Patent Application
20080220420
The present invention relates to a method, an apparatus and a reagent for detecting genome DNA polymorphism for plants and animals including humans, particularly SNP (single-nucleotide polymorphism), and a method and apparatus for diagnosing disease morbidity, the relationship between the type and effect or side effect of a drug administered, etc. by using the result of the above detection.
The method and apparatus for detecting gene polymorphism can be utilized in the study of gene analysis and in the clinical field.
A method and apparatus for estimating susceptibility to diseases, etc., by using gene polymorphism have been proposed as follows:
For determining whether a patient is susceptible to sepsis and/or rapidly develops sepsis, a nucleic acid sample is collected from the patient, a pattern 2 allelic gene or a marker gene which is in linkage disequilibrium with a pattern 2 allelic gene in the sample is detected, and if a pattern 2 allelic gene or a marker gene in linkage disequilibrium with a pattern 2 allelic gene is detected, the patient is judged to be susceptible to sepsis (see Patent Literature 1).
For diagnosis of one or more single-nucleotide polymorphisms in the human flt-1 gene, a sequence of one or more positions in human nucleic acid, that is, positions 1953, 3453, 3888 (which are respectively in accordance with numbering in EMBL Accession No. X51602), 519, 786, 1422, 1429 (which are respectively in accordance with numbering in EMBL Accession No. D64016), 454 (in accordance with Sequence No. 3) and 696 (in accordance with Sequence No.: 5) is determined, and by referring to the polymorphism in fl1-1 gene, the constitution of the human is determined (JP-A 2001-299366).
Many methods have been reported on typing, that is, discrimination of bases in SNP sites. A typical example of these methods is as follows:
For carrying out typing several hundred thousand SNP sites with a relatively small amount of genome DNA, a plurality of base sequences containing at least one single-nucleotide polymorphism are amplified simultaneously with a genome DNA and pairs of primer, and a plurality of base sequences thus amplified are used to discriminate bases in single-nucleotide polymorphic sites contained in the base sequences by a typing step. For the typing step, an invader method or TaqMan PCR is used (see Patent Literature 3).
For typing of SNPs, however, preparation of genome DNA is essential at a stage leading to the amplification step, which requires much time and high costs.
When attention is focused on PCR for amplifying DNA, a method wherein a sample such as blood is subjected to PCR directly without pre-treating the sample has also been proposed. In this nucleic acid synthesis method of amplifying a desired gene in a gene-containing sample, a gene inclusion body in the gene-containing sample, or the gene-containing sample itself, is added to a gene amplification reaction solution to amplify the desired gene in the gene-containing sample in the reaction solution at pH 8.5 to 9.5 (25° C.) after addition (see Patent Literature 4).
Non-Patent Literature 1: Hsu T. M., Law S. M, Duan S, Neri B. P., Kwok P. Y., “Genotyping single-nucleotide polymorphisms by the invader assay with dual-color fluorescence polarization detection”, Clin. Chem., 2001 August; 47(8):1373-7
In the previously constructed typing system, the amount of DNA first collected may be small because a plurality of SNP areas to be subjected to typing are amplified by PCR, but the pretreatment, that is, the extraction of DNA from a biological sample is required prior to amplification with PCR. The pretreatment is time-consuming and troublesome.
On one hand, a method of direct PCR amplification of a biological sample such as blood without extracting nucleic acid from the sample has also been previously established, but when the direct PCR method is combined with the typing method, an automatic system wherein a plurality of SNP sites as the object of typing are simultaneously amplified has still not been established. The object of the present invention is to enable typing for a plurality of objective SNP sites automatically from the stage of sample preparation.
The method of detecting gene polymorphism according to the present invention comprises an amplification step of amplifying genome DNA by allowing a biological sample not subjected to a nucleic acid extraction procedure to act directly on a gene amplification reaction solution containing a plurality of primers binding to a plurality of polymorphic sites by sandwiching each site between primers and a typing step of discriminating bases in a plurality of the polymorphic sites by allowing a typing reagent prepared so as to correspond to a plurality of the polymorphic sites to act on the genome DNA amplified in the amplification step.
The relationship between the polymorphic sites and primers is as follows: For amplifying one polymorphic site, a pair of primers binding to the polymorphic site by sandwiching it between primers is necessary. A plurality of kinds of polymorphic sites occur in a target biological sample, and when polymorphic sites occur in positions separated from one another, twice as many kinds of primers as kinds of polymorphic sites are necessary. However, when two polymorphic sites are close to each other, amplification thereof can be effected by binding the primers to each of the polymorphic sites by sandwiching each site between primers or by binding the primers to both sides of a sequence of the two polymorphic sites with no primer between the polymorphic sites. Accordingly, the types of necessary primers are not always twice as many as kinds of polymorphic sites. In the present invention, “a plurality of primers each binding to polymorphic sites by sandwiching each site between primers” is intended to refer to types of primers necessary for amplifying a plurality of polymorphic sites not only in the case where a pair of primers bind to one polymorphic site by sandwiching it between primers but also in the case where a pair of primers bind to two or more polymorphic sites by sandwiching a series of such polymorphic sites between primers.
The polymorphism includes mutation, deletion, overlap, transfer etc. A typical example is SNP.
The nucleic acid extraction procedure as used herein refers to a series of procedures ranging from decomposition of nucleic acid inclusion bodies (membrane structures containing nucleic acid therein, such as cells, microorganisms, fungi, viruses etc.) to extraction of a nucleic acid from the decomposed nucleic acid inclusion bodies. The decomposition of nucleic acid inclusion body is carried out by using, for example an enzyme, a surfactant, a chaotropic agent etc. Extraction of nucleic acid from the decomposed nucleic acid inclusion body is carried out by using, for example, phenol or phenol/chloroform etc.
Accordingly, the biological sample not subjected to the nucleic acid extraction procedure is a sample not subjected to a series of such procedures and includes a nucleic-acid-body-containing-biological sample, a biological sample of nucleic acid inclusion body in a state decomposed by heat treatment or freezing treatment, and a nucleic acid inclusion body recovered from a living sample. The method of recovering a nucleic acid inclusion body from a living sample includes, for example, methods using centrifugation or ultracentrifugation or with a coprecipitating agent such as ethylene glycol etc. or with an adsorbent carrier, etc.
As used herein, the biological sample refers to an animal or plant tissue, a body fluid, excrements, etc., and the body fluid includes blood and saliva. The genome DNA includes DNA such as human, animal or plant DNA, bacterial or viral DNA, and cDNA synthesized with RNA as a template.
In the amplification step, PCR or the like can be used. In this case, PCR is carried out preferably under the pH condition of 8.5 to 9.5 at 25° C.
The step of amplifying genome DNA from a biological sample not subjected to the nucleic acid extraction procedure described above is described in detail in Patent Literatures 4 and 5.
In the typing step, the invader method or TaqMan PCR can be used.
In the diagnostic method of the present invention, diagnostic values with respect to a specific polymorphism or a combination of polymorphisms are prepared as a database, and based on the result of polymorphism detected by the method of detecting gene polymorphism according to the present invention, diagnostic values are read out from the database. The diagnostic values can include disease morbidity, the relationship between the type and effect or side effect of a drug administered, etc.
One aspect of the apparatus for detecting gene polymorphism according to the present invention is constituted to detect gene polymorphism automatically by having a sample arrangement part for arranging a biological sample not subjected to a nucleic acid extraction procedure, an amplification reagent holding part for holding a gene amplification reaction solution containing a plurality of primers binding to a plurality of polymorphic sites by sandwiching each site between primers, a typing reagent holding part which holds a typing reagent prepared so as to correspond to a plurality of the polymorphic sites, an amplification part to control the temperature of a reaction solution having the biological sample added to the gene amplification reaction solution to amplify genome DNA in the reaction solution, a typing reaction part which has a probe fixing part for holding a probe emitting fluorescence corresponding to each of a plurality of the polymorphic sites and which controls the temperature of a reaction solution of the typing reagent and genome DNA amplified in the amplification part in order to react the reaction solution with each probe, a dispensing device which can transfer to the sample arrangement part, the amplification reagent holding part, the typing reagent holding part, the amplification part and the typing reaction part and which dispenses a sample, an amplification reagent, a typing reagent, and a reaction solution of the sample and these reagents to predetermined positions, a fluorescence detector for detecting fluorescence upon irradiation of each probe fixing part in the typing reaction part with an exciting light, and a control part for controlling the temperature of the amplification part and the typing reaction part, the dispensing operation of the dispensing device and the detection operation of the fluorescence detector.
Each probe fixing part can hold not only one kind of probe but also two or more kinds of probes. When two or more kinds of probes are held on one probe fixing part, the respective probes are arranged to be separated from one another so that two or more kinds of fluorescence generated from the respective probes can be distinguishably detected.
In one example of the typing reaction part, each of the probe fixing parts is provided with a concave part, the upper part of which is opened to feed a reaction solution. In this case, the apparatus for detecting gene polymorphism further has an oil holding part to hold oil for the prevention of evaporation of the reaction solution, and the dispensing device is preferably the one capable of dispensing the oil to the concave portion before or after dispensing the reaction solution to the concave portion.
In another example of the typing reaction part, a flow path through which a reaction solution is fed is arranged in each of the probe fixing parts. The flow path may be provided in each of the probe fixing parts with an inlet for feeding the reaction solution and an outlet for discharge and may be connected to a common inlet for feeding the reaction solution and a common outlet for discharge. In this case, the probe fixing part may be formed as a concave portion in the flow path.
An additional example of the typing reaction part is provided with a flow part having a plurality of the probe fixing parts formed therein.
The sample arrangement part and the amplification part may share a temperature regulation part.
The examination reagent kit of the present invention is a kit wherein an amplification reagent holding part in which a gene amplification reaction solution containing a plurality of primers binding to a plurality of polymorphic sites by sandwiching each site between primers has been held, a typing reagent holding part in which a typing reagent prepared so as to correspond to a plurality of the polymorphic sites has been held, and a plurality of probe fixing parts in which probes emitting fluorescence corresponding to each of a plurality of the polymorphic sites have been separately held, are integrally formed.
In the examination reagent kit, a diluent holding part for holding a diluent diluting a sample may further be integrally formed.
Another aspect of the apparatus for detecting gene polymorphism according to the present invention uses the examination reagent kit of the present invention, and comprises an examination reagent kit-mounting part for mounting the examination reagent kit, an amplification part for controlling the temperature of a reaction solution of the gene amplification reaction solution and a body fluid sample for amplifying genome DNA in the reaction solution in the amplification reagent holding part, a typing reaction part for controlling the temperature of a reaction solution of the typing reagent and the genome DNA amplified in the amplification part in order to react the reaction solution with the probe in the probe fixing part, a liquid transferring device for transferring liquid from the amplification reagent holding part to the typing reagent holding part and for transferring liquid from the typing reagent holding part to the probe fixing part, a fluorescence detector for detecting fluorescence upon irradiation of each of the probe fixing parts with an exciting light, and a control part for controlling the temperature of the amplification part and the typing reaction part, the liquid transferring operation of the liquid transferring device, and the detection operation of the fluorescence detector.
By way of example, the liquid transferring device is a dispensing device equipped with a dispensing nozzle and arranged so as to be movable to a necessary place.
Still another aspect of the apparatus for detecting gene polymorphism according to the present invention uses the examination reagent kit of the present invention wherein each of the holding parts is made of a soft material, and the liquid transferring device is a pressing device for pressing to deform each of the holding parts, thereby transferring the liquid.
The diagnostic device of the present invention comprises the apparatus for detecting gene polymorphism according to the present invention, a database in which diagnostic values such as disease morbidity, the relationship between the type and effect or side effect of a drug administered, etc., with respect to a specific SNP or a combination of SNPs are memorized, and a display unit for displaying diagnostic values by reading out from the database on the basis of the result of SNPs detected by the apparatus for detecting gene polymorphism.
In the PCR step, a PCR reaction solution 4 is added to a biological sample 2 such as blood, or alternatively, the biological sample 2 is added to the PCR reaction solution 4. For example, 1 μL of the sample 2 is collected, and about 10 μL of the PCR reaction solution 4 is added thereto. The PCR reaction solution 4 has been previously prepared, and contains a plurality of primers for SNP sites to be measured, a buffer solution for adjusting pH, 4 kinds of deoxyribonucleotides and other necessary reagents, and is prepared to exhibit pH 8.5 to 9.5 when mixed with the sample 2.
A mixed solution of sample 2 and PCR reaction solution 4 is subjected to PCR according to a predetermined temperature cycle. The PCR temperature cycle includes 3 steps, that is, denaturation, primer adhesion (annealing) and primer extension, and this cycle is repeated whereby DNA is amplified. In one example of the steps, the denaturation step is carried out at 94° C. for 1 minute, the primer adhesion step at 55° C. for 1 minute, and the primer extension at 72° C. for 1 minute. The sample is not subjected to genome extraction procedure, and at high temperatures of PCR temperature cycles, DNA is released from hemocytes and cells, and the reagents necessary for the PCR reaction are brought into contact with DNA, whereby the reaction proceeds.
After the PCR reaction is finished, an invader reagent 6 is added. A fluorescence-emitting FRET probe and cleavase (structure-specific DNA degradative enzyme) are contained in the invader reagent 6. The FRET probe is a fluorescent-labeled oligo having a sequence completely irrelevant to the genome DNA, and, irrespective of the type of SNP, its sequence is common.
Next, the reaction solution to which the invader reagent 6 has been added is reacted by addition to the probe fixing part 8 in the typing reaction part. At each site of the probe fixing parts 8, an invader probe and a reporter probe are individually held correspondingly to each of a plurality of SNP sites, and the reaction solution reacts with the invader probe to emit fluorescence if SNP corresponding to the reporter probe is present.
The invader method is described in detail in paragraphs [0032] to [0034] in Patent Literature 3.
Two reporter probes have been prepared depending on each base of SNP and can judge whether the SNP is a homozygote or heterozygote.
In the PCR method in the amplification step used in the present invention, a plurality of objective SNP sites are simultaneously amplified, while a plurality of genome DNAs containing the SNP sites are amplified by PCR directly from a biological sample not subjected to nucleic acid procedure. Accordingly, a gene amplification reaction solution containing a plurality of primers for the SNP sites is allowed to act directly on a biological sample and subjected to PCR under the condition of pH 8.5 to 9.5 at 25° C.
The PCR reaction solution contains a pH buffer solution, salts such as MgCl2, KCl etc., primers, deoxyribonucleotides and a thermostable synthase. In addition, substances such as a surfactant and protein can be added as necessary.
The pH buffer solution can use not only a combination of tris(hydroxymethyl) aminomethane and a mineral acid such as hydrochloric acid, nitric acid, sulfuric acid or the like, but also various pH buffer solutions. The buffer solution with adjusted pH is used preferably at a concentration of 10 mM to 100 mM in the PCR reaction solution.
The primer refers to an oligonucleotide acting as the starting point for DNA synthesis by the PCR reaction. The primers may be synthesized or isolated from biological sources.
The synthase is an enzyme for synthesis of DNA by primer addition and includes chemically synthesized synthases. Suitable synthase includes, but is not limited to, E. coli DNA polymerase I, E. coli DNA polymerase Klenow fragment, T4 DNA polymerase, Taq DNA polymerase, T. litoralis DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, Hot Start Taq polymerase, KOD DNA polymerase, EX Taq DNA polymerase, and a reverse transcriptase. The term “thermostable” refers to the property of a compound which maintains its activity even at high temperatures, preferably at 65 to 95° C.
The invader method used in the typing step is a method of typing SNP site by hybridizing an allele-specific oligo with DNA containing SNP as an object of typing, wherein DNA containing SNP as an object of typing, two kinds of reporter probes specific to the each allele of SNP as an object of typing, one kind of invader probe, and an enzyme having a special endonuclease activity by which a structure of DNA is recognized and cleaved are used (see Patent Literature 3).
In the method of detecting gene polymorphism according to the present invention, a plurality of objective polymorphic sites are simultaneously amplified from a biological sample not subjected to nucleic acid extraction procedure, thereby typing these polymorphic sites simultaneously, thus enabling the typing of polymorphism to be achieved in a short time in the simple process. The diagnostic method of the present invention can be used in the medical field because diagnostic values are read out from a database on the basis of the typing of polymorphisms.
In the apparatus for detecting gene polymorphism in the first aspect of the invention, the typing of a plurality of objective polymorphisms can be automatically carried out by merely initiating measurement after a biological sample not subjected to nucleic acid extraction procedure is arranged in the sample arrangement part.
In the apparatus for detecting gene polymorphism in the second and third aspects of the invention, the typing of a plurality of objective polymorphisms can be automatically carried out in the simple measurement apparatus by using the examination reagent kit having a gene amplification reaction solution, a typing reagent and a diluent accommodated therein, wherein probe fixing parts are integrally formed.
In the diagnostic apparatus of the present invention, processes ranging from the typing of polymorphisms to the indication of diagnostic values based on the typing can automatically be carried out.
The numeral 10 denotes a sample table serving as a sample arrangement part and a reagent holding part Blood collection tubes 12 are arranged as sample containers in the sample arrangement part. The sample table is loaded with blood collection tubes 12 with various sizes, for example those of 13 mm in diameter, 16 mm in diameter, etc., further loaded with a universal adaptor which can also be loaded with sample cups and is thus capable coping with various sample containers. Blood is collected in the blood collection tube 12 as a body fluid sample not subjected to genome extraction procedure and is mounted on the sample table 10.
The sample holding part in the sample table 10 is loaded with the PCR reaction solution 14 as an amplification reagent and the invader reaction reagent 15 as a typing reagent.
Hereinafter, the present invention is described in detail by reference to the composition of each reaction solution, but the technical scope of the present invention is not limited to the following examples.
PCR is carried out by using 24 μl of the PCR reaction solution for 1 μl of fresh human blood.
50 mmol each of 40 kinds of primers (20 pairs), 10 U EX-Taq DNA polymerase (manufactured by Takara Shuzo Co., Ltd.), 0.55 μg TaqStart (manufactured by CLONTECH Laboratories), and AmpDirect (manufactured by Shimadzu Corporation) are mixed in this PCR reaction solution. As the primers, for example, SNP ID Nos.:1 to 20 and Sequence Nos.:1 to 40 shown in Table 1 in Patent Literature 3 can be used.
As the invader reagent, an invader assay kit (manufactured by Third Wave Technology) is used. That is, a buffer, a FRET probe, a cleavase and distilled water were prepared by mixing them at the ratio of 3:3:3:50.
The numeral 20 denotes a reaction table, and a PCR area 22 is in the inside of the reaction table 20, and amplification reaction containers 24 are arranged. The PCR area 22 is provided with a temperature control part by which the temperature of the reaction solution becomes a temperature established for PCR amplification reaction. The amplification reaction containers 24 are disposable containers made of resin and are formed to have such a thin wall as to improve heat exchange. The temperature of the PCR area 22 is changed at 3 stages, for example at 94° C., 63° C. and 72° C., and established to repeat this cycle.
On the reaction table 20, the invader reaction area 28 is arranged for a typing reaction in a fashion concentric with the PCR area 22, at the side of the outer circumference of the PCR area 22. Typing reaction containers 30 are arranged in the invader reaction area 28, and minute wells 42 which are as many as or several times as many as SNPs to be detected are formed in the typing reaction containers 30. The capacity of well 42 is for example several 10 nL to several μL. The invader reaction area 28 is provided with a temperature control part independent of that in the PCR area 22, so as to make the invader reaction area 28 different in temperature from the PCR area 22. The temperature of the invader reaction area 28 is set for example at 63° C.
A sectional view of the typing reaction containers 30 is shown in
As a specific example of the reporter probe and invader probe, it is possible to employ, for example, primary probes 1, 2 and invader probe described in Table 1 in Non-Patent Literature 1.
The invader probe and reporter probe have been fixed to each well in an air-dried state.
In order to measure fluorescence from the typing reaction containers 30 in the bottom side of wells 42, the typing reaction containers 30 are made of a light-permeable resin with a low-spontaneous-fluorescent property (that is, a property of generating little fluorescence from it), for example a material such as polycarbonate.
By reference to
A dispensing probe 32 having a nozzle 34 is arranged between the sample table 10 and the reaction table 20. The nozzle 34 is transferred between the sample table 10 and the reaction table 20, and carries out the operation of suctioning a sample from the blood collection tube 12 arranged in the sample table 10 and dispensing it into the amplification reaction container 24 in the PCR area in the reaction table 20, the operation of dispensing the PCR reaction solution 14 arranged in the sample table 10 into the amplification reaction container 24, the operation of dispensing the invader reaction reagent 15 arranged in the sample table 10 into the amplification reaction container 24, and the operation of dispensing the reaction solution in the amplification reaction container 24 into well 42 in the typing reaction container 30. For manipulating the dispensing operation with the nozzle 34, a syringe pump 38 and washing water 40 are connected via a switching valve 36 to the nozzle 34. The washing water 40 is used in dispensing a fluid and in washing the nozzle 34.
To prevent the reaction solutions in the amplification reaction containers 24 or wells 42 from being dried during measurement of fluorescence, a mineral oil container 17 is also arranged in the sample table 10, and the mineral oil therein is dispensed via the nozzle 34 into the wells 42, and as shown in number 45, the surfaces of the reaction solutions are covered with the oil, whereby the reaction solutions can be prevented from being evaporated.
The movement in this example is described.
The dispensing probe 32 dispenses a sample in the blood collection tube 12, for example in an amount of 1 to several μL, into the amplification reaction container 24 in the PCR area, and then the dispensing probe 32 dispenses the PCR reaction solution 14, for example in an amount of 5 to 10 μL into the amplification reaction container 24 having the sample dispensed therein. Alternatively, the PCR reaction solution 14 may be first dispensed, and the sample then dispensed. In the amplification reaction container into which the sample and the PCR reaction solution were dispensed, PCR is carried out by repeating a predetermined temperature cycle for 1 to 1.5 hours for example. The sample and the reaction solution are also dispensed in series into other amplification reaction containers 24 in the PCR area, to repeat PCR.
In the amplification reaction container 24 in which PCR was finished, the invader reaction reagent 15 is added via the dispensing probe 32 to, and mixed in, the amplification reaction container 24. The mixture is dispensed by the dispensing probe 32 into a plurality of wells 42 in the typing reaction container 30 in the invader reaction area 28, and the invader reaction is carried out for several minutes to several hours. During the reaction or after conclusion of the reaction, fluorescence is measured by the fluorescence detector 50. After the reaction solution is dispensed into the typing reaction container 30, mineral oil may be dispensed onto the reaction solution in well 42 to prevent evaporation of the reaction solution.
In the apparatus for detecting gene polymorphism in this example, the amplification reaction container 24 and the typing reaction container 30 are transferred via the container transfer arm 72 to predetermined positions respectively. The amplification reaction containers 24 are transferred to, and held in, both the PCR area 22 and preheat area 22a respectively. First, the sample in the blood collection tube 24 is dispensed into the amplification reaction container 24 in the preheat area 22a and preheated at 94° C. At the beginning of PCR, the amplification reaction container in the preheat area 22a is transferred by the container transfer arm 72 to the PCR area 22.
Similarly to the movement shown in the examples in
In this example, the amplification reaction container 24 and the typing reaction container 30, in which the reaction has been finished, are transferred by the container transfer arm 72 to a disposal part and discarded, and new amplification reaction container 24 and typing reaction container 30 are set in predetermined positions in the reaction table.
The movement in this example is described.
A sample in the blood collection tube, for example in an amount of 1 to several μL, is collected, and dispensed into an amplification reaction container 24 and arranged in the PCR area. At the beginning of PCR, a PCR reaction solution 14, for example in an amount of 5 to 10 μL, is dispensed via a dispensing probe 32 into the amplification reaction container 24 into which the sample has been dispensed. Alternatively, the PCR reaction solution 14 may first be dispensed and the sample then dispensed. In the amplification reaction container into which the sample and the PCR reaction solution were dispensed, PCR is carried out by repeating a predetermined temperature cycle for 1 to 1.5 hours for example. The PCR reaction solution is dispensed in series into other amplification reaction containers 24 in the PCR area, in each of which the other sample has been dispensed, to repeat PCR.
In the amplification reaction container 24 in which PCR was finished, the invader reaction reagent 15 is added via the dispensing probe 32 to, and mixed in, the amplification reaction container 24. The mixture is dispensed by the dispensing probe 32 into a plurality of wells 42 in the typing reaction container 30 in the invader reaction area 28, and the invader reaction is carried out for several minutes to several hours. During the reaction or after conclusion of the reaction, fluorescence is measured by the fluorescence detector 50.
In a typing reaction container 30a in
The base material forming the flow path 74 consists of two substrates 76a and 76b bonded to each other. A groove for the flow path 74 is formed on the surface of one substrate 76a so that the flow path 74 is positioned in the inside of the substrate, while the other substrate 76b is bonded to the flow path-forming surface. Both ends of the flow path 74 are provided with an inlet 78a and outlet 78b for the reaction solution, each of which penetrates through the substrate 76b and is open to the surface of the substrate.
A typing reaction container 30b in
When the reaction solution is dispensed into the inlet 78a in the typing reaction containers 30a and 30b in
The typing reaction container 30c shown in
When the reaction solution is dispensed into the common inlet 80a in the typing reaction container 30c in
In the reaction container 30d shown in
When the reaction solution is dispensed into the common inlet 84a in the typing reaction container 30d in
Each examination reagent kit includes 3 holding parts, that is, a diluent holding part 88, a PCR reaction solution holding part 90 and an invader reaction reagent holding part 92, each of which is expanded in one side of the substrate, and a plurality of invader probe fixing parts 94 are arranged on the surface of the substrate.
The diluent, reaction solution and reaction reagent is held on the respective holding parts 88, 90 and 92, and the openings of the holding parts 88, 90 and 92 are sealed with a detachable film or plate so as to prevent leakage of the liquid before use. After the film or plate on the opening of the diluent holding part 88 is removed, the sample blood is dispensed by nozzle 95 into the diluent holding part 88. After the sample is dispensed, the opening of the diluent holding part 88 is closed again with the film or plate, and the examination reagent kit is attached to the detector.
Invader probes different from one another have been fixed to the invader probe fixing parts 94, and the material of the substrate at least at a part where the invader probe fixing part 94 is arranged is made of a low-spontaneous-fluorescent, light-permeable resin or the like so that emitted fluorescence can be detected on the back surface.
In the examination reagent kit in
Transfer of the liquid from the holding parts 88, 90 and 92 in this examination reagent kit is carried out by using a dispensing nozzle. Accordingly, the film or plate with which the openings of the holding parts 88, 90 and 92 have been sealed is preferably a film or plate into which the dispensing nozzle can easily penetrate.
After a sample, such as blood, is dispensed by the dispensing nozzle into the diluent holding part 88, this reagent kit is attached to an apparatus for detecting gene polymorphism (
The examination reagent kit shown in
The examination reagent kit shown in
In this examination reagent kit, dispensing of the reaction solution into the invader probe fixing part 94b can be accomplished by dispensing it into the inlet 96a only once. The reaction solution dispensed into the inlet 96a penetrates into the flow paths and reacts with the invader probes fixed in the flow paths, and fluorescence by the invader reaction is generated where the corresponding SNP is present in the sample, and the fluorescence is detected by the fluorescence detector in the apparatus for detecting gene polymorphism.
In the examination reagent kit shown in
In the examination reagent kit shown in
In this examination reagent kit, the reaction solution may be dispensed through a nozzle into one end of the invader probe fixing part 98, and the reaction solution is diffused through the material of the invader probe fixing part 98 thereby reacting with the probes fixed to respective positions.
The examination reagent kit shown in
In the examination reagent kits shown in
Before use, the seal or plate is removed, and the sample is dispensed into the diluent holding part 88. Thereafter, when the examination reagent kit is attached to the apparatus for detecting gene polymorphism, the liquid can flow through the grooves 108, 110 between the holding parts 88, 90 and 92 and pass through the groove 104, whereby the fluid can flow from the invader reaction reagent holding part 92 to the invader probe fixing parts 94b, 94c, 96b and 98.
Sending of liquid between the holding parts 88, 90 and 92 in the apparatus for detecting gene polymorphism and sending of liquid from the invader reaction reagent holding part 92 to the invader probe fixing parts 94b, 94c, 96b and 98 are carried out by squashing the holding parts 88, 90 and 92 in this order by mechanical pressing. That is, when the diluent holding part 88 is squashed, the liquid in the diluent holding part 88 passes through the groove 108 and transfers to the PCR reaction holding part 90. Then, when the PCR reaction solution holding part 90 is squashed, the liquid in the PCR reaction solution holding part 90 passes through the groove 110 and transfers to the invader reagent holding part 92. When the invader reaction reagent holding part 92 is further squashed, the liquid in the invader reaction reagent holding part 92 passes through the groove 104 and transfers to the invader probe fixing parts 94b, 94c, 96b and 98, to cause the invader reaction.
The examination reagent kit shown in
The examination reagent kit shown in
The examination reagent kit shown in
The examination reagent kit shown in
Because the invader probe fixing part 98 in the examination reagent kits in
The gene polymorphism detector 120 is provided with a mounting part for mounting a plurality of examination reagent kits 122, in which each of the examination reagent kits 122 is mounted in such a state that a sample has been dispensed into the diluent holding part. The detector 120 is also provided with a movable nozzle 124 for sending the liquid to the examination reagent kits 122 mounted in the mounting part.
Although not shown in the figure, the detector 120 is also provided with an amplification part controlling the temperature of reaction solutions, each consisting of the PCR reaction solution and a biological sample in the PCR reaction solution holding part 90 in the examination reagent kit 122, to amplify genome DNAs in the reaction solutions, and is also provided with a typing reaction part controlling the temperature of reaction solutions, each consisting of a typing reagent and genome DNA amplified in the amplification part, to react the reaction solutions with the probes in the probe fixing parts 94, 94a, 94c, 96b and 98.
The numeral 126 denotes a photometric part as a fluorescence detector and is arranged to detect fluorescence emitted from the invader probe fixing parts in a plurality of the examination reagent kits 122 by moving among the examination reagent kits 122. The typing result judged from the detected fluorescence is displayed on display 128.
The above example is an apparatus for detecting gene polymorphism, which can also be a diagnostic apparatus for disease morbidity, the type and effect or side effect of a drug administered and so on. In this case, the apparatus for detecting gene polymorphism is provided with a database in which diagnostic values such as disease morbidity and the type and effect or side effect of a drug administered with respect to a specific SNP or a combination of SNPs are memorized, or the apparatus is connected to such database in the outside. When the apparatus is connected to the database in the outside, the apparatus can be connected to the database via a special line or a general-purpose communication line. The apparatus for detecting gene polymorphism according to the present invention, when used as a diagnostic apparatus, detects SNP results on the basis of which the diagnostic values are read out from the database and displayed on the display unit.
The present invention can be used in detecting genome DNA polymorphism for plants and animals including humans, particularly SNP (single-nucleotide polymorphism) and can further be utilized, not only in diagnosing disease morbidity, the relationship between the type and effect or side effect of a drug administered and so on by using the results of the above detection, but also in judgment of the variety of animal, or plant, diagnosis of injections (judgment of the type of invader) etc.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-336607 | Nov 2004 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP05/21235 | 11/18/2005 | WO | 00 | 5/12/2008 |
