WO2013119049A1 - 생물학적 시료의 자동 분석 장치 및 방법 - Google Patents
생물학적 시료의 자동 분석 장치 및 방법 Download PDFInfo
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- WO2013119049A1 WO2013119049A1 PCT/KR2013/000985 KR2013000985W WO2013119049A1 WO 2013119049 A1 WO2013119049 A1 WO 2013119049A1 KR 2013000985 W KR2013000985 W KR 2013000985W WO 2013119049 A1 WO2013119049 A1 WO 2013119049A1
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6804—Nucleic acid analysis using immunogens
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
- C12M1/38—Temperature-responsive control
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
- C12Q2565/10—Detection mode being characterised by the assay principle
- C12Q2565/125—Electrophoretic separation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N35/1065—Multiple transfer devices
Definitions
- the present invention relates to an automated analysis apparatus and method for biological samples, and more particularly, the present invention purifies target nucleic acids from biological samples, PCR, RT-PCR, nested PCR and real-time quantitative PCR for quantitatively performing the above process.
- the present invention relates to a device for performing isothermal gene amplification and subsequent electrophoretic analysis in a single device.
- UV lamps are movably provided by moving parts to fundamentally prevent false positives caused by gene amplification products, so that the amplification reactions can be intensively investigated, such as PCR, reverse transcription PCR, nested PCR, and real-time quantitative PCR including the same.
- the present invention relates to an automated analysis apparatus and method for biological samples that can enhance analytical accuracy and efficiency while individually or continuously performing isothermal amplification and PCR-electrophoresis analysis.
- Gene amplification test is an in vitro diagnostic testing (IVD testing) technology that amplifies a specific sequence of genes to determine the presence of genes, food testing as well as pathogenic microorganisms, genotype testing of various animals, plants, humans, etc.
- GMO is used in various fields.
- nucleic acid extraction process is required to remove various reaction inhibitors contained in biological samples that inhibit gene amplification reaction from biological samples and obtain high purity target nucleic acid.
- the target nucleic acid may be DNA or RNA and mixtures thereof depending on the purpose of detection.
- the extracted target nucleic acid is mixed with the gene amplification solution, and the gene amplification reaction is performed. Then, the DNA amplification test is completed by confirming DNA corresponding to the length of the gene amplification product DNA through electrophoresis.
- the "biological sample” is a substance derived from an organism, including animals, plants, microorganisms, viruses, and fungi, and may be interpreted to include all substances defined as living things. have.
- RNA viruses are the most popular methods of amplifying DNA.
- Nested PCR has been widely used to perform a PCR reaction once more using a primer complementary to the primer sequence of the amplified DNA.
- Reverse transcription PCR or RT / PCR (Reverse Transcription PCR) is used to test mRNA expression levels of RNA viruses and specific genes.
- RT / PCR Reverse Transcription PCR
- the PCR test method has a limit in performing quantitative analysis because the deoxynucleotide triphosphate is depleted and reaches a limit where it is no longer amplified when DNA amplification is performed to some extent. For example, higher initial concentrations of template nucleic acid will saturate even shorter reactions within 20 cycles. Lower concentrations of 1000 will saturate after 30 cycles.
- a real-time quantitative PCR technology has been developed that can measure the concentration of nucleic acid after every cycle to measure the cycle at which a constant nucleic acid concentration is reached. This technology has been developed as a very useful technique for molecular diagnosis because it can measure the concentration of virus or pathogen quantitatively.
- Real-time quantitative PCR technology is dominated by methods using fluorescence that increases in proportion to the amount of DNA.
- This fluorescence method is divided into DNA sequence-specific method and non-specific method to be amplified.
- Non-amplified DNA sequence nonspecific methods use an intercalating dye that binds to all the DNA to be amplified and increases fluorescence, which increases the amount of fluorescence in proportion to the amount of all amplified DNA.
- non-specific amplification or trying to amplify two or more specific targets there is a problem that it is impossible to exactly know the initial amount of the target nucleic acid.
- methods using fluorescence probes specific for the DNA sequence to be amplified are capable of quantitatively detecting multiple target nucleic acids simultaneously while amplifying a plurality of target nucleic acids.
- multiple probes emitting different fluorescence are simultaneously hybridized to each amplification product DNA by amplifying targets simultaneously using primer pairs to fluoresce.
- PCR methods have been developed.
- this method also has a problem in that the performance is rapidly deteriorated when a multiprex quantitative PCR is performed on four or more targets, as the number of target nucleic acid primer pairs and probes, i.e., triple-fold oligos, must be included as the number of target nucleic acids increases.
- multiplex PCR 10 multiplex PCRs can be optimized to perform well, and thus multiplex PCR is advantageous for detecting multiple target nucleic acids.
- PCR method is not quantitative, a test method for quantitatively examining multiple targets is required.
- Another limitation of the real-time quantitative PCR method is that there is no method to determine the length of the DNA to be amplified, there is a problem that can not be used to analyze the DNA defect or DNA insertion or detect the number of repeat bases such as VNTR.
- the present invention is to provide a fully automatic device that can determine the length of the product amplified by general PCR after performing real-time quantitative PCR to solve this problem. This allows amplification of many targets simultaneously and quantitative analysis of each target at the same time, quantitative analysis of DNA defects or DNA insertions, or detection of the number of repeat bases such as VNTR while quantitatively determining the initial DNA amount of the target. It is to provide equipment that can be inspected.
- the gene amplification test can be detected with high sensitivity and specificity, it is used in various microorganisms, genetic tests.
- amplification contamination amplicon contamination
- methods for inactivating amplification products using ultraviolet rays or enzymatic reactions have been developed.
- 8-MOP (8-methoxypsoralene) is pre-mixed into the PCR reaction solution and irradiated with UV light after the amplification reaction to perform photochemical reaction with DNA. This is how you do it.
- Enzyme-based method uses deoxyuracil triphosphate (dUTP) and UDG (Uracil Deoxy-Glycosidase) enzyme, and adds dUTP to PCR reaction solution so that PCR reaction product contains deoxyuracil base. Thereafter, UDG enzyme is added to the PCR reaction to decompose and remove the contaminated PCR reaction product through the UDG enzyme reaction before the PCR amplification reaction.
- these methods cannot be used in the case of nested PCR, which requires a subsequent PCR as a PCR reaction product. Therefore, in general, an institution performing a gene amplification test prepares separate laboratories for nucleic acid extraction tablets, gene amplification reaction sample preparation, and electrophoretic analysis, and each task is performed here. It costs a lot. Nevertheless, there are always risks of false positive results from contamination by workers and false negative results from nucleic acid extraction and gene amplification reaction preparations. Examination is being conducted mainly by clinical testing institutions.
- a method of using magnetic particles is widely used.
- microparticles having a large surface area are used to rapidly attach target nucleic acids in a suspension state of a solution and apply a magnetic field to aggregate magnetic particles to which target nucleic acids are attached.
- various automated equipments related thereto have been developed.
- the component is a tubular one connected in series to the jet channel, the jet channel being defined as the flow inlet at the end of the tube and having a diameter smaller than the separation chamber;
- the first position is adjacent to the outside of the dividing wall and the second position is within the separation chamber range.
- the tubular shape is the second part connected in series with the cylindrical separation chamber.
- the separation chamber is separated from the jet channel, and the cylinder channel is equipped with a movable piston to suck and push liquid into the separation chamber.
- the suspension of the solution is difficult to be uniform and the cleaning solution is not completely removed even after the final step of purification, so that the remaining amount of the remaining amount during elution Since the washing solution is included there is a problem that can affect the process, such as gene amplification.
- PCR PCR
- Nested PCR RT / PCR
- isothermal nucleic acid amplification a preparation step for preparing a gene amplification reactant by mixing the extracted target nucleic acid with a gene amplification reaction solution and a reaction step for proceeding the reaction are required.
- a reaction may be prepared by mixing a predetermined amount of the gene amplification reaction solution containing a primer, a nucleic acid polymerase, a polymerization monomer, nucleotide triphosphate (dNTP or NTP), and a buffer, and an extracted target nucleic acid.
- dNTP or NTP nucleotide triphosphate
- the isothermal amplification only needs to maintain a constant temperature.
- a heating and cooling step is required for the temperature cycling reaction.
- the gene amplification reaction requires raising the temperature, so it is usually sealed to prevent evaporation.
- the gene amplification reaction container is sealed, after the gene amplification reaction, the gene amplification reaction product must be opened to transfer the amplification product to the electrophoretic gel 362 after performing the electrophoretic analysis.
- a complicated device is needed and contamination of gene amplification products occurs.
- nested PCR relies on spatial separation to perform the test because amplification product inactivation methods for preventing amplification product contamination cannot be used.
- agarose electrophoresis is used. This method has the advantages of low cost and simple analysis in the traditional way, but has a problem that the analysis takes a relatively long time and requires a lot of manual labor by skilled personnel. Recently, in order to solve these problems, methods using capillary electrophoresis capable of automatically performing electrophoresis in a short time have been developed, but the apparatus and consumables are expensive, and thus they are limitedly used. Since all electrophoresis steps are performed with gene amplification products at all times, fine aerosols generated from them are mixed in the gene amplification reaction solution and cause false positives.
- the real-time quantitative PCR and general PCR are performed continuously with high reliability without being contaminated by external contaminants.
- the object of the present invention is to perform a complex process of purifying, amplifying and analyzing the target nucleic acid from a biological sample using a single device can be performed automatically PCR It provides a convenient, reproducible and economical genetic test system by fully performing nested PCR, RT / PCR, quantitative real-time quantitative PCR and isothermal nucleic acid amplification.
- the present invention provides a biological sample automatic analysis device and method with high accuracy by solving the problem of false positives caused by repetitive testing and preventing contamination of the specimen.
- the case 100 is formed with an opening 110 capable of opening and closing a predetermined region;
- a plurality of wells 211 forms the first to the N-th column, the multi-well plate kit 210 is embedded in one or more samples or reagents necessary for biological samples, purification in a specific column of the first to N-th column, respectively
- Purifying unit 200 for purifying the target nucleic acid from the biological sample including;
- a plurality of pipettes 500 that form a column and move biological samples, samples or reagents used in the purification unit 200, purified target nucleic acids, and amplified DNA;
- a moving part 600 for moving the pipette 500 in the longitudinal direction and the height direction of the case 100 and for controlling the operation of the pipette 500;
- the biological sample automatic analysis device 1000 is detachably provided with the multi-well plate kit 210, the electrophoretic body 310, by the guide unit 130 fixed to the lower surface of the case 100 A base plate 700 which is guided and movable in the longitudinal direction of the case 100 through the opening 110; It is characterized in that the further provided.
- the biological sample automatic analysis device 1000 is characterized in that the first sterilization means 800 is fixed to the moving unit 600 is further provided.
- the first sterilization means 800 is an ultraviolet lamp, characterized in that it further comprises a reflecting means (810).
- the purification unit 200 includes a magnetic field applying unit 201 for applying a magnetic field to a specific row of the multi-well plate kit 210 including a magnet 221; And a heating unit 202 formed adjacent to the magnetic field applying unit 201 to heat the same specific heat. Characterized in that it comprises a.
- the purification unit 200 further includes a biological sample tube rack 280 including a biological sample tube 281 for putting a biological sample for extracting the target nucleic acid, the biological sample tube rack 280 is
- the base plate 700 is detachably provided.
- the magnetic field applying unit 201 includes a magnet mounting unit 220 on which the magnet 221 is mounted; It is mounted on the lower surface of the case 100, the lifting unit 270 for raising and lowering the magnet mounting portion 220; characterized in that it comprises a.
- one or more samples or reagents required for purification contained in the first to Nth columns of the multiwell plate kit 210 may include one or more of enzymes, cell lysis solutions, nucleic acid binding solutions, magnetic particle aqueous dispersions, and washing solutions. Characterized in that it comprises a.
- the magnet mounting portion 220 is formed with an inlet groove 222 so that the lower portion of the specific row of the multi-well plate kit 210 is introduced into the surface on which the magnet 221 is mounted, the base plate 700 is the When the magnet mounting portion 220 is raised, a first exposure hole 701 is formed in which a predetermined region is hollowed so that a lower portion of a specific row is seated in the inlet groove 222.
- the magnet mounting portion 220 is formed of a metal material
- the heating unit 202 is characterized in that the heating film formed in contact with the magnet mounting portion 220.
- the base plate 700 is provided in the protruding fixing part 730 and the protruding fixing part 730 protruding to be positioned between the plurality of wells 211 under the multiwell plate kit 210.
- An elastic means 731 in close contact with 211 is further characterized.
- the base plate 700 is characterized in that the waste container 740, which stores the solution discarded after use is detachably provided.
- the biological sample automatic analysis device 1000 includes a pipette block 630 in which the moving part 600 is mounted and detached from the pipette 500, and the pipette 500 is attached to the base plate 700.
- the pipette rack 510 that can be stored is characterized in that it is provided detachably.
- the pipette block 630 of the moving part 600 includes a pipette block fixing plate 644 which is formed at the bottom of the pipette fixing protrusion 644-1 on which the pipette 500 is mounted and movable in the height direction.
- the pipette 500 may be mounted to the pipette fixing protrusion 644-1 through a pipette mounting portion.
- pipette block 630 of the moving part 600, the pipette (631) and the piston 631 is fixed to the pipette through a pipette control unit including a piston fixing plate 634 which is movable in the height direction 500 is characterized in that the suction and discharge is controlled.
- the pipette block 630 of the moving part 600 has a hollow area corresponding to the pipette fixing protrusion 644-1, and when the pipette 500 is mounted, the pipette block fixing plate 644 and the lower surface (500) a length longer than the height of the pipette extrusion plate 638-1 positioned to abut between upper surfaces, the pipette extrusion pin fixing plate 639 positioned above the pipette block 630, and the pipette block 630.
- a pipette extrusion pin 638 connecting the pipette extrusion plate 638-1 and the pipette extrusion pin fixing plate 639, and the pipette extrusion pin fixing plate 639 is lowered by the pipette adjusting part.
- the pipette extrusion pin fixing plate 639, the pipette extrusion pin 638-1 and the pipette extrusion plate 638-1 is moved downward so that the pipette 500 is the pipette fixed protrusion 644-1 It is characterized in that it is detached from).
- the purification unit 200 is a vacuum module 291 that can be attached / detached to the moving unit 600, and the vacuum is installed on the inner lower surface of the case 100 to store the vacuum module 291 in a predetermined position
- a drying unit 290 including a modular rack 293 and a vacuum pump 295 connected to the vacuum module 291 and a hose.
- the convex portion 294 protrudes from the upper surface of the vacuum module rack 293, and the concave portion corresponding to the convex portion 294 is formed on the lower surface of the vacuum module 291. 292) is formed.
- drying unit 290 is provided with a support portion 293-1 for supporting one side of the vacuum module 291 in the vacuum module rack 293, the support portion 293-1 and the vacuum module 291. ) Is characterized in that the magnet is built in the surface in contact with each other.
- Electrophoretic part 300 is seated on the electrophoretic body fixing plate 750 installed on the base plate 700, the hole type electrical connection terminal 311 is connected to each of the electrode line (311a) forming a positive electrode and a negative electrode, respectively.
- Electrophoretic body 310 is formed; A protruding electrical connection terminal 341 inserted into the hole type electrical connection terminal 311 of the electrophoretic body 310 to connect a power source; A power supply unit 320 fixed to the lower inner surface of the case 100 to supply power to the electrophoretic body 310; An excitation light irradiation unit 330 for irradiating excitation light to the electrophoretic body 310; And a photographing unit 350 photographing the electrophoretic state.
- the electrophoretic part 300 has a lower surface of the power connection terminal 340 and a first surface of the power supply unit 320 contacted with each other.
- the contact part 342 and the second contact part 322 are formed to be inclined, and the contact part 342 and the second contact part 322 are in close contact with each other by the elastic means 321.
- the excitation light irradiation unit 330 is provided with LEDs for irradiating light of the excitation light wavelength band toward the electrophoretic body 310 is arranged at equal intervals on the inner bottom surface of the case 100, the base plate 700
- the electrophoretic body fixing plate 750 on which the electrophoretic body 310 is mounted is formed of a material for scattering excitation light, and the bottom plate of the electrophoretic body 310 absorbs light of a nucleic acid fluorescence light emitting band and excites light band.
- the light is characterized in that formed of a passing material.
- the photographing unit 350 is formed to include an image sensor, a lens, a short wavelength filter, and is fixed to the moving unit body 610 of the moving unit 600.
- the electrophoretic unit 300 may include a marker tube 920 having a marker and a fluorescent dye and a marker tube rack 910 on which the marker tube 920 is mounted. 900 is further provided, the marker tube rack 910 is characterized in that it is provided detachably to the base plate (700).
- the electrophoretic body 310 is provided with a gel tray (360, 360-1), the gel tray (360, 360-1) is overlapped with the electrophoretic loading well 363 and the electrophoretic movement path on the bottom It is characterized in that a plurality of electrophoretic gel fixing part (361) is formed in a position not supported.
- the nucleic acid amplifier 400 includes an amplification tube 411 to form one or more columns;
- An amplifying block 410-1 formed of a metal having a concave mounting recess 421 formed in close contact with a lower portion and having a temperature sensor inserted therein;
- An amplifying block cover 410 made of a heat insulating material having a first fixed magnet 422; Peltier element 423; A heat transfer part 420 transferring heat generated from the Peltier element 423 to the outside;
- second holes formed in a smaller hole than an upper surface of the amplification tube 411, and are formed to surround a predetermined region on an upper side of the amplification tube 411, and corresponding to the first fixed magnet 422.
- the amplification tube fixing part 430 is inserted to fix the amplification tube 411, the amplification tube fixing portion 430 having a height spaced a predetermined distance from the surface formed with the amplification block cover 410; Characterized in that it comprises a.
- the base plate 700 has a hollow second exposure hole 702 is formed so that the amplification block 410-1 and the amplification tube fixing portion 430 corresponding to the amplification tube fixing portion 430 is formed. It features.
- nucleic acid amplification unit 400 is characterized in that it is located adjacent to the opening (110).
- the nucleic acid amplifier 400 is a light irradiation unit 440 for irradiating the fluorescent excitation light toward the amplification tube (411); And a fluorescence detector 450 which is provided to be movable in the vertical direction so as to be in close contact with the upper part of the amplifying tube 411 and can measure the amount of fluorescence in the amplifying tube 411 in real time.
- the base plate 700 is characterized in that the handle 710 is formed on the upper surface of one side adjacent to the opening (110).
- case 100 may further include an air flow unit 142 that forms a flow path through which air flows on the outer lower surface, and a blower 141 that blows air to the air flow unit 142. do.
- the biological sample automatic analysis device 1000 includes a first position fixing part 720 at the other end of the base plate 700; And the second position fixing unit 120 fixed to the case 100 at a position corresponding to the first position fixing unit 720; Is further formed, the first position fixing portion 720 and the second position fixing portion 120 is characterized by being fixed to each other by the magnetism of the third fixed magnet 721.
- the automatic analysis method of the biological sample according to the present invention, the analysis method using the biological sample automatic analysis device having the characteristics as described above, the purification step of obtaining a target nucleic acid from the biological sample (S10); An amplification step (S20) of amplifying the target nucleic acid having completed the purification step (S10 ); And an electrophoresis step (S30); Characterized in that it comprises a.
- the automatic analysis method of the biological sample is taken out of the base plate 700 to the outside of the case 100 before the purification step (S20), the electrophoretic body 310, the pipette to the base plate 700
- a second mounting step of fixing the amplifying tube 411 by the second fixed magnet 431 of the amplifying tube fixing part 430 It characterized in that the preparation step (S40) comprising a.
- the purification step (S10) is a pipette mounting step of mounting the pipette 500 in the pipette block 630 by moving the moving unit 600; And a mixture excluding magnetic particles by moving / mixing the moving unit 600 to move / mix the biological sample, one or more samples or reagents necessary for purification, respectively, embedded in the first to Nth rows of the multiwell plate kit 210.
- a target nucleic acid-containing solution obtaining step of obtaining a target nucleic acid-containing solution includes, but the magnetic field applying step of applying a magnetic field to a specific row of the multiwell plate kit 210 by the magnetic field applying unit 201 and the heating unit 202 of the multiwell plate kit 210 as necessary Characterized in that a heating step of heating the specific heat is carried out.
- the target nucleic acid-containing solution acquisition step is a sample lysis step of dissolving the nucleic acid by dissolving the biological sample in the purification unit 200; Attaching the magnetic particles to the target nucleic acid and removing the filtrate from the target nucleic acid; A magnetic particle washing step of removing impurities by washing the magnetic particles to which the target nucleic acid is attached; The pipette 500 mounted on the moving unit body 610 is detached, and the vacuum module 291 is fixed to the moving unit body 610 of the moving unit 600, and the multiwell plate kit 210 Magnetic particle drying step of drying the magnetic particles by sucking the washing solvent of a specific heat; And an acquisition step of obtaining a target nucleic acid by adding an elution buffer to the dried magnetic particles.
- the amplifying step (S20) is a mixing step of moving and mixing the target nucleic acid-containing solution obtained through the purification step (S10) to the amplification tube (411); And a temperature adjusting step of controlling and amplifying the temperature by the heat transfer unit 420.
- the electrophoresis step (S30) is a marker mixing step of mixing the amplification product and the marker solution; Performing electrophoresis to separate the amplification product by supplying power to the electrophoretic body 310 by the power supply unit 320 and the power connection terminal 340; And an analysis step of irradiating light by the excitation light irradiation unit 330, measuring an electrophoretic pattern of the amplified product using the photographing unit 350, and analyzing molecular weight; Characterized in that it comprises a.
- the automatic analysis method of the biological sample further comprises an inactivation step (S50) of inactivating the amplified nucleic acid by operating the first sterilization means 800 between the amplification step (S20) and the electrophoresis step (S30). Characterized in that it is carried out.
- the amplifying step (S20) detects the point of time when the threshold fluorescent value is detected by measuring the amount of fluorescence generated for a predetermined time or every cycle with the fluorescent detector 450 while irradiating a constant excitation light from the light irradiation unit 440 It is characterized in that the real-time quantitative amplification analysis to calculate the initial nucleic acid concentration.
- the automatic analysis method of the biological sample is capable of correcting and analyzing the result of the quantitative amplification analysis obtained in the amplification step (S20) through a result including the number of molecular weight and amplification products obtained through the electrophoresis step (S30). It features.
- the antigen concentration acquisition method using the quantitative immunity PCR of the present invention is capable of quantitative test of the concentration of the antigen contained in the biological sample by performing a quantitative immunity PCR using the automatic biological sample analysis device 1000 having the characteristics as described above Do.
- the biological sample automatic analysis device and method of the present invention is a device for purifying, amplifying, and analyzing target nucleic acids from biological samples, including PCR, Nested PCR, RT / PCR, and real-time quantitative PCR.
- PCR PCR
- Nested PCR RT / PCR
- real-time quantitative PCR PCR
- the biological sample automatic analysis apparatus and method of the present invention can quantitatively analyze nucleic acid through real-time quantitative PCR and then perform electrophoresis to analyze the length of the amplified DNA to quantitatively identify VNTR, gene insertion, deletion, mutation, and the like.
- This advantage can be applied to many diagnostic fields. For example, in order to treat various viral diseases, it is expensive and time-consuming to test the blood concentration of the virus by quantitative PCR and to perform the virus variant through the genetic base sequence determination test.
- the equipment of the present invention after performing real-time quantitative PCR with multiplex PCR tubes containing primer pairs designed to yield PCR reactions of selective and different length for each strain, the fluorescence value for quantifying viruses can be measured.
- the quantitative test and the strain test can be performed at once. Since all these operations are performed automatically, there is an advantage in that virus quantification and strain testing can be easily and quickly performed using a single clinical sample.
- the automatic biological sample analysis device and method of the present invention is provided with a UV lamp to be movable in the mobile unit to inactivate the gene amplification product immediately after the gene amplification can prevent the false positive results due to contamination of the gene amplification product.
- Sterilization means for total sterilization may be further provided to prevent equipment contamination of pathogens contained in the biological sample, and as all processes are performed inside the case, the source of contamination may be blocked from the outside, thereby increasing the reliability of the analysis. There is this.
- the automatic biological material analysis apparatus and method of the present invention the multi-well plate kit, waste fluid bottle, electrophoretic body, biological sample tube rack, pipette rack, and marker tube rack are formed detachably on the base plate, respectively.
- the module is easy to mount, and there is an advantage that the analysis can be prepared by a simple method of mounting and inserting each component.
- the magnetic field can be applied to the specific heat and heated at the same time. There is an advantage that can be further increased.
- the biological sample automatic analysis apparatus and method of the present invention is provided with a drying unit, the residual solvent can be removed in a short time to prevent the adverse effect on the amplification process as the washing solvent remains in the purified target nucleic acid solution
- the moving unit body for detaching and moving the vacuum module of the drying unit there is an advantage that can simplify the configuration of the entire apparatus, and facilitate the operation.
- the biological sample automatic analysis device and method of the present invention is formed on the upper surface of the temperature control means is formed a seating groove in which the amplification tube is seated, and the fixing portion is formed to support the upper portion of the amplification tube to surround the amplification tube
- the amplification tube is stably fixed, there is an advantage to increase the amplification efficiency by preventing the temperature change.
- the biological sample automatic analysis device and method of the present invention has the advantage that the photographing unit is fixed to the moving part to shorten the shooting distance, to obtain a precise electrophoretic image with high sensitivity.
- the biological sample automatic analysis device and method of the present invention can be easily fixed to the base plate as the first position fixing and the second position fixing is formed, when the base plate is inserted into the base plate, the power supply As the first inclined portion of the connection terminal and the second inclined portion of the power supply unit are brought into contact with each other, the elastic means is compressed, and the first contact portion and the power supply portion of the power connection terminal at the final insertion position of the base plate.
- the second contact portion of the can be in contact with each other there is an advantage that the operation of the battery moving part is easy.
- the automatic biological sample analyzing apparatus and method of the present invention have an advantage that the Peltier cooling device can be efficiently used by appropriately cooling the bottom of the case by using the bottom of the case as a heat sink and forming an air flow unit and a blower.
- FIG. 1 and 2 are a perspective view, a bottom perspective view showing a biological sample automatic analysis device according to the present invention.
- FIG. 3 and 4 are each a perspective view of the automatic biological sample analysis device according to the present invention.
- FIG. 3 is the outer cover of the case of the deleted form
- Figure 4 is the outer cover and the skeleton partially deleted form
- FIG. 5 is a view showing a moving body and the moving unit of the biological sample automatic analysis device according to the present invention.
- 6 to 8 are each a pipette mounting, suction and discharge control, desorption operation of the automatic biological sample analysis device according to the present invention.
- FIG. 9 is a view showing a base plate spaced apart state of the biological sample automatic analysis device according to the invention.
- 10 and 11 are a perspective view and an exploded perspective view of the base plate upper coupling of the automatic biological sample analysis apparatus according to the present invention.
- FIG. 12 is an exploded perspective view showing the base plate and the multiwell plate kit fixed state of the automated biological sample analysis device according to the present invention.
- 13 and 14 are a perspective view and a bottom perspective view showing a magnetic field applying unit and a heating unit of the automatic biological sample analysis device according to the present invention.
- 15 and 16 are views showing a drying unit of the biological sample automatic analysis device according to the present invention.
- 17 is a view for explaining the use of the drying unit of the automatic biological sample analysis apparatus according to the present invention.
- 18 and 19 are a perspective view and a cross-sectional view showing a nucleic acid amplifier for real-time nucleic acid amplification of the biological sample automatic analysis device according to the present invention.
- 20 and 21 are another view showing the nucleic acid amplification unit of the biological sample automatic analysis device according to the present invention.
- Figure 22 and Figure 23 is a view showing the electrophoretic unit of the automatic biological sample analysis apparatus according to the present invention, respectively.
- 24 is a view showing the electrophoresis unit electrical connection form of the biological sample automatic analysis device according to the present invention.
- 25 and 26 are schematic diagrams showing an automatic analysis method of a biological sample according to the present invention.
- magnet mounting portion 221 magnet
- lifting unit 271 lifting motor
- drying unit 291 vacuum module
- electrophoretic body 311 hole type electrical connection terminal
- 360, 360-1 Gel Tray 361: Electrophoretic Gel Fixation
- nucleic acid amplification unit 410 amplification block cover
- amplification tube fixing part 431 second fixed magnet
- moving part 610 moving part body
- piston belt 634 piston fixing plate
- piston guide rod 638 pipette extrusion pin
- pipette extrusion plate 639 pipette extrusion pin fixing plate
- Pipette fixing protrusion 645 Z-axis screw nut
- base plate 701 first exposure hole
- first sterilization means 810 reflection means
- marker storage unit 910 marker tube rack
- Automatic biological sample analysis device 1000 of the present invention includes a case (100); Purification unit 200 for purifying the target nucleic acid from the biological sample; A nucleic acid amplification unit 400 for amplifying the target nucleic acid purified by the purification unit 200; An electrophoretic unit 300 including an electrophoretic body 310 for examining an amplification product through the nucleic acid amplification unit 400; A biological sample, a sample or reagent used in the purification unit 200, a pipette 500 for moving a target nucleic acid purified and amplified target nucleic acid; A moving part 600 for controlling the operation of the pipette 500 and moving the pipette 500 in the longitudinal direction and the height direction of the case 100; It is formed including a first sterilization means 800 and a control unit (not shown).
- the case 100 is a basic body constituting the biological sample automatic analysis device 1000 of the present invention, and may include a skeleton and an outer cover.
- the case 100 is formed with an opening 110 capable of opening and closing a predetermined area so that the base plate 700 can be pulled out and drawn out in the longitudinal direction of the case 100.
- the base plate 700 is a configuration that can be added to the biological sample automatic analysis device 1000 of the present invention, and will be described again below.
- FIG. 1 an example in which the opening part 110 is formed to open and close a predetermined area of the left side of the case 100 is illustrated.
- the opening part 110 when the opening part 110 is opened, the base plate 700 is opened. It should be formed in such a size that withdrawal is possible.
- the case 100 is positioned to be spaced apart from the ground on which the outer lower surface is mounted, and blows air to the air flow unit 142 and the air flow unit 142 which form a flow path through which air flows on the outer lower surface.
- Blower 141 may be further provided.
- the air flow unit 142 may be provided with a plurality of dense heat radiation fins formed long in the longitudinal direction of the case 100 to form a plurality of air flow paths.
- the blower 141 is configured to blow air into the air flow unit 142.
- the air flow unit 142 and the blower 141 may be formed to efficiently cool the lower surface of the case 100, and the case 100 to increase the cooling efficiency.
- the bottom surface may be made of a material having high thermal conductivity (for example, aluminum).
- the purification unit 200 is a component for purifying target nucleic acid from a biological sample.
- the purification unit 200 includes a multiwell plate kit 210, a magnetic field applying unit 201, and a heating unit 202.
- the multiwell plate kit 210 has a plurality of wells 211 forming the first to Nth rows, one or more samples or reagents required for biological samples, purification, respectively, in a specific column of the first to Nth columns. It is built.
- the multiwell plate kit 210 includes a plurality of wells 211 provided in a lateral direction to form a plurality of rows.
- Each of the first to the Nth columns may be a biological sample or at least one sample required for purification. Or reagents are embedded.
- the sample or reagent required for purification may include an enzyme, a cell lysis solution, a nucleic acid binding solution, a magnetic particle aqueous dispersion, and a washing solution, wherein some rows of the multiwell plate kit 210 are used. May be empty.
- the multiwell plate kit 210 is provided with a biological sample, a reagent or a sample necessary for purification, in a detachable manner in the base plate 700, so that the user can use the multiwell plate kit 210 for purification.
- a biological sample a reagent or a sample necessary for purification
- the base plate 700 a detachable manner in which the user can use the multiwell plate kit 210 for purification.
- there is no need for a separate process has the advantage of very convenient use.
- the magnetic field applying unit 201 includes a magnet 221 to apply a magnetic field to a specific row of the multiwell plate kit 210 to separate magnetic particles having a target nucleic acid attached thereto from a solution.
- the purification unit 200 may include a heating unit 202 to accelerate various reactions to further increase the purification efficiency.
- the heating unit 202 is formed adjacent to the magnetic field applying unit 201 to heat the same specific heat.
- the same specific column means a column selected from the first to Nth columns formed in the multiwell plate kit 210, and the magnetic field applying unit 201 and the heating unit 202 are respectively arranged in a specific column.
- the magnetic field may be applied or the magnetic field and heating may be applied simultaneously.
- the method of applying the magnetic field is made through the lifting unit 270 to raise and lower the magnet mounting portion 220 on which the magnet 221 is mounted, in order to increase magnetic field application efficiency and heat transfer during heating.
- An inlet groove 222 in which a lower portion of a specific row is seated may be formed in the 220.
- the base plate 700 of the portion where the specific row of the multiwell plate kit 210 is located is hollowed out in a predetermined area so that the specific row can be directly seated in the inlet groove 222 of the magnetic field applying unit 201.
- the first exposure hole 701 should be formed.
- the heating unit 202 may be formed in various forms for heating a specific heat
- the magnet mounting portion 220 is formed of a metal material
- the magnetic field is applied to the specific heat. And heating can be performed at the same time there is an advantage that can further increase the purification efficiency.
- the lifting unit 270 is mounted inside the lower surface of the case 100 and is configured to raise and lower the magnet mounting unit 220.
- the lifting unit 270 may be formed in various ways, and the exemplary embodiments illustrated in FIGS. 13 and 14 will be described. do.
- the lifting unit 270 is a lifting motor 271; A first lifting shaft 272 connected to the lifting motor 271 at one end thereof and rotating; One end of the elevating cam 273 integrally connected to the other end of the first elevating shaft 272 and the one end of the elevating cam 273 so as to move in a height direction (up and down directions) in a circular motion. It may include a second lifting shaft 274 connected to the lifting cam (273).
- the magnetic field applying unit 201 may include a magnet mounting unit support 230 for supporting the magnet mounting unit 220, and a guide rod 240 may be connected to the bottom surface of the magnet mounting unit support 230.
- the guide rod 240 is inserted into the guide hole of the guide block 250, the guide hole is formed to be slidable in the height direction.
- a tension spring 260 is provided between the magnet mounting portion 220 and the support so that the magnet mounting portion 220 can be stably seated on the multiwell plate kit 210.
- the lifting unit 270 is in one embodiment, the biological sample automatic analysis device 1000 of the present invention is not limited thereto.
- the purification unit 200 is equipped with a pipette 500 to the moving unit body 610 is moved / operated by the moving unit 600, by the suction and discharge of the pipette 500, the multi-well
- the biological sample contained in the plate kit 210, a sample or reagent necessary for purification are transported and mixed, a magnetic field is applied by the magnetic field applying unit 201 during the above process, and is heated by the heating unit 202. Purification takes place.
- the operation of the lifting unit 270 may be linked with the height sensor 275 including the sensing unit 275-1 and the sensing target unit 275-2.
- the purification unit 200 may further include a biological sample tube rack 280 including a biological sample tube 281 for putting a biological sample for extracting the target nucleic acid, the biological sample tube rack 280 ) Is preferably formed detachably on the base plate 700 to facilitate the transfer.
- a biological sample tube rack 280 including a biological sample tube 281 for putting a biological sample for extracting the target nucleic acid, the biological sample tube rack 280 ) Is preferably formed detachably on the base plate 700 to facilitate the transfer.
- the biological sample automatic analysis device 1000 of the present invention, the magnetic field applying unit 201 and the heating unit in order to perform a process of applying a magnetic field and heat to the multi-well plate kit 210 in the purification unit 200 202 may be positioned adjacent to the plurality of wells 211 while moving in the height direction through the lifting unit 270, wherein the multiwell plate kit 210 is stably attached to the base plate 700.
- the protruding fixing portion 730 and the protruding fixing portion 730 protruding to be positioned between the plurality of wells 211 in the base plate 700 on which the multiwell plate kit 210 is seated Is provided in the elastic means 731 in close contact with the well 211 may be further formed.
- the elastic means 731 is a structure fixed to the protrusion fixing portion 730, a material that can provide a friction force, such as a rubber ring is used to stably fix the multi-well plate kit 210.
- the multiwell plate kit 210 is also fixed at the correct position to facilitate control of the moving unit 600. Accordingly, there is an advantage that can be easily performed purification.
- Automatic biological sample analysis device 1000 of the present invention is provided with a detachable pipette 500 on the moving body 610 of the moving part 600, the pipette 500 is replaced when one analysis is completed Alternatively, a pipette rack 510 capable of storing the pipettes 500 may be provided to facilitate the cleaning and reuse of the pipettes 500.
- the pipette rack 510 with the pipette 500 is mounted on the base plate 700 and inserted into the case 100, and the moving part 600
- the pipette 500 is preferably fixed to the moving part body 610 so that the pipette 500 can be detachably moved / operated.
- the pipette rack 510 for fixing the pipette 500 is detachably mounted to the base plate 700.
- the purification unit 200 may be further formed with a drying unit 290 for exhausting the air of a specific heat of the multi-well plate kit 210 in order to increase the washing efficiency, (see Fig. 15 to 17)
- the pipette rack 510 stores the pipettes 500 when the moving part 600 performs operations related to the operation of the drying part 290.
- the drying unit 290 is mounted on the vacuum module 291, the inner surface of the case 100, the vacuum module rack 293 for storing the vacuum module 291 in a predetermined position, and the vacuum module 291 And a vacuum pump 295 connected to the hose.
- the vacuum module rack 293 is configured to store the vacuum module 291 at a predetermined position when the vacuum module 291 is not used, and a convex portion 294 is formed on an upper surface of the vacuum module rack 293.
- a protrusion 292 corresponding to the convex portion 294 is formed on the bottom surface of the vacuum module 291 so that the vacuum module 291 is stored at the correct position in the vacuum module rack 293. It can be made easier.
- the convex portion 294 is preferably formed to be inclined to widen the diameter in the downward direction so that the vacuum module 291 can be easily seated.
- drying unit 290 is a support portion 293-1 for supporting one side of the vacuum module 291 to the vacuum module rack 293 in order to further maximize the movement preventing effect of the vacuum module 291.
- the support unit 293-1 and the vacuum module 291 may have a stator magnet 296 embedded in a surface in contact with each other.
- FIG. 16 shows the lower part of the vacuum module 291, and (b) shows the vacuum module rack 293 part.
- the vacuum module 291 has a hole formed in the upper portion so as to be detached / attached to the moving part body 610 of the moving part 600, when the vacuum drying is necessary, the moving part body 610 of the moving part 600. Is moved to the correct position in the vacuum module rack 293 to attach the vacuum module 291 and is moved to a specific row of the multiwell plate kit 210, and the air is exhausted to the specific column by the operation of the vacuum pump 295 Let's do it.
- the vacuum module 291 is again the vacuum module rack 293. ) Is moved to the position and detachable from the moving body 610, and is stored in the vacuum module rack (293).
- the moving part body 610 of the moving part 600 is moved to the pipette rack 510 position and the pipettes 500 fixed to the pipette block 630 are fixed again.
- the drying unit 290 fixes the vacuum module 291 and uses a configuration of the moving unit 600 to operate the pipette 500, rather than using a separate configuration for moving it.
- the biological sample automatic analysis apparatus 1000 of the present invention can simplify the configuration of the entire apparatus and facilitate the operation.
- the biological sample automatic analysis device 1000 of the present invention is provided with the drying unit 290 to drastically reduce the time to dry the washing solvent inside the purified target nucleic acid to quickly remove the washing solvent that adversely affects the amplification process There is an advantage to this.
- the nucleic acid amplifier 400 is configured to amplify the target nucleic acid purified by the purification unit 200, amplify the target material purified by the purification unit 200. 20 and 21.
- the nucleic acid amplifier 400 may include an amplification tube 411, an amplification block cover 410, a heat transfer part 420, and an amplification tube fixing part 430. .
- the amplification tube 411 is a space in which reagents for nucleic acid amplification are built, and is positioned to correspond to the same position as the wells 211 positioned in the horizontal direction forming a single row of the multiwell plate kit 210, respectively. .
- the amplification tube 411 is shown as an example formed in two rows, the biological sample automatic analysis device 1000 and method of the present invention is not limited thereto, and may be formed in one or more various ways as necessary.
- the amplification tube 411 When the amplification tube 411 is formed in two rows, one row of tubes are used for PCR and isothermal amplification. After amplification, the amplification products embedded in one column are moved to the electrophoretic unit 300, and the amplification products embedded in the remaining columns are moved to the electrophoretic unit 300 again to be retested, used for another test, or stored separately. Can be.
- gene amplification may be performed using two rows of amplification tubes 411 having different materials embedded therein, and each row may be adjusted to a temperature suitable for each reaction step.
- the reverse transcription reaction solution is contained in the first column of the amplification tube 411, and mixed with the target nucleic acid-containing solution to perform reverse transcription reaction.
- PCR reaction solution can be carried out by mixing the reactants of the reverse transcription reaction in the heat.
- the first PCR reaction solution is contained in the first column of the amplification tube 411, and the first PCR reaction is performed by mixing with the target nucleic acid-containing solution, and the second PCR reaction solution is carried out in the second column of the amplification tube 411. It contains the reaction of the first PCR reaction can be carried out nested PCR reaction.
- the amplification process may be prepared by a simple process of seating on the seating groove 421 of the amplification block cover 410 and fixing the amplification tube fixing part 430.
- the heat transfer part 420 is configured to heat and cool the amplification tube 411, and is formed at a lower portion of the amplification block cover 410 in which a seating groove 421 in which the amplification tube 411 is seated is formed.
- the heat transfer part 420 may be used in various forms, and the Peltier element 423 and a temperature sensor are used to facilitate temperature control.
- the nucleic acid amplifier 400 has a first fixed magnet 422 is inserted into the amplification block cover 410 to fix the amplification tube 411, a second corresponding to the first fixed magnet 422
- An amplifying tube fixing part 430 into which the stator magnet 431 is inserted is provided.
- the amplification tube fixing part 430 is supported on the amplification tube 411 to surround the amplification tube 411, the amplification tube 411 is stably in close contact with the seating groove 421 temperature. It facilitates transfer and prevents temperature changes, resulting in better amplification efficiency.
- the lower surface of the amplification tube fixing part 430 is spaced (S) spaced a predetermined distance from the surface formed with the seating groove 421 of the amplification block cover 410 so that the amplification tube 411 is in close contact It is preferably formed to have a height to form a.
- the base plate 700 has a hollow second exposure hole 702 is formed on the upper surface of the amplification block cover 410 so that the amplification tube 411 and the amplification tube fixing part 430 can be located. .
- the second exposure hole 702 is formed in a region where the heat transfer part 420 is located when the base plate 700 is inserted into the case 100, the amplification tube. It is formed to a size larger than the fixing portion 430.
- the amplification tube 411 and the amplification tube fixing part 430 complete insertion of the base plate 700 into the case 100. After that, it is fixed to the heat transfer part 420 through the second exposure hole 702 of the base plate 700.
- the biological sample automatic analysis device 1000 of the present invention as shown in Figure 18 and 19, the light irradiation unit 440 for irradiating the excitation light to the nucleic acid amplifier 400 to the amplification tube 411 side. ; And a fluorescence detector 450 fixed to the moving part 600 to measure the inside of the amplification tube 411 in real time.
- the light irradiation unit 440 and the fluorescence detector 450 are configured to enable real-time PCR, and the light irradiation unit 440 may use an LED.
- the fluorescent detector 450 is fixed to the moving part 600 and configured to be movable. As shown in FIGS. 3 and 4, the fluorescent detector 450 may be formed to be individually adjustable in height.
- the fluorescent detector 450 is a means capable of measuring the amount of fluorescence through imaging, and may include a photodiode, a lens, and a filter.
- the first sterilization means 800 may be located at the front side of the fluorescent detector 450 in the moving part 600 region.
- the electrophoretic unit 300 is a portion for examining the amplification product through the nucleic acid amplification unit 400, and examines the length and amount of DNA.
- the electrophoretic unit 300 is formed to include an electrophoretic body 310, a power supply unit 320, an excitation light irradiation unit 330, and a photographing unit 350, the base plate 700
- the power connection terminal 340 is formed.
- the electrophoretic body 310 is a part in which an electrophoretic process is performed by supplying a material (eg, Agarose gel) necessary for electrophoresis and separating the amplified DNA according to its length.
- the power supply unit 320 is provided on the case bottom surface 140 to supply DC power to the electrophoretic body 310.
- the power connection terminal 340 is fixed to the base plate 700 and is formed in contact with the power supply 320 to connect power to the electrophoretic body 310.
- a protrusion 341 is formed on the power connection terminal 340 to protrude toward the electrophoretic body 310 so that the power connection terminal 340 and the electrophoretic body 310 can be simultaneously supplied with power.
- the electrophoretic body 310 has a hole type electrical connection terminal 311 into which the protrusion 341 is inserted.
- the excitation light irradiation unit 330 is provided on the inner lower surface of the case 100 at the position where the electrophoretic body 310 is seated, and irradiates excitation light toward the electrophoretic body 310.
- a predetermined space on which the electrophoretic body 310 and the base plate 700 of the electrophoretic body 310 is seated is formed of a material through which the excitation light irradiated by the excitation light irradiation unit 330 is transmitted.
- the excitation light irradiation unit 330 preferably irradiates uniform light to the entire electrophoretic body 310, when formed of a plurality of LEDs, the electrophoretic body of the base plate 700 (
- the electrophoretic body fixing plate 750 which is a predetermined space in which 310 is seated, preferably has a material for scattering excitation light, for example, a light scattering material layer such as milky acrylic.
- the excitation light irradiation unit 330 is used to emit a large amount of light in the excitation light wavelength band, the bottom plate of the electrophoretic body 310 nucleic acid contained in the excitation light dispersed while passing through the base plate 700
- the light absorbs all of the fluorescence emission band and the excitation band is made of a material that passes as much as possible to minimize the background light during fluorescence detection to maximize the detection of nucleic acid.
- a blue material layer that passes light is used.
- the photographing unit 350 is configured to photograph the electrophoretic state, a lens capable of obtaining an image on an electrophoretic gel 362 on a CCD or CMOS sensor, and blocks excitation light entering the lens and generates a fluorescent wavelength band generated from DNA. It is configured as a short wavelength cut-off filter for passing the above light.
- the photographing unit 350 may photograph and transmit the progress and final result of the electrophoresis. (See FIG. 5)
- the photographing unit 350 may be formed on the upper surface of the case 100, but more preferably. Is preferably formed in the moving unit body 610 of the moving unit 600 to be able to check the state of the electrophoretic unit 300 in a more adjacent position.
- the pipette 500 is stored in the pipette rack 510. Since the photographing unit 350 is fixed to the moving unit body 610 of the moving unit 600, the photographing distance is shortened, and the photographing unit 350 can take a photograph with high sensitivity.
- the power supply unit 320 and the power connection terminal 340 is the elastic means 321 is interposed below the power supply unit 320 to be stably connected by the insertion of the base plate 700, the base As the plate 700 is inserted into the case 100, the first contact part 342 and the second contact part 322 are in contact with each other on the lower surface of the power connection terminal 340 and the lower surface of the power supply unit 320. Is preferably formed.
- the first contact portion 342 of the power connection terminal 340 is such that the first contact portion 342 of the power connection terminal 340 and the second contact portion 322 of the power supply unit 320 are in perfect contact.
- a first inclined portion 343 having an edge portion inclined toward the center of the first contact portion 342 in the insertion direction of the base plate 700 is formed, and the second contact portion 322 of the power supply 320 is Preferably, a second inclined portion 323 having an edge portion inclined toward the center of the second contact portion 322 in a direction opposite to the insertion of the base plate 700 in the longitudinal direction of the case 100 is formed.
- the base plate 700 When the base plate 700 is inserted, the first inclined portion 343 of the power connection terminal 340 and the second inclined portion 323 of the power supply unit 320 are guided in contact with each other, the elastic The means 321 is compressed, and the first contact portion 342 of the power connection terminal 340 and the second contact portion 322 of the power supply 320 are strongly in contact with each other at the final insertion position of the base plate 700. There is an advantage that can be easily transferred to the electrical transmission.
- the biological sample automatic analysis device 1000 of the present invention may use a gel tray 360 that can be electrophoresis in the entire region of the electrophoretic body 310, as shown in FIG. As shown in FIG. 23, only 1/2 samples may be electrophoresed using the gel tray 360-1 in which two electrophoretic gels 362 enter the electrophoretic body 310.
- one half biological sample and a pipette 500 may be mounted, and only one half sample may be used for the purification portion and the amplification portion, and the rest may be used later.
- the electrophoretic body 310 may be a jaw formed on the upper side so as not to overflow the solution required for electrophoresis inside.
- the gel trays 360 and 360-1 show an example in which an electrophoretic gel fixing part 361 is formed to protrude an electrophoretic gel 362 inside the lower surface of the gel trays 360 and 360-1.
- the electrophoretic gel 362 can be easily prepared by one of ordinary skill in the art and boils a solution containing 1 to 5% of agarose so that the solution may be loaded in the same position as the pipette 500 before loading. 363) is installed and the gel tray (360, 360-1) is placed on the horizontal plane with the both sides of the gel tray (360, 360-1) closed, and then the liquid gel solution is poured and cooled Form a gel. At this time, the hollow part of the gel corresponding to the electrophoretic gel fixing part 361 is formed to prevent the gel from moving in the forward and rearward directions, thereby accurately positioning the amplification product by loading the pipette 500 into the loading well 363. There is an advantage to maintain.
- the biological sample automatic analysis apparatus 1000 of the present invention may use various types of electrophoretic gels 362, and thus the shape of the electrophoretic body 310 may be variously modified in addition to the examples shown in the drawings.
- the biological sample automatic analysis device 1000 of the present invention may be further provided with a marker storage unit 900 in which a marker and a fluorescent dye are embedded in the electrophoretic unit 300.
- the marker storage unit 900 includes a marker tube 920 in which a marker and a fluorescent dye are embedded, and a marker tube rack 910 in which the marker tube 920 is mounted.
- Rack 910 is preferably provided detachably to the base plate 700 to facilitate the embedding of the marker.
- a plurality of pipettes 500 are mounted to form a column, biological samples in the entire region of the purification unit 200, nucleic acid amplification unit 400 and the electrophoretic unit 300, the sample used in the purification unit 200 or It is a structure for moving a reagent, a purified target nucleic acid, and amplified DNA.
- the pipette 500 is configured to suck and discharge the biological sample, the sample or reagent used in the purification unit 200, the purified target nucleic acid, and the amplified DNA.
- Purification is performed while moving the first to the Nth columns of the well plate kit 210, and the purified target nucleic acid is transferred to the nucleic acid amplification unit 400, and the amplified DNA is transferred to the marker tube, the electrophoretic unit 300, and the like. Perform the transfer with.
- the pipette 500 is a configuration for transferring various substances in the biological sample automatic analysis device 1000 of the present invention, a plurality of heat is formed, the pipette 500 is provided in the horizontal direction to form a single row
- the number of is used equal to the number of wells 211 positioned in the transverse direction of the multiwell plate kit 210.
- the amplification tube 411 is also preferably used to match the number of wells 211 of the pipette 500 and the multiwell plate kit 210 forming a single row of the pipette 500.
- 12 wells 211 formed in the horizontal wells 211 of the multiwell plate kit 210 are formed, and the pipette 500 shows an example of forming a single row, but the biological sample of the present invention
- the automatic analyzer 1000 is not limited thereto. More specifically, a plurality of rows may be used and 12 or more wells may be used to increase the number of test treatments.
- the moving unit 600 is a pipette block 630 through the pipette 500, the suction, discharge, removable, removable configuration, below, a detailed configuration for implementing the above operation will be described.
- the moving unit 600 may be configured using various forms, and the forms illustrated in FIGS. 3 to 8 will be described below, but the biological sample automatic analysis apparatus 1000 of the present invention is not limited thereto.
- the moving part 600 is moved in the longitudinal direction of the case 100.
- the support rod 150 is installed in the longitudinal direction inside the case 100, and the moving part body 610.
- the slider 621 is formed in the guide rod 150 can be moved in the longitudinal direction.
- the moving part 600 may be formed with an X-axis moving motor 622 and an X-axis moving belt 623 connected to the X-axis moving motor 622 to move the moving part 600. have.
- the biological sample automatic analysis apparatus 1000 includes a pipette mounting part including a pipette block fixing plate 644. 500 may be mounted on the pipette block 630.
- FIG. 6 is a view illustrating a state in which the pipette 500 is fixed to the pipette fixing protrusion 644-1 by the operation of the pipette block fixing plate 644, and the pipette fixing protrusion 644-1 is covered by the pipette 500.
- Invisible The configuration of the pipette fixing protrusion 644-1 is shown in FIG. 8)
- Pipette mounting portion is a pipette fixed protrusion 644-1, the pipette fixed protrusion 644-1, the pipette block fixing plate 644-1 is provided with a pipette 500, and the pipette block fixing plate 644 can be moved in the height direction Include configuration to ensure
- Z-axis screw 641 As a configuration to move the pipette block fixing plate 644 in the height direction, in Figure 6 Z-axis screw 641, Z-axis screw nut 645, Z-axis guide rod 646, Z-axis guide rod slide ( 647), a Z-axis motor 642 fixed to the moving unit body 610, and Z for transmitting the rotational movement of the Z-axis motor 642 to the Z-axis screw 641 to rotate the Z-axis screw 641.
- the shaft shift belt 643 An example in which the shaft shift belt 643 is formed is illustrated.
- the biological sample automatic analysis apparatus 1000 of the present invention moves the pipette block fixing plate 644 in the vertical direction as the Z-axis motor 642 is rotated, and moves by the downward direction of the pipette block fixing plate 644.
- the pipette fixing protrusion 644-1 is inserted into the upper side of the pipette 500, through which the pipette 500 may be mounted.
- pipette block fixing plate 644 moving up and down of the moving part 600 is preferably installed in the device related to the operation of the pipette 500 to enable the pipette 500 to operate stably.
- the pipette block 630 is mounted on the moving part body 610 of the pipette block fixing plate 644 is a vertical movement reference point to control the suction and discharge operation of the pipette 500, the pipette 500 to the case It can move in the longitudinal direction (X axis) and the height direction (Z axis) of 100, the configuration for adjusting the suction and discharge of the pipette 500 will be described with reference to FIG.
- Automatic biological sample analysis apparatus 1000 of the present invention through the pipette control unit including a piston 631 and the piston 631 is fixed to the upper side of the pipette block 630 and the piston fixing plate 634 which is movable in the height direction. Suction and discharge of the pipette 500 can be controlled.
- the pipette block 630 includes a pipette control unit to maintain the airtightness of the pipette 500 in a configuration to control the suction and discharge operation of the pipette 500, wherein the pipette control unit is a piston 631, the piston fixing plate 634 and means (movable means for compressing the piston 631) for moving the piston fixing plate 634 in the height direction.
- the pipette control unit is a piston 631, the piston fixing plate 634 and means (movable means for compressing the piston 631) for moving the piston fixing plate 634 in the height direction.
- Means for moving the piston fixing plate 634 in the height direction is the piston motor 632 for moving the piston 631 up and down and the piston moving screw 635, the piston fixing plate 634 rotated by the piston belt 633.
- the piston moving screw nut 636 which moves the piston fixing plate 634 up and down by the rotation of the piston moving screw 635, the piston 631 and the piston moving screw nut on the upper part of the piston 631. It may be formed including a piston fixing plate 634 for fixing the (636).
- the piston motor 632 may be provided on the uppermost plate supported by the piston guide rod 647 installed on the piston fixing plate 634.
- a piston fixing plate 634 is installed on the upper part of the piston 631, the piston 631 and the piston moving screw nut 636 ) Is fixed.
- FIG. 8 An example for implementing detachment of the pipette 500 is illustrated in FIG. 8, and the example illustrated in FIG. 8 includes a pipette extrusion plate 638-1, a pipette extrusion pin fixing plate 639, and a pipette extrusion pin 638. I use it.
- the pipette extrusion plate 638-1 has a hollow area corresponding to the pipette fixing protrusion 644-1 and when the pipette 500 is mounted to the pipette fixing protrusion 644-1, The pipette block fixing plate 644 is positioned in contact with the upper surface of the pipette 500.
- the pipette extrusion pin fixing plate 639 is in the form of a plate located on the upper side of the pipette block 630, the pipette extrusion pin 638 has a length longer than the height of the pipette block 630 and the pipette extrusion plate ( 638-1) and the pipette extrusion pin fixing plate 639.
- the biological sample automatic analysis device 1000 of the present invention when the pipette extrusion pin fixing plate 639 is moved downward by the operation of the pipette control unit, the pipette extrusion pin fixing plate 639, pipette extrusion pin 638 -1) and the pipette extrusion plate 638-1 is moved downward to push the pipette 500, so that the pipette 500 is detached from the pipette fixing protrusion 644-1.
- the pipette extrusion plate 638-1 and the pipette extrusion pin 638 are pushed by the pipette 500, as shown in FIG. 6.
- the pipette extrusion pin fixing plate 639 is pushed to the top of the pipette block 630 and positioned at a predetermined distance from the top of the pipette block 630.
- the piston moving screw 635 is rotated to move the piston fixing plate 634 downward in contact with the pipette block 630.
- the first sterilization means 800 is fixed to the moving part 600, and may use an ultraviolet lamp.
- the first sterilization means 800 is preferably fixed to the fluorescence detection unit 450 among the entire configurations forming the moving unit 600 in order to be easily positioned and close to the sterilization target site. Do. In FIG. 5, the first sterilization means 800 is fixed to the side of the fluorescence detection unit 450, and shows an example formed to irradiate UV or ozone in a downward direction. In addition, the first sterilization means 800 may be further provided with a reflecting means 810 so as to concentrate the specific effect on a specific location. The first sterilization means 800 is used for inactivation of the gene amplification product and can be used in close proximity to the gene amplification reaction vessel by the operation of the mobile unit 600 for inactivation of the gene amplification reaction product. Inactivation can be maximized.
- the biological sample automatic analysis device 1000 may further include a second sterilization means 820 made of one of an ultraviolet lamp and an ozone generator.
- the first sterilization means 800 is moved to the mobile unit 600 to sterilize each component of the purification unit 200, nucleic acid amplification unit 400, electrophoresis unit 300 and the second sterilization Means 820 may be used to sterilize the whole.
- the biological sample automatic analysis device 1000 of the present invention may be further provided with a base plate 700.
- the base plate 700 is detachably provided with the multiwell plate kit 210 and the electrophoretic body 310, and is guided by a guide part 130 fixed to the bottom surface of the case 100 to open the opening part.
- the configuration is movable in the longitudinal direction of the case 100 through 110.
- the base plate 700, as described above, the biological sample tube rack 280, pipette rack 510, and marker tube rack 910 may be provided to be mounted and detachable.
- the multiwell plate kit 210 includes a sample or a reagent necessary for purification along with a biological sample
- the electrophoretic body 310 contains a material necessary for electrophoresis
- the marker tube 920 is Markers and DNA fluorescent dyes for electrophoresis are embedded.
- the user has a multiwell plate kit 210, an electrophoretic body 310, a biological sample tube rack 280, a pipette rack 510, and a marker tube rack 910 on each base plate 700. Since the mounting and detachment are made possible, the analysis can be prepared by a simple method of mounting and inserting each component. Meanwhile, the amplification tube 411 and the amplification tube fixing part 430 are directly fixed to the heat transfer part 420 through the second exposure hole 702 of the base plate 700, and the base plate 700 ) Is inserted into the case 100 and then fixed.
- a guide part 130 is formed on the inner bottom surface of the case 100 to move the base plate 700 in the longitudinal direction, and a handle 710 is formed on one side surface adjacent to the opening part 110 so that the user The base plate 700 can be easily taken out to mount and insert necessary components.
- the biological sample automatic analysis device 1000 of the present invention is the other end of the base plate 700 in order to accurately position the base plate 700 is inserted into the case 100 is fixed (the opening portion)
- the first position fixing part 720 is formed at the side opposite to the side adjacent to the 110, and the second position fixing part fixed to a position corresponding to the first position fixing part 720 of the case 100 ( 120 is formed, the first position fixing part 720 and the second position fixing part 120 may be fixed to each other by magnetic.
- a third stationary magnet 721 is formed in the first position fixing unit 720, and the third stationary magnet 721 is fixed to the second position fixing unit 120. It was.
- the third stationary magnet 721 is located at the second position fixing unit 120, or the first position fixing unit 720 and the second position fixing unit.
- Third fixing magnets 721 coupled to the government 120 may be located, respectively, and the second position fixing part 120 may have a groove corresponding to the shape of the first position fixing part 720. It may have a form having.
- the base plate 700 may be detachably provided with a waste container 740 in which used waste liquids are stored.
- the fluids are wastes discarded in the process of purifying a biological sample, and the waste fluid container 740 is detachably provided on the base plate 700 to facilitate use.
- the controller controls the purification unit 200, the nucleic acid amplification unit 400, the electrophoretic unit 300, and the moving unit 600, and controls all components that require the remaining control.
- the biological sample automatic analysis device method of the present invention uses the biological sample automatic analysis device 1000 as described above.
- the method for automatically analyzing biological samples of the present invention qualitatively analyzes the size of amplification products through purification, amplification and quantitative electrophoresis using one device (automatic biological sample analyzing apparatus 1000 of the present invention).
- a purification step (S10) of obtaining a target nucleic acid from a biological sample An amplification step (S20) of amplifying the target nucleic acid having completed the purification step (S10); And an electrophoresis step (S30); Characterized in that it comprises a.
- the purification step (S10) is a pipette mounting step of moving the moving part 600 to insert the pipette 500 into the pipette block 630 of the fixed body; And the moving unit 600 is moved to move / mix one or more samples or reagents required for purification, each of which is embedded in the first to the Nth columns of the multiwell plate kit 210, from the biological sample included in the biological sample tube. And a target nucleic acid-containing solution obtaining step of obtaining a target nucleic acid-containing solution.
- the obtaining of the target nucleic acid-containing solution may include: a sample dissolving step of eluting the target nucleic acid by melting a biological sample containing the target nucleic acid; A target nucleic acid attachment step of attaching the target nucleic acid contained in the homogenized solution of the biological sample to the magnetic particles and removing other biological material solutions; Magnetic particle washing step to remove other impurities of the magnetic particles to which the target nucleic acid is attached; Magnetic particle drying step of removing the washing solvent contained in the magnetic particles; An acquisition step of applying an elution solution to remove the target nucleic acid attached to the magnetic particles; It includes.
- the heating unit 202 is characterized in that a heating step of heating a specific heat of the multi-well plate kit 210 is performed.
- the magnetic particle drying step is to remove the pipette 500 inserted into the moving unit body 610 of the moving unit 600, the pipette 500 is moved to insert a vacuum module 291, After moving so as not to contact the inside of the well containing the magnetic particles of the multi-well plate kit 210 is to suck the air into the vacuum pump 295 to discharge. At the same time, by heating the wells by the heating unit 202, it is possible to shorten the drying time of the cleaning solvent.
- the amplifying step (S20) is a mixing step of moving and mixing the target nucleic acid-containing solution obtained through the purification step (S10) to the amplification tube (411); And a temperature adjusting step of controlling and amplifying the temperature by the heat transfer unit 420. It may include.
- the mixing step is obtained through the purification step (S10) by fixing the pipette 500 fixed to the moving unit body 610 to move the moving unit 600
- the target nucleic acid-containing solution is transferred to the amplification tube 411 of the amplification unit and mixed with the reaction solution.
- the temperature control step is a step of controlling and amplifying the temperature by the heat transfer unit 420.
- the nucleic acid amplification unit 400 in which the amplification step (S20) is performed may be equipped with up to two rows of amplification tubes 411, using one row of amplification tubes 411, or two rows of amplification tubes ( 411) can be used later for other analysis.
- each column may be adjusted to a temperature suitable for each reaction step.
- the reverse transcription reaction solution is contained in the first column of the amplification tube 411, and the reverse transcription reaction is performed by mixing with the target nucleic acid-containing solution. It can be carried out by mixing the reaction of the reverse transcription reaction PCR.
- the first PCR reaction solution is contained in the first column of the amplification tube 411, and the first PCR reaction is performed by mixing with the target nucleic acid-containing solution, and the second PCR reaction solution is carried out in the second column of the amplification tube 411. It contains the reaction of the first PCR reaction can be carried out nested PCR reaction.
- a sealing step may be further performed between the mixing step and the temperature control step to prevent evaporation of the reaction solution.
- the sealing step is to prevent evaporation generated during the reaction because the upper portion of the amplification tube 411 is open, it may be performed by forming an evaporation preventing material on the upper portion of the reaction solution.
- the evaporation preventing material should not evaporate even at 100 ° C. and should not affect the gene amplification reaction, and should be lighter in weight than the reaction solution.
- Mineral oil may be used as an example of the anti-vaporization material, and paraffin may also be used at about 60 to 70 ° C.
- the sealing step may be omitted.
- the electrophoresis step (S30) is a marker mixing step; Performing electrophoresis; And analysis step; It is carried out including.
- the marker mixing step is moved to the amplification tube 411 containing the amplified product finally amplified through the moving unit 600, the amplified product inhaled a certain amount, the marker containing a solution containing a marker and a fluorescent dye Moving to the tube 910, it can be carried out by mixing the amplification product and the marker solution.
- the electrophoresis step is to move the amplification product mixed with a marker to the agarose gel loading well 363 of the electrophoretic body 310 to load the amplification product mixture in the agarose gel loading well, and then the power supply unit ( 320), power is supplied to the electrophoretic body 310 by the power connection terminal 340 to perform electrophoresis.
- the analyzing step (S30) is a step of irradiating the light by the excitation light irradiation unit 330, and confirming this by using the imaging unit 350.
- the preparation step (S40) may be performed before the purification step (S10).
- the preparation step (S40) to draw out the base plate 700 to the outside of the case 100, the electrophoretic body 310, pipette rack 510, multi-well plate kit 210 to the base plate 700 ),
- a second mounting step of fixing the amplification tube 411 Characterized in that it comprises a.
- an inactivation step S40 may be further performed between the amplification step S20 and the electrophoresis step S30.
- the inactivation step (S40) is for inactivating the nucleic acid amplified before the end of the gene amplification step (S20) and before the marker mixing step for performing the electrophoresis step (S30), by moving the moving unit 600 1) the step of inactivating the nucleic acid amplification product by photochemical reaction by moving the fluorescent detector 450 attached to the sterilization means 800 to the upper portion of the amplification tube 411, and irradiating ultraviolet rays by operating the first sterilization means 800. to be.
- the biological sample automatic analysis device 1000 and the method of the present invention can use a single device for the entire process of purifying, amplifying and analyzing the target nucleic acid from the biological sample, and improve the analysis reliability and efficiency. There is an advantage.
- the present invention can perform the antigen concentration acquisition method using a quantitative immunity PCR using the biological sample automatic analysis device 100, through which, by performing a quantitative immunity PCR, the concentration of the antigen contained in the biological sample It is possible.
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Abstract
Description
Claims (42)
- 일정 영역을 개폐 가능한 개방부(110)가 형성된 케이스(100);복수개의 웰(211)이 제1열 내지 제N열을 형성하되, 상기 제1열 내지 제N열 중 특정한 열에 각각 생물학적 시료, 정제에 필요한 하나 이상의 시료 또는 시약이 내장된 멀티웰플레이트 키트(210)를 포함하여 상기 생물학적 시료로부터 타겟핵산을 정제하는 정제부(200);상기 정제부(200)에 의해 정제된 타겟핵산을 증폭하는 핵산증폭부(400);전기영동몸체(310)를 포함하여 상기 핵산증폭부(400)를 통해 증폭산물을 검사하는 전기영동부(300);복수개가 열을 이루며, 생물학적 시료, 상기 정제부(200)에서 이용되는 시료 또는 시약, 정제된 타겟핵산, 증폭된 DNA를 이동하는 피펫(500);상기 피펫(500)을 상기 케이스(100)의 길이방향 및 높이방향으로 이동하고, 상기 피펫(500)의 작동을 조절하는 이동부(600); 및상기 정제부(200), 핵산증폭부(400), 전기영동부(300), 및 이동부(600)를 제어하는 제어부(미도시); 를 포함하는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제1항에 있어서,상기 생물학적 시료 자동 분석 장치(1000)는상기 멀티웰플레이트 키트(210), 전기영동몸체(310)가 탈착가능하게 구비되며, 상기 케이스(100) 하면에 고정된 가이드부(130)에 의해 안내되어 상기 개방부(110)를 통해 상기 케이스(100)의 길이방향으로 이동가능한 베이스 플레이트(700); 가 더 구비되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제1항에 있어서,상기 생물학적 시료 자동 분석 장치(1000)는상기 이동부(600)에 고정되는 제1멸균수단(800)이 더 구비되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제3항에 있어서,상기 제1멸균수단(800)은 자외선램프이고, 반사수단(810)을 더 포함하는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제2항에 있어서,상기 정제부(200)는자석(221)을 포함하여 상기 멀티웰플레이트 키트(210)의 특정 열에 자기장을 인가하는 자기장 인가부(201); 및상기 자기장 인가부(201)에 인접하게 형성되어 동일한 특정 열을 가열하는 히팅부(202); 를 포함하는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제5항에 있어서,상기 정제부(200)는 타겟핵산을 추출하기 위한 생물학적 시료를 넣어주는 생물학적 시료 튜브(281)를 포함하는 생물학적 시료 튜브랙(280)을 더 포함하며,상기 생물학적 시료 튜브랙(280)이 상기 베이스 플레이트(700)에 탈착가능하게 구비되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제5항에 있어서,상기 자기장 인가부(201)는 자석(221)이 장착되는 자석장착부(220);상기 케이스(100)의 하면에 장착되어 상기 자석장착부(220)를 상승 및 하강시키는 승강부(270);를 포함하는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제5항에 있어서,상기 멀티웰플레이트 키트(210)의 제1열 내지 제N열에 내장된 정제에 필요한 하나 이상의 시료 또는 시약은 효소, 세포용해용액, 핵산결합용액, 자성입자 수분산액, 및 세척용액 중 하나 이상을 포함하는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제7항에 있어서,상기 자석장착부(220)는 상기 자석(221)이 장착된 면에 상기 멀티웰플레이트 키트(210) 특정열의 하부가 인입되도록 인입홈(222)이 형성되고,상기 베이스 플레이트(700)는 상기 자석장착부(220)의 상승 시 특정열의 하부가 상기 인입홈(222)에 안착되도록 일정영역이 중공되는 제1노출공(701)이 형성되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제9항에 있어서,상기 자석장착부(220)는 금속재질로 형성되고, 상기 히팅부(202)는 상기 자석장착부(220)에 접촉 형성되는 발열필름인 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제5항에 있어서,상기 베이스 플레이트(700)는상기 멀티웰플레이트 키트(210) 하측에서 복수개의 웰(211) 사이에 위치되도록 돌출형성되는 돌출고정부(730) 및 상기 돌출고정부(730)에 구비되어 웰(211)과 밀착되는 탄성수단(731)이 더 형성되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제8항에 있어서,상기 베이스 플레이트(700)는 사용 후 페기되는 용액이 저장되는 폐액통(740)이 탈착가능하게 구비되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제5항에 있어서,상기 생물학적 시료 자동 분석 장치(1000)는상기 이동부(600)가 상기 피펫(500)을 장착 및 탈착가능한 피펫블록(630)을 포함하며,상기 베이스 플레이트(700)에 상기 피펫(500)을 보관 가능한 피펫랙(510)이 탈착가능하게 구비되는 것을 특징으로 하는 생물학적 시료의 자동 분석 장치.
- 제13항에 있어서,상기 이동부(600)의 피펫블록(630)은하측에 상기 피펫(500)이 장착되는 피펫고정돌출부(644-1)가 형성되고 높이방향으로 이동가능한 피펫블록고정판(644)을 포함하는 피펫장착부를 통해 상기 피펫(500)이 상기 피펫고정돌출부(644-1)에 장착가능한 것을 특징으로 하는 생물학적 시료의 자동 분석 장치.
- 제14항에 있어서,상기 이동부(600)의 피펫블록(630)은상측에 피스톤(631) 및 상기 피스톤(631)이 고정되며 높이방향으로 이동가능한 피스톤고정판(634)을 포함하는 피펫조절부를 통해 상기 피펫(500)의 흡입 및 토출이 조절되는 것을 특징으로 하는 생물학적 시료의 자동 분석 장치.
- 제15항에 있어서,상기 이동부(600)의 피펫블록(630)은상기 피펫고정돌출부(644-1)에 대응되는 영역이 중공되며 상기 피펫(500)이 장착되었을 때, 피펫블록고정판(644) 하면과 피펫(500) 상면 사이에 맞닿게 위치되는 피펫압출판(638-1),상기 피펫블록(630)의 상측에 위치되는 피펫압출핀고정판(639), 및상기 피펫블록(630)의 높이보다 긴 길이를 가지며 상기 피펫압출판(638-1)과 상기 피펫압출핀고정판(639)를 연결하는 피펫압출핀(638)을 포함하고,상기 피펫조절부의 작동에 의해 상기 피펫압출핀고정판(639)이 하측방향으로 이동되면, 상기 피펫압출핀고정판(639), 피펫압출핀(638-1) 및 피펫압출판(638-1)이 하측으로 이동되어 상기 피펫(500)이 상기 피펫고정돌출부(644-1)로부터 탈착되는 것을 특징으로 하는 생물학적 시료의 자동 분석 장치.
- 제13항에 있어서,상기 정제부(200)는상기 이동부(600)에 탈/부착 가능한 진공모듈(291)과, 상기 케이스(100) 내부 하면에 장착되어 상기 진공모듈(291)을 일정위치에 보관하는 진공모듈랙(293)과, 상기 진공모듈(291)과 호스로 연결되는 진공펌프(295)를 포함하는 건조부(290)를 더 포함하는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제12항에 있어서,상기 건조부(290)는 상기 진공모듈랙(293)의 상면에 볼록부(294)가 돌출형성되며, 상기 진공모듈(291)의 하면에 상기 볼록부(294)에 대응되는 오목부(292)가 형성되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제18항에 있어서,상기 건조부(290)는상기 진공모듈랙(293)에 상기 진공모듈(291)의 일측을 지지하는 지지부(293-1)가 구비되며,상기 지지부(293-1)와 진공모듈(291)은 서로 맞닿는 면에 자석이 내장되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제2항에 있어서,상기 전기영동부(300)는상기 베이스 플레이트(700)에 설치된 전기영동몸체고정판(750)에 안착되며, 각각 양극과 음극을 형성하는 전극선(311a)이 각각 연결된 구멍형전기접속단자(311)들이 형성된 전기영동몸체(310); 전원을 연결하기 위해 전기영동몸체(310)의 구멍형전기접속단자(311)에 삽입되는 돌출형전기접속단자(341); 상기 케이스(100) 하부 내면에 고정되어 상기 전기영동몸체(310)에 전원을 공급하는 전원공급부(320); 상기 전기영동몸체(310)에 여기광을 조사하는 여기광조사부(330); 및 전기영동 상태를 촬영하는 촬영부(350);를 포함하여 형성되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제20항에 있어서,상기 전기영동부(300)는상기 베이스 플레이트(700)가 상기 케이스(100)에 삽입됨에 따라 상기 전원연결단자(340)의 하면과 상기 전원공급부(320)의 상면이 서로 접촉 연결되는 제1접촉부(342) 및 제2접촉부(322)가 경사지게 형성되며,상기 전원공급부(320)의 제2접촉부(322) 하측에 탄성수단(321)에 의해 밀착되는 것을 특징으로 하는 생물학적 시료의 자동 분석 장치.
- 제20항에 있어서,상기 여기광조사부(330)는 상기 전기영동몸체(310) 측으로 여기광 파장대의 빛을 조사하는 LED가 상기 케이스(100) 내부 하면에 등간격으로 배열되어 구비되며,상기 베이스 플레이트(700)의 상기 전기영동몸체(310)가 안착되는 전기영동몸체고정판(750)이 여기광을 산란하는 소재로 형성되고,상기 전기영동몸체(310)의 바닥판은 핵산형광 발광대의 빛을 흡수하며 여기광대의 빛은 통과하는 재질로 형성된 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제20항에 있어서,상기 촬영부(350)는 이미지센서, 렌즈, 단파장필터를 포함하여 형성되고,상기 이동부(600)의 이동부몸체(610)에 고정되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제20항에 있어서,상기 전기영동부(300)는마커(marker)와 형광염색물질이 내장된 마커튜브(920)와, 상기 마커튜브(920)가 장착된 마커튜브랙(910)을 포함하는 상기 마커저장부(900)가 더 구비되며,상기 마커튜브랙(910)은 상기 베이스 플레이트(700)에 탈착가능하게 구비되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제20항에 있어서,상기 전기영동몸체(310)는 겔트레이(360, 360-1)가 구비되며,상기 겔트레이(360, 360-1)는 바닥에 전기영동 로딩웰(363) 및 전기영동 이동로와 겹쳐지지 않는 위치에 복수개의 전기영동겔고정부(361)가 형성된 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제2항에 있어서,상기 핵산증폭부(400)는하나 이상의 열을 형성하는 증폭튜브(411);하부와 밀착되는 오목한 형태의 안착홈(421)들이 형성되고 온도센서가 삽입된 금속으로 이루어진 증폭블록(410-1);제1고정자석(422)이 구비된 단열재질로 된 증폭블록커버(410);펠티어소자(423);펠티어소자(423)에서 발생되는 열을 외부로 전달하는 열전달부(420); 및상기 증폭튜브(411)의 상부면보다 작은 구멍들이 형성되며, 상기 증폭튜브(411)의 상측 일정 영역을 감싸도록 형성되고, 상기 제1고정자석(422)과 대응되는 제2고정자석(431)이 삽입되어 상기 증폭튜브(411)를 고정하되, 상기 증폭블록커버(410)가 형성된 면과 일정거리 이격되는 높이를 갖는 증폭튜브고정부(430); 를 포함하는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제26항에 있어서,상기 베이스 플레이트(700)는 상기 증폭블록(410-1) 및 증폭튜브고정부(430)가 상기 증폭튜브고정부(430)에 대응되도록 중공된 제2노출공(702)이 형성되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제26항에 있어서,상기 핵산증폭부(400)가 상기 개방부(110)에 인접하게 위치되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제26항에 있어서,상기 핵산증폭부(400)는상기 증폭튜브(411) 측으로 형광여기광을 조사하는 빛조사부(440); 및상기 증폭튜브(411) 상부에 밀착될수 있도록 상하방향으로 이동 가능하게 구비되어 증폭튜브(411)내의 형광량을 실시간으로 측정가능한 형광디텍터(450)를 더 포함하는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제2항에 있어서,상기 베이스 플레이트(700)는 상기 개방부(110)에 인접한 일측 상면에 손잡이(710)가 형성되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제1항에 있어서,상기 케이스(100)는 외측 하면에 공기가 유동되는 유로를 형성하는 공기유동부(142)와, 상기 공기유동부(142)로 공기를 송풍하는 송풍기(141)가 더 구비되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제2항에 있어서,상기 생물학적 시료 자동 분석 장치(1000)는상기 베이스 플레이트(700)의 타측 단부에 제1위치고정부(720); 및상기 제1위치고정부(720)에 대응되는 위치의 상기 케이스(100)에 고정되는 상기 제2위치고정부(120); 가 더 형성되어,상기 제1위치고정부(720) 및 제2위치고정부(120)는 제3고정자석(721)의 자성에 의해 서로 고정되는 것을 특징으로 하는 생물학적 시료 자동 분석 장치.
- 제1항 내지 제32항 중 선택되는 어느 한 항의 생물학적 시료 자동 분석 장치(1000)를 이용한 분석 방법에 있어서,생물학적 시료로부터 타겟핵산을 획득하는 정제 단계(S10);상기 정제 단계(S10)를 완료한 타겟핵산을 증폭하는 증폭 단계(S20); 및전기영동 단계(S30); 를 포함하는 생물학적 시료의 자동 분석 방법.
- 제33항에 있어서,상기 생물학적 시료의 자동 분석 방법은상기 정제 단계(S20) 이전에상기 케이스(100)의 외부로 베이스 플레이트(700)를 인출하고, 상기 베이스 플레이트(700)에 전기영동몸체(310), 피펫랙(510), 멀티웰플레이트 키트(210), 생물학적 시료 튜브랙(280), 폐액통(740), 마커튜브랙(910) 및 마커튜브(920)를 장착하는 제1장착 단계;상기 제1위치고정부(720)와, 제2위치고정부(120)가 서로 맞닿아 고정되도록 상기 베이스 플레이트(700)를 인입하는 인입 단계; 및베이스 플레이트(700)의 제2노출공(702)을 통해 노출된 증폭블록(410-1)에 증폭튜브(411)를 안착하고, 상기 증폭블록커버(410)의 제1고정자석(423)과, 증폭튜브고정부(430)의 제2고정자석(431)에 의해 증폭튜브(411)를 고정하는 제2장착 단계; 를 포함하는 준비 단계(S40)가 수행되는 것을 특징으로 하는 생물학적 시료의 자동 분석 방법.
- 제34항에 있어서,상기 정제 단계(S10)는이동부(600)가 이동되어 피펫블록(630)에 피펫(500)을 장착하는 피펫 장착 단계; 및상기 이동부(600)가 이동되어 상기 멀티웰플레이트 키트(210)의 제1열 내지 제N열에 각각 내장된 생물학적 시료, 정제에 필요한 하나 이상의 시료 또는 시약을 이동/혼합하여 자성입자를 제외한 혼합물인 타겟핵산 함유용액 획득하는 타겟핵산 함유용액 획득 단계; 를 포함하되,필요에 따라 자기장 인가부(201)에 의해 상기 멀티웰플레이트 키트(210)의 특정 열에 자기장을 인가하는 자기장 인가 단계 및 히팅부(202)에 의해 상기 멀티웰플레이트 키트(210)의 특정 열을 가열하는 가열 단계가 수행되는 것을 특징으로 하는 생물학적 시료의 자동 분석 방법.
- 제35항에 있어서,상기 타겟핵산 함유용액 획득 단계는상기 정제부(200)에서 생물학적 시료를 용해하여 핵산을 용출하는 시료 용해 단계;타겟핵산에 자성입자를 붙이고 여액을 제거하는 타겟핵산 부착 단계;상기 타겟핵산이 부착된 자성입자를 세척하여 불순물을 제거하는 자성입자세척 단계;상기 이동부몸체(610)에 장착된 피펫(500)이 탈착되고, 상기 이동부(600)의 이동부몸체(610)에 진공모듈(291)이 고정되며, 상기 멀티웰플레이트 키트(210)의 특정 열의 세척용매를 흡입하여 자성입자를 건조하는 자성입자 건조 단계; 및건조된 자성입자에 용출버퍼를 가하여 타겟핵산을 획득하는 획득 단계;를 포함하는 생물학적 시료의 자동 분석 방법.
- 제33항에 있어서,상기 증폭 단계(S20)는상기 정제 단계(S10)를 통해 획득된 타겟핵산 함유용액을 증폭튜브(411)에 이동하여 혼합하는 혼합 단계; 및열전달부(420)에 의해 온도를 조절하여 증폭하는 온도 조절 단계;를 포함하는 것을 특징으로 하는 생물학적 시료의 자동 분석 방법.
- 제33항에 있어서,상기 전기영동 단계(S30)는 증폭산물과 마커용액을 혼합 마커 혼합 단계;전원공급부(320), 전원연결단자(340)에 의해 전기영동몸체(310)에 전원을 공급하여 증폭산물을 분리하는 전기영동 수행 단계; 및상기 여기광조사부(330)에 의해 빛을 조사하며, 촬영부(350)를 이용하여 증폭산물의 전기영동 패턴을 측정하고 분자량을 분석하는 분석 단계; 를 포함하는 것을 특징으로 하는 생물학적 시료의 자동 분석 방법.
- 제33항에 있어서,상기 생물학적 시료의 자동 분석 방법은상기 증폭 단계(S20)와 전기영동 단계(S30) 사이에 제1멸균수단(800)을 가동하여 증폭된 핵산을 불활화하는 불활화 단계(S50)가 더 수행되는 것을 특징으로 하는 생물학적 시료의 자동 분석 방법.
- 제37항에 있어서,상기 증폭 단계(S20)는빛조사부(440)에서 일정한 여기광을 조사하면서 일정시간 동안 또는 매 사이클마다 발생되는 형광량을 형광디텍터(450)로 측정하여 임계형광값이 검출되는 시점을 검출하여 초기의 핵산농도를 계산하는 실시간 정량증폭분석이 가능한 것을 특징으로 하는 생물학적 시료의 자동 분석 방법.
- 제40항에 있어서,상기 생물학적 시료의 자동 분석 방법은상기 증폭 단계(S20)에서 얻어진 정량증폭 분석값을 상기 전기영동 단계(S30)를 통해 얻어진 분자량 및 증폭산물의 개수를 포함하는 결과를 통해 보정 및 분석 가능한 것을 특징으로 하는 생물학적 시료의 자동 분석 방법.
- 제1항 내지 제32항 중 선택되는 어느 한 항의 생물학적 시료 자동 분석 장치(1000)를 이용한 정량면역PCR을 수행함으로써 생물학적 시료에 함유된 항원의 농도를 정량검사하는 정량면역PCR을 이용한 항원농도획득방법.
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CN106676002A (zh) * | 2017-03-08 | 2017-05-17 | 深圳市爱康生物科技有限公司 | 一种应用于自动核酸提取仪的自动磁力架 |
CN108277148A (zh) * | 2018-04-25 | 2018-07-13 | 江苏硕世生物科技股份有限公司 | 核酸提取分液装置 |
CN108277148B (zh) * | 2018-04-25 | 2024-05-28 | 江苏硕世生物科技股份有限公司 | 核酸提取分液装置 |
CN111235008A (zh) * | 2020-02-29 | 2020-06-05 | 深圳市朗司医疗科技有限公司 | 一种封闭性一体化病原体检测卡盒 |
CN111624355A (zh) * | 2020-06-10 | 2020-09-04 | 波音特生物科技(南京)有限公司 | 一种胰岛素检测用易于大量筛查的诊断试剂盒 |
Also Published As
Publication number | Publication date |
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EP2813579B1 (en) | 2017-04-05 |
US9765383B2 (en) | 2017-09-19 |
IN2014MN01572A (ko) | 2015-05-08 |
CA2864260C (en) | 2018-05-01 |
CN104204229A (zh) | 2014-12-10 |
JP5908613B2 (ja) | 2016-04-26 |
RU2014136713A (ru) | 2016-04-10 |
EP2813579A1 (en) | 2014-12-17 |
BR112014019798A2 (pt) | 2021-06-01 |
US20150037803A1 (en) | 2015-02-05 |
CN104204229B (zh) | 2016-09-07 |
JP2015506710A (ja) | 2015-03-05 |
EP2813579A4 (en) | 2015-09-16 |
RU2610687C2 (ru) | 2017-02-14 |
AU2013218402A1 (en) | 2014-09-04 |
BR112014019798B1 (pt) | 2022-03-03 |
KR101762295B1 (ko) | 2017-08-04 |
CA2864260A1 (en) | 2013-08-15 |
AU2013218402B2 (en) | 2016-07-07 |
KR20130092185A (ko) | 2013-08-20 |
BR112014019798A8 (pt) | 2017-07-11 |
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