzle 28. A disposable tip 70 is detachably attached to the tip of the nozzle 28. (A) shows a state where the tip 70 is not attached to the nozzle 28, and (B) shows a state where the tip 70 is attached to the nozzle 28. The nozzle 28 sucks and discharges the liquid into the chip 70. Therefore, the nozzle 28 has a mounting portion 28a having an outer dimension for mounting the tip 70 at the tip thereof and a length at the tip of the mounting portion 28a that is not in contact with the liquid to be dispensed and has a larger outer dimension than the mounting portion 28a. And a small protrusion 28b.

  As shown in FIG. 10, a fluorescence detection unit 64 that performs fluorescence detection is disposed below the heat block 60, and the fluorescence detection unit 64 passes through the opening 152 of the heat block 60 from the lower surface side of the reaction vessel 41. The probe placement portion is irradiated with excitation light, and fluorescence from the probe placement portion is detected through the opening 152 of the heat block 60 on the lower surface side of the reaction vessel 41. The fluorescence detection unit 64 detects the fluorescence from the probe placement unit 18 by moving in the arrow X direction 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. 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 distribution arm 66 of the dispensing unit 112. Controls note behavior.
When using a reaction vessel 41 that does not include a gene amplification reaction unit, such as the reaction vessel of FIG. 2, an amplification reaction temperature control unit that controls the temperature of the gene amplification reaction unit is not necessary, and the control unit 118 However, it is not necessary to provide a function for controlling the temperature of the amplification reaction temperature controller.

FIG. 12 shows the fluorescence detection unit 64 in detail. Illustration of the heat blocks 60 and 62 and the cover 154 is omitted. In order to detect the presence or absence of an SNP corresponding to an Invader (registered trademark) probe, the probe placement unit 18 holds a reporter probe corresponding to the base of the SNP, and the reporter probe is labeled with a phosphor .
Each probe placement unit 18 is dispensed with a reaction solution 170 for reacting with an Invader (registered trademark) probe, and the surface of the reaction solution 170 is covered with mineral oil 40 to prevent the reaction solution from evaporating during the reaction. ing.

  The fluorescence detection unit 64 constitutes an optical unit including an excitation light irradiation unit and a fluorescence light reception unit. In order to detect fluorescence from the labeled phosphor, an LED 92 is provided as an excitation light source, and a pair of lenses 94 and 96 that irradiate the emitted light of the LED 92 as excitation light on the bottom surface of the probe placement portion 18 of the reaction vessel 41. And a dichroic mirror 98. Moreover, the filter 94 for selecting light of 488 nm among light emission of LED92 as excitation light is also provided. The lens 94 condenses the excitation light from the LED 92 into parallel light, and the lens 96 is an objective lens that irradiates the excitation light collimated and reflected by the dichroic mirror 98 on the bottom surface of the reaction vessel 41. It is.

The objective lens 96 also functions as a lens that collects the fluorescence from the labeled phosphor generated from the reaction vessel 41. The dichroic mirror 98 has a wavelength characteristic that reflects excitation light of 488 nm and transmits fluorescence of 525 nm. On the optical path of the transmitted light (fluorescence) of the dichroic mirror 98, a filter 99 that removes the excitation light component and transmits the fluorescent component is disposed. A condenser lens 102 is disposed on the optical path that has passed through the filter 99, and the fluorescence condensed by the condenser lens 102 is guided to the photodetector 104. As the photodetector 104, a photomultiplier tube, a photodiode, or the like is used.
The LED 92, the lens 94, the filter 95, the dichroic mirror 98, and the lens 96 constitute an excitation light irradiation unit, and the lens 96, the dichroic mirror 98, the filter 99, and the lens 102 constitute a fluorescence light receiving unit.

  The fluorescence detection operation of the fluorescence detection unit 64 is controlled by the control unit 118. The LED 92 is controlled to blink. During the operation of the fluorescence detection unit 64, the optical unit is scanned relative to the reaction container 41 with the LED 92 blinking and lit. When the optical axis comes under the probe placement portion 18 of the reaction vessel 41, the detection signal of the photodetector 104 is sampled and held.

Here, a case where one type of fluorescence is detected is shown, but two types of fluorescence detection by two optical units having different fluorescence wavelengths to be detected are performed on an Invader (registered trademark) probe fixed at each probe arrangement position. The presence or absence of the corresponding SNP and whether the SNP is a homozygote or a heterozygote can be detected. Furthermore, when it is necessary to detect three or more types of fluorescence, it is possible to detect three or more types of fluorescence in the same manner.
As the labeling phosphor, FAM, Redmond Red, ROX, VIC, TAMRA, or the like can 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 gene amplification reaction part in the same reaction container as a sectional view in the YY line position of FIG. 5 (B), (A) is the state in which the reaction liquid was inject | poured, (B) collects the reaction liquid. It is in a state to do. 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 sectional drawing which shows the typing reaction temperature control part in the Example. It is the front view showing the nozzle front-end | tip part in the Example, (A) represents the state in which the chip | tip is not mounted to the nozzle, and (B) represents the state in which the chip | tip was mounted to the nozzle. 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 (registered trademark) 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 28a Chip mounting part 28c Protrusion part 30 Gene amplification reagent storage part 31 PCR completion liquid injection part 32 Gene amplification reaction part 41 Reaction vessel 60, 62 Heat block 64 Fluorescence detection part 66 Liquid supply arm 70 Chip 92 LED
94, 96, 102 Lens 104 Photodetector 110 Typing reaction temperature control unit 112 Dispensing unit 116 Chip position sensor unit 118 Control unit

Claims (3)

At least one reaction part formed on a flat substrate for causing the sample to react, and at least one reagent housed in the substrate as a recess and used for the sample reaction and sealed with a film And a plurality of probe placement portions each holding a probe that emits fluorescence corresponding to each of a plurality of polymorphic sites, including at least a typing reagent storage portion as the reagent storage portion. A reaction vessel mounting part for mounting a reaction vessel for genetic polymorphism diagnosis ;
A dispensing unit that includes a mechanism for suction and discharge by a nozzle and transfers and dispenses the liquid;
A heat block having an opening only at a position corresponding to the probe placement portion is provided, and the temperature of the probe placement portion is reacted with the probe of the probe placement portion with the reaction solution of the genomic DNA of the sample and the typing reagent by the heat block. A typing reaction temperature controller that controls the temperature to be
Irradiating the probe placement unit with excitation light through the opening of the heat block, and detecting fluorescence from the probe placement unit through the opening of the heat block ;
The detection operation of the fluorescence detection unit, the dispensing part of the dispensing operation, and comprises at least a control unit for controlling at least the temperature control of the typing reaction temperature control unit,
The fluorescence detection unit includes a light emitting element composed of an LED or an LD as an excitation light source, and detects the fluorescence while the light emitting element is blinking and lit so as to increase the light emission intensity than continuous light emission. Processing equipment. The reaction vessel is formed as a recess in the substrate, the reaction of claim 1 wherein the reaction mixture in which further comprises a non-volatile liquid containing portion sealed with a film containing a low volatile liquid specific gravity than Container processing equipment. A gene amplification reagent containing a gene amplification reagent containing a plurality of primers that bind to each of a plurality of polymorphic sites and a mixture of the gene amplification reagent and the sample; A reaction vessel for diagnosis of genetic polymorphism further comprising an amplification reaction part for performing
The reaction vessel processing apparatus further includes an amplification reaction temperature control unit that controls the temperature of the amplification reaction unit to a temperature for gene amplification that amplifies DNA in the reaction solution of the sample and the gene amplification reagent,
The reaction vessel processing apparatus according to claim 1 , wherein the control unit also performs temperature control of the amplification reaction temperature control unit.

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