WO2016104601A1 - Method for analyzing nucleic acid, and fluorescence/turbidity measurement device used therein - Google Patents

Method for analyzing nucleic acid, and fluorescence/turbidity measurement device used therein Download PDF

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Publication number
WO2016104601A1
WO2016104601A1 PCT/JP2015/086024 JP2015086024W WO2016104601A1 WO 2016104601 A1 WO2016104601 A1 WO 2016104601A1 JP 2015086024 W JP2015086024 W JP 2015086024W WO 2016104601 A1 WO2016104601 A1 WO 2016104601A1
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WIPO (PCT)
Prior art keywords
container
fluorescence
nucleic acid
measurement
dna
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PCT/JP2015/086024
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French (fr)
Japanese (ja)
Inventor
一興 五十島
真樹 伊澤
文典 牧
雅和 木谷
晋治 金山
祐康 米田
Original Assignee
立山マシン株式会社
株式会社ニッポンジーン
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Application filed by 立山マシン株式会社, 株式会社ニッポンジーン filed Critical 立山マシン株式会社
Priority to JP2016566440A priority Critical patent/JP6751352B2/en
Publication of WO2016104601A1 publication Critical patent/WO2016104601A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M1/00Apparatus for enzymology or microbiology
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS 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
    • C12M1/00Apparatus for enzymology or microbiology
    • C12M1/34Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01Arrangements or apparatus for facilitating the optical investigation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated

Definitions

  • the present invention relates to a nucleic acid analysis method and a fluorescence / turbidity measurement apparatus used therefor. More specifically, the present invention relates to a nucleic acid analysis method that can be used for genetic testing and a fluorescence / turbidity measurement apparatus used therefor. More specifically, the present invention is a method of amplifying deoxyribonucleic acid (DNA) extracted from a living body, detecting DNA amplification, and DNA single nucleotide polymorphism (SNP or SNPs). The present invention relates to a device capable of both detecting SNPs), a method for detecting DNA amplification using this device, and a nucleic acid analysis method including a method for detecting SNPs.
  • DNA deoxyribonucleic acid
  • SNP DNA single nucleotide polymorphism
  • a method of detecting DNA using a DNA extracted from a living body such as a method using ultraviolet light or a method using electric peristalsis, is used.
  • DNA extracted from a living body is used as it is, the number of DNA is small, so detection sensitivity is low and the possibility of erroneous detection is high. Therefore, generally, DNA extracted from a living body is amplified before DNA is detected. The method is taken.
  • a method for amplifying DNA and a method for detecting DNA a plurality of methods have been put into practical use according to the type of DNA, and devices corresponding to each method are manufactured and sold.
  • LAMP Loop-Mediated Isometric Amplification
  • DNA amplification process the double strands of DNA are cut into two, and substances are bound to the respective strands to create DNA having the same double-stranded structure as the original DNA. By repeating this, DNA is amplified. At this time, as the DNA is amplified, the light intensity of the fluorescence from the fluorescent substance that binds to the DNA increases, or the solution becomes cloudy due to pyrophosphate generated in the DNA synthesis reaction. Whether or not DNA amplification is possible is determined by detecting changes in the fluorescence intensity of the solution, changes in the fluorescence intensity, or a decrease in light intensity or a change in light intensity due to turbidity.
  • the catalyst is deactivated by heating at a predetermined temperature (any temperature from 80 ° C. to 100 ° C., which differs for each DNA) for a predetermined time (any time from 2 minutes to 10 minutes, which differs for each DNA).
  • the FLP method uses a substance that binds a phosphor to a substance that binds to DNA and a substance that binds a substance that quenches the phosphor to a substance that binds to DNA, and regularly binds both to DNA.
  • FLP Fluorescence Loop Primer
  • the method for regularly binding both to DNA is as follows. It is.
  • a substance in which a phosphor is bound to a substance that binds to DNA a substance (referred to as FLP) in which a phosphor is bound to a substance that binds to a single strand of DNA during DNA amplification is used.
  • a substance in which a substance that quenches a substance that binds to the base of the SNPs to be detected is used as a substance that binds a substance that quenches the phosphor to a substance that binds to DNA.
  • QP Quencher Probe
  • the DNA double strand is separated into two by lowering or raising the temperature from high temperature (about 100 ° C.) to low temperature (about 40 ° C.) and DNA
  • QP binds instead of FLP and the fluorescence is quenched.
  • the bonding temperature differs depending on the type of SNPs, the type of SNPs is determined by detecting the fluorescence light intensity or the change in fluorescence light intensity.
  • Non-patent Document 1 I-densy / Quenching Probe method
  • Non-patent Document 2 ABI Prism 7900 HT / TaqMan method
  • Non-patent Document 3 -LightCycler / melting curve analysis
  • Non-patent Document 3 -LightCycler 480 / High-resolution Melting Analysis, Hybridization Probe method, Invader-plus method, TaqMan method
  • Patent Document 3 ABI Prism 7700 / LAMP method
  • Patent Document 1 Japanese Unexamined Patent Publication No. 2013-31463
  • Non-Patent Document 1 Sensors 2012, 12, 16614-16627
  • Non-Patent Document 2 Cancer Epidemiol Biomarkers Prev 2007
  • Non-Patent Document 3 JOURNAL OF CLINICAL MICROBIOLOGY, May 2011, p. 1853-1860
  • the entire descriptions of Patent Document 1 and Non-Patent Documents 1 to 3 are specifically incorporated herein by reference.
  • Devices using the LAMP method for amplifying DNA are manufactured and sold (see Patent Document 1).
  • amplification of DNA cannot be confirmed, and after use of the device, the state of the reagent is visually confirmed by a human.
  • devices that can automatically detect that DNA exceeds a predetermined amount are manufactured and sold, but the types are small and expensive.
  • either the method of detecting the light intensity of a fluorescent substance mixed with DNA or the intensity of transmitted light of a turbid substance mixed with DNA is detected. There is no device that can detect automatically at the same time.
  • Devices using the FLP method for detecting DNA SNPs are manufactured and sold. However, in these devices, human beings visually check the measured characteristics with numerical data or graphs to determine SNPs, and for that purpose, specialized knowledge is required. Furthermore, there are few types of equipment and they are expensive. At present, there is no device that can automatically detect SNPs. Furthermore, in combination with the LAMP method for amplifying DNA, after DNA amplification, there are few devices that can detect DNA SNPs without removing reagents, and DNA amplification detection / confirmation and DNA SNPs detection There is no equipment that can do both automatically.
  • Non-Patent Documents 1 to 3 SNPs detection detects fluorescence, but detection and confirmation of DNA amplification as a detection target is performed visually. There is no known apparatus capable of performing both detection and confirmation of DNA (nucleic acid) amplification and detection of SNPs with fluorescence.
  • an apparatus for nucleic acid analysis that can be used in combination of the LAMP method for amplifying DNA and the FLP method for detecting SNPs of DNA, which does not require specialized knowledge and can be handled with simple operations.
  • an apparatus that enables the above-mentioned.
  • provision of an automatic detection device for DNA SNPs using this apparatus is also desired.
  • the present invention has been made for the purpose of solving the above-mentioned problems. While amplifying the DNA, it is detected that the amount of the predetermined DNA has been exceeded, and then the catalyst for amplifying the DNA is lost. It is an apparatus that automatically performs a process of detecting and detecting SNPs of DNA by a simple operation. Hereinafter, each claim will be described.
  • a sample holder having at least one container storage unit for storing a container for containing a measurement sample, wherein the container storage unit of the sample holder has one vertical penetration in the depth direction of the container storage unit at the bottom
  • the side wall near the bottom and the bottom has two lateral through-holes whose openings are opposed to each other in the direction perpendicular to the depth direction of the container storage unit,
  • a light source for irradiating light to the container stored in the container storage unit, An element for measuring turbidity of a measurement sample in a container stored in the container storage unit, An element for fluorescence measurement emitted from a measurement sample in a container stored in the container storage unit,
  • a temperature adjusting device for adjusting the temperature of the container stored in the container storage unit,
  • a fluorescence and turbidity measuring device comprising: The light source and the container storage are in communication via one of the two lateral through-holes; The container storage and the turbidity measuring element communicate with each other through the other of the two lateral through
  • the light source, the container storage and the turbidity measuring element are positioned in a substantially horizontal relationship;
  • the fluorescence measuring element is located substantially below the container storage;
  • the light source is an LED;
  • the fluorescence measuring element is a photo IC;
  • the turbidity measuring element is a photodiode;
  • the LED has a peak wavelength of 465 nm, a half width of about 20 nm
  • the photo IC has a wavelength band of 500 to 580 nm
  • the photodiode has a wavelength band of 450 to 500 nm
  • the wavelength filter is a filter that cuts a wavelength of 520 nm or less.
  • the temperature adjusting device includes a heating unit provided inside the sample holder, a blower for blowing air toward the sample holder, and a temperature measuring unit provided inside the sample holder or adjacent to the sample holder. Including, The device according to any one of [1] to [4]. [6] A container lid pressing member for pressing the lid of the container stored in the container storage part of the sample holder, which can approach and separate from the sample holder, at a position facing the opening of the container storage part of the sample holder. The device according to any one of [1] to [5], wherein the container lid pressing member includes a temperature adjusting device.
  • [7] The apparatus according to any one of [1] to [6], which is used to measure turbidity and fluorescence in the container simultaneously or sequentially.
  • the method wherein the nucleic acid is dissociated by observing fluorescence emission or quenching.
  • Fluorescence measurement of the solution in the container is performed by changing the temperature of the solution from a high temperature to a low temperature to hybridize single-stranded nucleic acids and observe fluorescence emission or quenching, [10] the method of.
  • the fluorescence measurement of the solution in the container is performed by observing fluorescence emission or quenching by dissociating the double-stranded nucleic acid by changing the temperature of the solution from a low temperature to a high temperature.
  • nucleic acid amplification using the measurement sample as a template is performed by an isothermal nucleic acid amplification method.
  • isothermal nucleic acid amplification method is a LAMP method.
  • Nucleic acid amplification by the isothermal nucleic acid amplification method is performed at a temperature of 40 to 70 ° C., and after completion of the amplification, the solution is heated to 90 to 100 ° C. to dissociate the amplified double-stranded nucleic acid into a single-stranded nucleic acid.
  • the present invention it is possible to provide an apparatus having both a function of detecting and confirming DNA amplification by the LAMP method and a function of detecting SNPs.
  • the apparatus of the present invention it is possible to provide a nucleic acid analysis method for detecting and confirming amplification of DNA by the LAMP method and detecting ability and SNPs with one apparatus. It can also be performed automatically, including confirmation of completion of amplification.
  • FIG. 1 is a perspective view of a measuring unit 10 of the apparatus of the present invention.
  • FIG. 2 is a perspective view of the measuring unit 10 and the fan 50 of the apparatus of the present invention.
  • FIG. 3 is a perspective sectional view of the measuring unit 10 of the apparatus of the present invention.
  • FIG. 4 is a schematic cross-sectional explanatory view of the measuring unit 10 of the device of the present invention.
  • FIG. 5 is an explanatory view of the sample holder 11 of the apparatus of the present invention.
  • FIG. 6 is an explanatory view of the container (tube) lid pressing portion 14 of the apparatus of the present invention.
  • FIG. 7 is a diagram showing a DNA amplification process, a DNA amplification detection process, a catalyst deactivation process, and a DNA SNP detection process using the solution containing the DNA of the embodiment of the present invention. It is.
  • FIG. 8 is a temperature graph at the time of measurement.
  • FIG. 9 is a light intensity graph at the time of turbidity measurement.
  • FIG. 10 is a turbidity graph.
  • FIG. 11 is a fluorescence light intensity graph at the time of fluorescence measurement.
  • FIG. 12 is a fluorescence change graph.
  • FIG. 13 is a list of detection results.
  • the device of the present invention A sample holder 11 having at least one container storage portion 11a for storing a container 1 for storing a measurement sample; A light irradiating light source 20 for the container 1 stored in the container storage portion 11a, The element 30 for measuring turbidity of the measurement sample in the container 1 stored in the container storage unit 11a, The fluorescence measuring element 40 emitted from the measurement sample in the container 1 stored in the container storage unit 11a; A temperature adjusting device for heating and cooling the container 1 stored in the container storage section 11a; including.
  • the sample holder 11 is a sample holder 11 having at least one container storage portion 11a for storing the container 1 for storing the measurement sample.
  • the container 1 for storing the measurement sample is light transmissive at least in the wavelength range related to the measurement of turbidity and fluorescence from the viewpoint of not hindering the turbidity and fluorescence measurement of the measurement sample accommodated in the container 1.
  • the light from the light source is light-transmitting with respect to light in a wavelength range related to the measurement of turbidity and fluorescence.
  • An example of such a container is a reaction tube that is transparent in the visible light region.
  • the container holder 11a of the sample holder 11 has one vertical through hole 11c in the depth direction of the container storage portion at the bottom and an opening in the container storage portion in a direction perpendicular to the depth direction of the container storage portion in the side wall near the bottom.
  • one of the horizontal through-holes is for communicating between the light source 20 and the container storage portion 11a, so that light irradiation from the light source 20 to the container storage portion 11a can be ensured without excess or deficiency. It can have a shape and dimensions.
  • the other lateral through-hole is for communicating between the container storage portion 11a and the turbidity measuring element 30, and the turbidity measurement of the measurement sample in the container stored in the container storage portion 11a is performed.
  • the element 30 can be shaped and dimensioned so that the light transmitted through the measurement sample in the container can reach the turbidity measuring element 30 without excess or deficiency so that measurement can be performed.
  • the vertical through hole is for communicating between the container storage portion 11a and the fluorescence measurement element 40, and the fluorescence emitted from the measurement sample in the container stored in the container storage portion 11a is used for fluorescence measurement.
  • the element 40 can have a shape and a dimension so that the fluorescence emitted from the measurement sample in the container can reach the fluorescence measurement element 40 without excess or deficiency so that measurement can be performed.
  • the number of container storage portions provided in the sample holder 11 is not particularly limited, and can be appropriately determined according to the number of measurement samples to be simultaneously measured in the apparatus of the present invention.
  • the number of container storage units provided in the sample holder 11 is, for example, in the range of 1 to 20, and can be in the range of 2 to 10. However, it is not intended to be limited to these ranges, but matters that can be determined as appropriate.
  • each container storage portion has one vertical through hole 11c in the depth direction of the container storage portion at the bottom and an opening in the container storage portion in a direction perpendicular to the depth direction of the container storage portion in the side wall near the bottom. There are two lateral through-holes 11b and 11d facing each other.
  • the shape and size of the container storage unit can be appropriately determined according to the shape and size of the container stored in the container storage unit.
  • the container can be a plastic tube that is widely used for experiments on biological samples, and the shape and dimensions of the container storage portion can be appropriately determined according to the shape and dimensions of the plastic tube.
  • the light irradiation light source 20 is for irradiating the container 1 stored in the container storage unit 11a with light for measuring turbidity and fluorescence.
  • the light source 20 for light irradiation is not particularly limited as long as it is a light source having a wavelength and a light amount, but it is an LED from the viewpoint that energy conversion efficiency is high and light irradiation in a desired wavelength region can be relatively easily performed.
  • the LED preferably has a peak wavelength of 465 nm and a full width at half maximum of about 20 nm from the viewpoint of both turbidity measurement and fluorescence measurement.
  • the present invention is not intended to be limited to such an LED, and an LED having another peak wavelength and a full width at half maximum can be appropriately used as long as turbidity measurement and fluorescence measurement are possible.
  • the light irradiation light source 20 is housed in a hermetically sealed light source unit light shielding unit 21, and the light source light shielding unit 21 stores therein the light irradiation light source 20, and the opening of the light source unit light shielding unit 21 is a container storage unit. It is connected to the external opening of the lateral through hole 11b of 11a. Irradiation light from the light irradiation light source 20 is applied to the container 1 stored in the container storage portion 11a through the lateral through hole 11b of the container storage portion 11a.
  • the turbidity measuring element 30 is an element for measuring the turbidity of the measurement sample in the container 1 stored in the container storage portion 11a.
  • the light irradiated from the light irradiation light source 20 is irradiated to the container 1 stored in the container storage portion 11a through the horizontal through hole 11b, passes through the measurement sample in the container 1, and is used for turbidity measurement.
  • the light intensity is measured by the element 30.
  • the turbidity measuring element 30 is housed in a sealed turbidity measuring light shielding unit 31, and the opening of the turbidity measuring light shielding unit 31 faces the external opening of the lateral through hole 11d of the container storage portion 11a. Connected.
  • the light from the container storage portion 11a reaches the turbidity measuring element 30 accommodated in the turbidity measurement light-shielding unit 31 through the lateral through hole 11d of the container storage portion 11a.
  • the light source 20, the container storage portion 11a, and the turbidity measuring element 30 are positioned on a substantially straight line. Thereby, the light source 30 and the container storage part 11a communicate with each other through one lateral through hole 11b. Further, the container storage portion 11a and the turbidity measuring element 30 communicate with each other through the other lateral through hole 11d. This state is shown in FIGS. More specifically, in the device of the present invention, the light source 20, the container storage portion 11a, and the turbidity measuring element 30 can be arranged in a substantially horizontal positional relationship.
  • the turbidity measuring element 30 can be a photodiode.
  • incident light is converted into electric charge, so that the light intensity can be measured as a voltage.
  • the photodiode has high sensitivity and can measure the light intensity with high accuracy.
  • the measurement sample in the container 1 is transparent, the light intensity is high, and when the measurement sample in the container 1 is cloudy, the light intensity is low.
  • the degree of change in light intensity is measured as turbidity.
  • the wavelength band of the photodiode can be, for example, 450 to 500 nm. However, it is not the intention limited to this.
  • the fluorescence measurement element 40 is an element for measuring fluorescence emitted from the measurement sample in the container 1 stored in the container storage portion 11a.
  • the light irradiated from the light irradiation light source 20 is irradiated to the container 1 stored in the container storage portion 11a through the horizontal through hole 11b, and the measurement sample in the container 1 emits fluorescence by this irradiation light. Measured by the fluorescence measuring element 40.
  • the fluorescence measurement element 40 is housed in a sealed fluorescence measurement light-shielding unit 41, and the opening of the fluorescence measurement light-shielding unit 41 is connected to face the external opening of the vertical through hole 11c of the container storage portion 11a. .
  • the fluorescence from the container storage portion 11a reaches the fluorescence measurement element 40 housed in the fluorescence measurement light-shielding unit 41 via the vertical through hole 11c of the container storage portion 11a.
  • the container storage portion 11a and the fluorescence measurement element 40 communicate with each other through the vertical through hole 11c.
  • a straight line connecting the light source 20 and the container storage unit 11a and a straight line connecting the container storage unit 11a and the element 40 for measuring fluorescence are in a substantially vertical relationship. Is located. More specifically, in the device of the present invention, the fluorescence measurement element 40 can be arranged so as to be positioned substantially below the container storage portion 11a.
  • the fluorescence measurement element 40 can be a photo IC.
  • incident light is converted into electric charge, so that the light intensity can be measured as a voltage. Since the photo IC amplifies charges by itself, it is possible to measure even weak fluorescence, and fluorescence measurement with higher accuracy and higher sensitivity is possible.
  • the fluorescent substance is contained in the measurement sample in the container 1, the light excited by irradiating the fluorescent substance with the light from the light source LED emits light in all directions.
  • the photo IC When the peak wavelength of the light source LED is 465 nm, for example, when the excitation wavelength of the phosphor is around 465 nm and the emission wavelength of the phosphor is around 530 nm, the photo IC has a wavelength band of, for example, 500 to 580 nm. be able to. However, it is not the intention limited to this.
  • ⁇ Wavelength filter 44 In the device of the present invention, a wavelength filter that cuts at least part of the light of the light source and transmits at least part of the fluorescence emitted from the measurement sample in the container 1 between the container storage portion 11a and the fluorescence measurement element 40. 44. Thereby, it becomes possible to measure only the fluorescence emitted from the measurement sample in the container 1, and the measurement accuracy can be improved.
  • the wavelength filter 44 can be a filter that cuts a wavelength of 520 nm or less, for example, when the peak wavelength of the light source LED is 465 nm.
  • the wavelength filter 44 can be appropriately selected in consideration of the peak wavelength of the light source LED and the peak wavelength of fluorescence emitted from the measurement sample in the container 1.
  • the device of the present invention includes a temperature adjusting device for adjusting the temperature of the container 1 stored in the container storage portion 11a. More specifically, as an apparatus for heating for temperature control, the sample holder 11 can incorporate a heater, for example, and when the sample holder 11 has a plurality of container storage portions 11a, As shown in FIG. 1, a rod heater 12 can be built in the longitudinal direction of the sample holder 11.
  • the rod heater 12 may be a single heater or a plurality of heaters.
  • a heater may be provided outside the sample holder 11 or in place of the internal heater. In that case, a heater having a shape other than a rod shape may be used.
  • a fan 50 for blowing air for blowing air toward the measuring unit 10 can be provided.
  • the fan 50 for blowing air can be provided, for example, in the lower part of the measuring unit 10, and the fan 50 sends air outside the apparatus to the sample holder 11 and the like to cool the sample holder 11 efficiently and uniformly. be able to. That is, when the fan 50 is operated, air outside the apparatus is sucked into the apparatus and blown in the direction of the measurement unit 10 to cool the sample holder 11.
  • the temperature adjusting device may include temperature measuring means, for example, a temperature sensor, provided inside the sample holder 11 or adjacent to the sample holder 11.
  • the apparatus according to the present invention is configured so that the container storage portion 11a of the sample holder 11 has a lid for the container stored in the container storage portion of the sample holder at a position facing the described opening for inserting and storing the container 1 into the container storage portion 11a.
  • the container lid pressing part 14 for pressing can be further provided.
  • the container lid holding part 14 can approach and separate from the sample holder.
  • the container cover holding part 14 takes a position away from the sample holder and inserts the container 1 into the container storage part 11a. After the storage, the sample holder can be approached and the lid of the container 1 stored in the container storage unit 11a can be pressed.
  • the container lid pressing part 14 can include a temperature adjusting device.
  • the temperature adjusting device here is a heater, and more specifically, a heater provided inside the container lid pressing portion 14.
  • the container lid holding portion 14 has a shape corresponding to the planar shape of the sample holder 11 to hold the lid of all the containers 1 (in a plurality of cases) stored in the container storage portion 11 a provided in the sample holder 11. It is preferable from the viewpoint that When the sample holder 11 has a plurality of container storage portions 11 a and the container lid pressing portion 14 has a corresponding shape, the heater provided inside the lid pressing portion 14 can be a rod-shaped heater 15.
  • the lid holding part 14 includes a heater, so that the lid of the container 1 can be heated, thereby preventing evaporation of the solution in the container and preventing water droplets from adhering to the upper part of the container or the lid.
  • the reaction conditions inside can be kept constant.
  • a heater may be provided outside the lid pressing portion 14 or in place of the internal heater. In that case, a heater having a shape other than a rod shape may be used.
  • FIGS. 4 to 6 a transparent tube for reaction is shown as the container 1.
  • FIG. 5 is a view showing the upper surface of the sample holder 11 and shows an example of a shape into which eight tubes 1 can be inserted.
  • a temperature sensor 13 measures the temperature of the sample holder around the tube.
  • the temperature sensors 13 are arranged between the holes for inserting the tubes 1 on the side surface of the sample holder 11, thereby measuring four temperatures of the temperature sensors 13 and eight temperatures of the tubes 1.
  • the bar heater 12 generates heat when a voltage is applied, and the amount of heat generated changes when the voltage is changed.
  • the temperature of the sample holder 11 is controlled by periodically measuring the temperature sensor 13 and controlling the voltage applied to the rod heater 13 according to the measured temperature.
  • FIG. 6 is a view showing the upper surface of the container (tube) lid pressing portion 14.
  • the container (tube) lid pressing portion 14 is a member that presses the lid of the tube 1 from above.
  • the tube lid holding portion 14 includes a rod-shaped heater 15 that can heat the lid of the tube 1, suppresses evaporation of the solution in the tube, and prevents water droplets from adhering to the tube.
  • FIG. 6 is a view showing the upper surface of the tube lid pressing portion 14.
  • Reference numeral 16 denotes a temperature sensor, which measures the temperature of the tube lid pressing portion 14.
  • the bar heater 15 generates heat when a voltage is applied, and the amount of generated heat changes when the voltage is changed.
  • the temperature of the tube lid holding part 14 is controlled by measuring the temperature sensor 16 periodically and controlling the voltage applied to the rod heater 15 according to the measured temperature.
  • FIG. 4 is a cross-sectional view of the measurement unit.
  • the LED substrate 23 is attached to the light source unit light shielding unit 21, and the light emitted from the LED 22 travels inside the light source unit light shielding unit 21 to irradiate the solution 1 containing the tube 1 and DNA.
  • the light emitted from the LED 22 is shielded by the light source unit light shielding unit 21 and is not irradiated in a direction deviating from the traveling direction of the light.
  • the turbidity measurement light-shielding unit 31 is attached to the turbidity measurement part light-shielding unit 31, and the light transmitted or diffused through the solution 2 containing the DNA is transmitted through the tube 1 and the interior of the turbidity measurement part light-shielding unit 31.
  • the turbidity measuring optical sensor 32 receives light. In this case, the external light is shielded by the turbidity measuring unit light shielding unit 31.
  • the fluorescence measurement light-shielding unit 41 and the wavelength filter 44 are attached to the fluorescence measurement unit light-shielding unit 41. The light emitted by the solution 2 containing DNA passes through the tube 1, passes through the wavelength filter 44, and is fluorescent.
  • the light travels inside the measurement unit light shielding unit 41 and is received by the fluorescence measurement optical sensor 42. In this case, external light is shielded by the fluorescence measurement unit light shielding unit 41. Further, only light having a transmission wavelength of the wavelength filter 44 is incident on the fluorescence measurement optical sensor 42, and light of other wavelengths is shielded.
  • the light emitted from the LED 22 travels inside the light source light blocking unit 21, passes through one horizontal hole at the tip of the sample holder 11, passes through the tube 1, and hits a substance contained in the solution 2 containing DNA. , The light is transmitted or diffused, part of the transmitted light and diffused light travels to the opposite side in the horizontal direction, further passes through the tube 1, and faces the front end of the container storage portion 11 a of the sample holder 11 in the horizontal direction. The light passes through another hole in the turbidity measurement unit, passes through the inside of the turbidity measurement unit light-shielding unit 31, and is received by the turbidity measurement optical sensor 32.
  • the light emitted from the LED 22 travels inside the light source block unit 21, passes through one horizontal hole at the tip of the sample holder 11, passes through the tube 1, and is contained in the solution 2 containing DNA. Fluorescent light is emitted upon hitting the substance, and further, a part of the fluorescent light travels downward in the sample holder 11, passes through the tube 1, passes through the downward hole in the distal end portion of the sample holder 11, and the fluorescence measuring unit The light passes through the light blocking unit 41 and is received by the fluorescence measuring optical sensor 42.
  • the light intensity received by the turbidity measurement optical sensor 32 and the light intensity received by the fluorescence measurement optical sensor 42 can be acquired simultaneously. Thereby, turbidity measurement and fluorescence measurement can be performed simultaneously.
  • the apparatus of the present invention is used to measure turbidity and fluorescence in the container 1 simultaneously or sequentially. Furthermore, the device of the present invention can be used to detect amplification of nucleic acids contained in the solution 2 containing DNA in the container 1 by turbidity measurement. More specifically, it is used to detect hybridization of single-stranded nucleic acid or dissociation of double-stranded nucleic acid using fluorescence emission or quenching after nucleic acid amplification in solution 2 containing DNA in container 1. Can do. This point will be described later.
  • the nucleic acid analysis method of the present invention is a nucleic acid analysis method using the apparatus of the present invention, and includes the following steps (1) to (3).
  • a solution containing a measurement sample and a material for nucleic acid amplification is placed in the container.
  • the turbidity of the solution in the container is measured to detect the degree of nucleic acid amplification using the measurement sample as a template.
  • the fluorescence of the solution in the container is measured to confirm the sequence of the amplified nucleic acid.
  • the fluorescence measurement of the solution in the container is performed by changing the temperature of the solution to hybridize single-stranded nucleic acid in the solution and observing fluorescence emission or quenching,
  • the double-stranded nucleic acid in the solution is dissociated by observing fluorescence emission or quenching.
  • a solution containing a measurement sample and a material for nucleic acid amplification is placed in the container 1.
  • the container 1 is as described in the above-described device of the present invention.
  • the measurement sample can be, for example, DNA extracted from a living body (for example, an animal or a plant).
  • a nucleic acid other than DNA may be used as long as it can be amplified by the LAMP method.
  • the material for nucleic acid amplification can be appropriately selected according to the type of nucleic acid to be amplified and the amplification method.
  • a solution containing a measurement sample and a material for nucleic acid amplification used in a reaction system for nucleic acid analysis a known solution can be used as it is.
  • the reaction system described as the LAMP-FLP method described in WO2014 / 167377 (for example, see paragraphs 0019 to 0036) can be used.
  • the measurement sample reference can be made to, for example, the descriptions in paragraphs 0060 to 0064 of WO2014 / 167377. The entire description of WO2014 / 167377 is hereby specifically incorporated by reference.
  • the container 1 containing the solution is set in the sample holder of the device of the present invention.
  • the apparatus of the present invention can have a plurality of sample holders, and by setting a container containing different solutions (particularly solutions containing different measurement samples) in each sample holder, nucleic acid analysis of these measurement samples is simultaneously performed. can do.
  • Step (2) The turbidity of the solution in the container is measured to detect the degree of nucleic acid amplification using the measurement sample as a template. Turbidity measurements can be made continuously or intermittently. The measured turbidity can be recorded as an electrical signal, and can be displayed on a display or a paper medium, if necessary. Furthermore, a threshold value is set for the measured turbidity based on known data, and when this threshold value is exceeded, it can be recognized that the nucleic acid amplification has progressed to a predetermined (desired) degree. Furthermore, it is possible to notify the measurer that the threshold value has been exceeded by displaying it on a display or a paper medium.
  • the fluorescence of the solution in the container is measured to confirm the sequence of the amplified nucleic acid. Fluorescence measurements can be made continuously or intermittently. The measured fluorescence can be recorded as a signal, and can be displayed on a display or a paper medium, if necessary.
  • the fluorescence measurement can be performed, for example, by changing the temperature of the solution from a high temperature to a low temperature to hybridize single-stranded nucleic acids and observe fluorescence emission or quenching.
  • fluorescence measurement can be performed by observing fluorescence emission or quenching by dissociating double-stranded nucleic acids by changing the temperature of the solution from a low temperature to a high temperature.
  • the “high temperature” and “low temperature” of the solution temperature can be appropriately set according to the sequence of the single-stranded nucleic acid to be hybridized and the sequence of the double-stranded nucleic acid to be dissociated.
  • Amplification of a nucleic acid using a measurement sample as a template can be performed by an isothermal nucleic acid amplification method.
  • the isothermal nucleic acid amplification method can be, for example, the LAMP method.
  • the nucleic acid amplification method by the LAMP method for example, the above-mentioned WO2014 / 167377 can be referred to.
  • Nucleic acid amplification by the isothermal nucleic acid amplification method is performed, for example, at a temperature of 40 to 70 ° C. After the amplification is completed, the solution is heated to 90 to 100 ° C. to dissociate the amplified double-stranded nucleic acid into a single-stranded nucleic acid, and then The solution can be cooled and fluorescence emission or quenching due to hybridization of the single-stranded nucleic acid can be observed. However, it is not intended to be limited to this condition.
  • the nucleic acid analysis method of the present invention can be used for measuring single nucleotide polymorphisms (SNPs) of nucleic acids contained in the measurement sample. This point will be described more specifically below.
  • SNPs single nucleotide polymorphisms
  • FIG. 7 is a diagram showing a DNA amplification process, a DNA amplification detection process, a catalyst deactivation process, and a DNA SNP detection process using the solution containing the DNA of the embodiment of the present invention. It is.
  • Reference numeral 1 denotes a tube for containing the solution.
  • Reference numeral 2 denotes a solution in which DNA extracted from a living body, a catalyst necessary for DNA amplification, a fluorescent substance that binds to DNA, and a substance that becomes turbid when bound to DNA are mixed.
  • the mixed solution 2 containing DNA is put into the transparent tube 1 and the following treatment is performed.
  • a predetermined time any temperature ranging from 50 ° C. to less than 70 ° C., which varies depending on the DNA
  • a predetermined time a time ranging from several minutes to several hours, which varies depending on the DNA
  • a predetermined time at a predetermined temperature any temperature between 80 ° C. and 100 ° C., which differs for each DNA
  • a predetermined time between 2 minutes and 10 minutes, which differs for each DNA Heating deactivates the catalyst used to amplify the DNA and stabilizes the DNA in the solution.
  • the DNA double strand is separated at one temperature and separated into two single strands, and another temperature.
  • a predetermined temperature range 100 ° C. to 40 ° C.
  • a measurement example will be given to explain a method for detecting DNA amplification and a method for detecting DNA SNPs.
  • Artificial DNA was used as a sample, and a mixture of a catalyst necessary for DNA amplification, a fluorescent substance that binds to DNA, and a substance that becomes turbid when bound to DNA was used. This time, No. 1 to No. Eight samples of eight were measured.
  • FIG. 8 is a temperature graph at the time of measurement.
  • the vertical axis is temperature (° C.), and the horizontal axis is time (seconds).
  • the measurement was completed at 35 ° C.
  • the period during which the temperature is maintained at 63 ° C. for 30 minutes is during turbidity measurement, and the turbidity light intensity of the sample is measured. During the period when the temperature is lowered from 98 ° C.
  • the fluorescence intensity of the sample which is the time of fluorescence measurement, is measured. Note that, here, an example in which fluorescence is measured when the temperature falls is described, but fluorescence measurement can also be performed when the temperature rises.
  • FIG. 9 is a light intensity graph at the time of turbidity measurement.
  • the vertical axis represents the voltage value (mV) of the 32 turbidity measuring photosensor, and the horizontal axis represents time (seconds).
  • LAMP the natural logarithm
  • the nth turbidity T n from the start of turbidity measurement is the denominator of the voltage value V 0 of the turbidity measurement photosensor immediately after the start of turbidity measurement, and the voltage of the nth turbidity measurement photosensor from the start of turbidity measurement. This is a value obtained by LN calculation using the value V n as a numerator (formula 1).
  • T n LN (V 0 / V n ) Equation 1
  • T n nth turbidity from the start of turbidity measurement
  • V 0 voltage value (mV) of the turbidity measuring photosensor immediately after the start of turbidity measurement
  • V n n-th turbidity voltage value of the measuring light sensor from the turbidity measurement start (mV)
  • the turbidity threshold varies depending on the conditions used, for example, the primer sequence in the LAMP method, and therefore an arbitrary value is set based on the measurement result using DNA.
  • the threshold is 1000.
  • the elapsed time Tt from the start of turbidity measurement until the threshold value is exceeded, and the turbidity peak value Df are calculated.
  • the threshold is set so that the turbidity always exceeds this value once when the amplification is positive, and is always below this value when the amplification is negative, and never exceeds this value. Furthermore, when turbidity above the threshold is shown, amplification positive is displayed at the end of the amplification reaction, and in the case of amplification negative where turbidity below the threshold is shown, it is also displayed on the apparatus main body. The Specifically, at the end of the amplification reaction, the LED lamp of the DET1 (detection 1) of the well is used to display the determination result. For example, green is lit when amplification is positive, and red is lit when amplification is negative. Can be. As a result, the presence / absence of amplification (whether the threshold is exceeded or not exceeded) can be immediately confirmed.
  • FIG. 10 is a light intensity graph at the time of fluorescence measurement.
  • the vertical axis represents the voltage value (mV) of the 42 fluorescence measurement photosensor, and the horizontal axis represents the temperature (° C.).
  • FIG. 11 is a fluorescence change graph at the time of fluorescence measurement.
  • the vertical axis represents the voltage difference (mV) of the 42 fluorescence measurement optical sensor, and the horizontal axis represents the temperature (° C.).
  • the amount of change in fluorescence is the difference between the measured voltage value of the 42 fluorescence measurement optical sensor and the voltage value measured once before. Since the influence of noise is large if the voltage difference is used as it is, an averaging process is performed to smooth the fluorescence change graph (FIG. 12).
  • a plurality of peaks in the fluorescence change graph are detected, and their temperatures are detected. If the peak temperature is within a predetermined temperature range, it is determined as the corresponding SNP. Since the temperature range of the peak differs depending on the probe used, first, for the automatic detection setting of the peak, each type of peak is determined using artificial DNA having the SNP as a major type and artificial DNA having the SNP as a minor type. Check the temperature. If the actually detected peak is similar to the major type, the major type is determined. If the detected peak is similar to the minor type, the minor type is determined. The same level is, for example, ⁇ 3 ° C.
  • a temperature range at which a peak is formed that is, a temperature range where the fluorescence of the probe is quenched is set in advance.
  • a probe designed to produce a difference in peak formation temperature between major allyl and minor allyl By using a probe designed to produce a difference in peak formation temperature between major allyl and minor allyl, a maximum of two peaks can be formed in SNPs typing. This peak position is determined by verification using artificial DNA as described above, and the temperature range error is within ⁇ 3 ° C.
  • the apparatus main body is previously provided with a temperature range setting item for detecting these two peaks. For example, by setting the peak on the high temperature side to 55 to 64 ° C. and the peak on the low temperature side to 45 to 54 ° C., it can be determined whether or not there is a peak in each range. However, in order not to detect noise as a peak, it is also possible to set a threshold here.
  • two temperature ranges first range from 40.5 ° C to 46.5 ° C (major type), second range from 50.0 ° C to 56.0 ° C (minor type)
  • the three types of SNPs can be detected: a major type SNP, a minor type SNP, and a SNP in which the major type and minor type coexist.
  • a peak is detected in one or both of the two ranges described above, and there are three combinations.
  • the display of the three results can be performed as follows, for example.
  • the LED of DET1 of the well is lit in green.
  • the DET2 LED of the well is lit in blue.
  • the LED of the well is lit both in green for DET1 and blue for DET2. If no peak is detected, the NG LED of the well can be set to light red.
  • the SNPs determination result can be easily visually recognized by displaying it on the apparatus main body after the reaction is completed.
  • FIG. 13 is a list of detection results.
  • No. 1 and No. 2 is a sample which has not been amplified.
  • No. 3 and no. No. 6 is a major type SNP (peak detection at the time of fluorescence measurement only in a temperature range of 40.5 ° C. to 46.5 ° C.).
  • 4 and no. No. 7 is a minor type SNP (peak detection at the time of fluorescence measurement only in a temperature range of 50.0 ° C. to 56.0 ° C.)
  • No. 7 5 and No. 8 is a sample of SNP (peak detection at the time of fluorescence measurement in both temperature range 40.5 ° C to 46.5 ° C and temperature range 50.0 ° C to 56.0 ° C) in which major type and minor type coexist. . It was confirmed that the target SNPs can be detected.
  • the present invention is useful in the field of nucleic acid analysis devices and analysis methods.

Abstract

The present invention relates to a device that allows, in a single device, turbidity measurement for detecting and confirming DNA amplification and fluorescence measurement for SNPs detection. Automatic detection of SNPs of DNA is also possible using this device. This device for analyzing nucleic acid is capable of being used in combination with the LAMP method for amplifying DNA and the FLP method for detecting SNPs of DNA. The present invention provides a device having both a function for detecting SNPs and a function capable of detecting and confirming DNA amplification by the LAMP method, and provides a method for analyzing nucleic acid using the device.

Description

核酸分析方法及びそれに用いる蛍光・濁度測定装置Nucleic acid analysis method and fluorescence / turbidity measuring apparatus used therefor
本発明は、核酸分析方法及びそれに用いる蛍光・濁度測定装置に関する。より具体的には、本発明は、遺伝子検査に利用できる核酸分析方法及びそれに用いる蛍光・濁度測定装置に関する。より詳細には、本発明は、生体から抽出したデオキシリボ核酸(DNA)を増幅させる方法において、DNAの増幅を検出すること、及びDNAの一塩基多型(SNP、あるいはSNPsと呼ぶが、ここではSNPsと呼ぶ)を検出することの両方が可能な装置、及びこの装置を用いた、DNAの増幅を検出する方法及びSNPsを検出する方法を含む核酸分析方法に関する。この核酸分析方法においては、DNAの増幅が所定の量を越えたことを自動で検出することが可能であり、かつSNPsの検出を自動で行うことも可能である。
関連出願の相互参照
 本出願は、2014年12月26日出願の日本特願2014-265940号の優先権を主張し、その全記載は、ここに特に開示として援用される。
The present invention relates to a nucleic acid analysis method and a fluorescence / turbidity measurement apparatus used therefor. More specifically, the present invention relates to a nucleic acid analysis method that can be used for genetic testing and a fluorescence / turbidity measurement apparatus used therefor. More specifically, the present invention is a method of amplifying deoxyribonucleic acid (DNA) extracted from a living body, detecting DNA amplification, and DNA single nucleotide polymorphism (SNP or SNPs). The present invention relates to a device capable of both detecting SNPs), a method for detecting DNA amplification using this device, and a nucleic acid analysis method including a method for detecting SNPs. In this nucleic acid analysis method, it is possible to automatically detect that DNA amplification has exceeded a predetermined amount, and it is also possible to automatically detect SNPs.
This application claims the priority of Japanese Patent Application No. 2014-265940 filed on Dec. 26, 2014, the entire description of which is specifically incorporated herein by reference.
 遺伝子検査において、生体から抽出したDNAを用いて、紫外線を用いる方法や、電気詠動を用いる方法などで、DNAを検出する方法が利用されている。しかし、生体から抽出したDNAをそのまま用いるとDNAの数が少ないため検出感度が低く誤検出する可能性が高いことから、一般的には、生体から抽出したDNAを増幅させてからDNAを検出する方法が採られている。DNAを増幅させる方法やDNAを検出する方法は、DNAの種類に応じて複数の方法が実用化されており、それぞれの方法に対応した機器が製造・販売されている。 In a genetic test, a method of detecting DNA using a DNA extracted from a living body, such as a method using ultraviolet light or a method using electric peristalsis, is used. However, if DNA extracted from a living body is used as it is, the number of DNA is small, so detection sensitivity is low and the possibility of erroneous detection is high. Therefore, generally, DNA extracted from a living body is amplified before DNA is detected. The method is taken. As a method for amplifying DNA and a method for detecting DNA, a plurality of methods have been put into practical use according to the type of DNA, and devices corresponding to each method are manufactured and sold.
これまでは、DNAなどの遺伝子を専門に扱う大学、研究機関、製薬会社などの専門機関に限り、DNAを用いた研究が盛んに行われていたが、近年、病院や保健所などの一般的な機関でもDNAを用いた検査が実施されるようになってきた。しかしながら、現状、製造・販売されている機器の多くが研究用途の為、機器の使用にはDNAの専門知識を必要とする。そのため、DNAの専門知識が無くても簡単に使用できる検査機器が望まれている。 Until now, research using DNA has been actively conducted only in specialized institutions such as universities, research institutions, and pharmaceutical companies that specialize in genes such as DNA. Tests using DNA have also been implemented in institutions. However, since many of the devices that are currently manufactured and sold are for research purposes, the use of the devices requires specialized knowledge of DNA. Therefore, there is a demand for a testing device that can be easily used without having specialized knowledge of DNA.
 DNAを増幅させる方法の1つにLoop-Mediated Isothermal Amplification(LAMP)法がある。LAMP法では、生体から抽出したDNAを、DNA増幅に必要な溶液に混合する。この時に混合したDNAと結合する蛍光物質や、DNAの合成反応で生成されたピロリン酸塩による白濁により、DNA増幅の可否を確認できる。DNAの入った混合溶液を、所定の温度(DNAごとに異なる50℃から70℃弱のいずれかの温度)で所定の時間(DNAごとに異なる数分から数時間のいずれかの時間)加熱することにより、DNAが増幅する。DNAの増幅過程では、DNAの二本鎖が2つに切り離され、それぞれの鎖に物質が結合し、元のDNAと同様の二本鎖の構造を持つDNAが作成される。これを繰り返し行うことにより、DNAが増幅する。この時に、DNAの増幅に合わせて、DNAに結合する蛍光物質からの蛍光の光強度が大きくなったり、DNA合成反応で生成されたピロリン酸塩により溶液が濁ってきたりする。溶液の蛍光の光強度や蛍光の光強度の変化や、濁りによる光強度の減光量や光強度の変化を検出することにより、DNA増幅の可否を判別する。 One of the methods for amplifying DNA is a Loop-Mediated Isometric Amplification (LAMP) method. In the LAMP method, DNA extracted from a living body is mixed with a solution necessary for DNA amplification. Whether or not DNA amplification is possible can be confirmed by the white turbidity caused by the fluorescent substance that binds to the mixed DNA and the pyrophosphate generated by the DNA synthesis reaction. Heating the mixed solution containing DNA at a predetermined temperature (a temperature ranging from 50 ° C. to less than 70 ° C., which varies depending on the DNA) for a predetermined time (a time ranging from a few minutes to a few hours depending on the DNA). As a result, DNA is amplified. In the DNA amplification process, the double strands of DNA are cut into two, and substances are bound to the respective strands to create DNA having the same double-stranded structure as the original DNA. By repeating this, DNA is amplified. At this time, as the DNA is amplified, the light intensity of the fluorescence from the fluorescent substance that binds to the DNA increases, or the solution becomes cloudy due to pyrophosphate generated in the DNA synthesis reaction. Whether or not DNA amplification is possible is determined by detecting changes in the fluorescence intensity of the solution, changes in the fluorescence intensity, or a decrease in light intensity or a change in light intensity due to turbidity.
DNAを増幅させた後、DNAを増幅するために用いた触媒(酵素)を失活させ、溶液中のDNAを安定させる必要がある。所定の温度(DNAごとに異なる80℃から100℃のいずれかの温度)で所定の時間(DNAごとに異なる2分から10分のいずれかの時間)加熱して、触媒を失活させる。 After amplifying the DNA, it is necessary to deactivate the catalyst (enzyme) used to amplify the DNA to stabilize the DNA in the solution. The catalyst is deactivated by heating at a predetermined temperature (any temperature from 80 ° C. to 100 ° C., which differs for each DNA) for a predetermined time (any time from 2 minutes to 10 minutes, which differs for each DNA).
 DNAのSNPsを検出する方法の1つにFluorescence Loop Primer(FLP)法がある。FLP法は、DNAに結合する物質に蛍光体を結合させた物質と、DNAに結合する物質に蛍光体を消光する物質を結合させた物質を使用し、規則的に両者をDNAへ結合させることにより、DNAの結合状態と蛍光の光強度の相関を導き、蛍光の光強度から遺伝子の結合状態を検出する方法である。 One of the methods for detecting DNA SNPs is the Fluorescence Loop Primer (FLP) method. The FLP method uses a substance that binds a phosphor to a substance that binds to DNA and a substance that binds a substance that quenches the phosphor to a substance that binds to DNA, and regularly binds both to DNA. Thus, the correlation between the DNA binding state and the fluorescence light intensity is derived, and the gene binding state is detected from the fluorescence light intensity.
 DNAに結合する物質に蛍光体を結合させた物質と、DNAに結合する物質に蛍光体を消光する物質を結合させた物質を使用し、規則的に両者をDNAへ結合させる方法は次の通りである。DNAに結合する物質に蛍光体を結合させた物質として、DNAの増幅時にDNAの一本鎖と結合する物質に蛍光体を結合した物質(FLPと呼ぶ)を使用する。DNAに結合する物質に蛍光体を消光する物質を結合させた物質として、検出対象となるSNPsの塩基と結合する物質に消光する物質を結合させた物質(Quencher Probe(QP)と呼ぶ)を使用する。DNAの増幅が停止し安定した状態で、高温(100℃程度)から低温(40℃程度)に温度を下降、又は、上昇させることにより、DNAの二本鎖が2つに切り離される状態とDNAの二本鎖が生成される状態が発生し、通常はFLPが結合して蛍光発光するDNAが、SNPsの塩基が結合する温度では、FLPに代わりQPが結合して蛍光が消光する。さらに、SNPsの型によって、結合する温度が異なるため、蛍光の光強度又は蛍光の光強度の変化を検出することにより、SNPsの型を判別する。 Using a substance that binds a phosphor to a substance that binds to DNA and a substance that binds a substance that quenches the phosphor to a substance that binds to DNA, the method for regularly binding both to DNA is as follows. It is. As a substance in which a phosphor is bound to a substance that binds to DNA, a substance (referred to as FLP) in which a phosphor is bound to a substance that binds to a single strand of DNA during DNA amplification is used. A substance (called Quencher Probe (QP)) in which a substance that quenches a substance that binds to the base of the SNPs to be detected is used as a substance that binds a substance that quenches the phosphor to a substance that binds to DNA. To do. When DNA amplification is stopped and stable, the DNA double strand is separated into two by lowering or raising the temperature from high temperature (about 100 ° C.) to low temperature (about 40 ° C.) and DNA In the temperature at which DNA that normally fluoresces when FLP binds to the SNPs base binds, QP binds instead of FLP and the fluorescence is quenched. Furthermore, since the bonding temperature differs depending on the type of SNPs, the type of SNPs is determined by detecting the fluorescence light intensity or the change in fluorescence light intensity.
DNAの増幅検出とSNPsの検出を行うための装置及び方法を開示する文献として、例えば、以下の文献がある。機器/検出方法(参照文献)の順で記載する。
・i-densy/Quenching Probe法(非特許文献1)
・ABI Prism 7900 HT/TaqMan法(非特許文献2)
・LightCycler/融解曲線分析(非特許文献3)
・LightCycler 480/High-resolution Melting Analysis、Hybridization Probe法、Invader-plus法、TaqMan法(非特許文献3)
・ABI Prism 7700/LAMP法(特許文献1)
For example, the following documents disclose devices and methods for performing amplification detection of DNA and detection of SNPs. List in order of device / detection method (references).
I-densy / Quenching Probe method (Non-patent Document 1)
ABI Prism 7900 HT / TaqMan method (non-patent document 2)
-LightCycler / melting curve analysis (Non-patent Document 3)
-LightCycler 480 / High-resolution Melting Analysis, Hybridization Probe method, Invader-plus method, TaqMan method (Non-patent Document 3)
ABI Prism 7700 / LAMP method (Patent Document 1)
特許文献1:日本特開2013-31463号公報 Patent Document 1: Japanese Unexamined Patent Publication No. 2013-31463
非特許文献1:Sensors 2012, 12, 16614-16627
非特許文献2:Cancer Epidemiol Biomarkers Prev 2007;16(6)
非特許文献3:JOURNAL OF CLINICAL MICROBIOLOGY, May 2011, p. 1853-1860
特許文献1及び非特許文献1~3の全記載は、ここに特に開示として援用される。
Non-Patent Document 1: Sensors 2012, 12, 16614-16627
Non-Patent Document 2: Cancer Epidemiol Biomarkers Prev 2007; 16 (6)
Non-Patent Document 3: JOURNAL OF CLINICAL MICROBIOLOGY, May 2011, p. 1853-1860
The entire descriptions of Patent Document 1 and Non-Patent Documents 1 to 3 are specifically incorporated herein by reference.
DNAを増幅させるためのLAMP法を用いた機器は製造・販売されている(特許文献1参照)。しかしながら、多くの機器では、DNAの増幅を確認することはできず、機器の使用後に、試薬の状態を人が目視確認して判別している。また、DNAが所定の量を越えたことを自動で検出できる機器は製造・販売されているが、種類は少なく、高価である。また、DNAが所定の量を越えたことを自動で検出できる機器では、DNAと混合させた蛍光物質の光強度を検出する方法か、DNAと混合させた濁る物質の透過光の光強度を検出する方法のいずれか一方による方法であり、同時に自動で検出できる機器は無い。 Devices using the LAMP method for amplifying DNA are manufactured and sold (see Patent Document 1). However, in many devices, amplification of DNA cannot be confirmed, and after use of the device, the state of the reagent is visually confirmed by a human. In addition, devices that can automatically detect that DNA exceeds a predetermined amount are manufactured and sold, but the types are small and expensive. For devices that can automatically detect that the amount of DNA exceeds a predetermined amount, either the method of detecting the light intensity of a fluorescent substance mixed with DNA or the intensity of transmitted light of a turbid substance mixed with DNA is detected. There is no device that can detect automatically at the same time.
DNAのSNPsを検出するためのFLP法を用いた機器は製造・販売されている。しかしながら、これらの機器では、測定した特性を数値データやグラフなどで人が目視確認してSNPsを判別しており、そのためには、専門的な知識を必要とする。さらに、機器は種類が少なく、高価である。また、現状、SNPsを自動で検出できる機器は無い。さらに、DNAを増幅させるためのLAMP法と組み合わせて、DNA増幅をさせた後、試薬を取りだすことなく、DNAのSNPsを検出できる機器はほとんど無く、DNA増幅の検出・確認とDNAのSNPs検出の両方を自動でできる機器は無い。 Devices using the FLP method for detecting DNA SNPs are manufactured and sold. However, in these devices, human beings visually check the measured characteristics with numerical data or graphs to determine SNPs, and for that purpose, specialized knowledge is required. Furthermore, there are few types of equipment and they are expensive. At present, there is no device that can automatically detect SNPs. Furthermore, in combination with the LAMP method for amplifying DNA, after DNA amplification, there are few devices that can detect DNA SNPs without removing reagents, and DNA amplification detection / confirmation and DNA SNPs detection There is no equipment that can do both automatically.
前記非特許文献1~3に記載の機器及び方法でも、SNPs検出は蛍光を検出しているが、検出対象であるDNA増幅の検出・確認は、目視で行っている。DNA(核酸)増幅の検出・確認とSNPsの蛍光での検出の両方を実施できる装置は知られていない。 Even in the devices and methods described in Non-Patent Documents 1 to 3, SNPs detection detects fluorescence, but detection and confirmation of DNA amplification as a detection target is performed visually. There is no known apparatus capable of performing both detection and confirmation of DNA (nucleic acid) amplification and detection of SNPs with fluorescence.
 そのため、専門的な知識を必要とせず、簡単な操作で取り扱いが可能な、DNAを増幅させるためのLAMP法とDNAのSNPsを検出するためのFLP法を組み合わせて使用できる核酸分析用装置であって、LAMP法によるDNAの増幅を検出及び確認できる機能とSNPsを検出する機能を併せ持った装置、より具体的には、DNA増幅の検出及び確認のための濁度測定と蛍光測定を1つの装置で可能とする装置が望まれている。さらに、この装置を用いた、DNAのSNPsの自動検出機器の提供も望まれている。 Therefore, it is a device for nucleic acid analysis that can be used in combination of the LAMP method for amplifying DNA and the FLP method for detecting SNPs of DNA, which does not require specialized knowledge and can be handled with simple operations. An apparatus having both a function capable of detecting and confirming amplification of DNA by the LAMP method and a function of detecting SNPs, more specifically, one apparatus for measuring turbidity and measuring fluorescence for detecting and confirming DNA amplification. There is a need for an apparatus that enables the above-mentioned. Furthermore, provision of an automatic detection device for DNA SNPs using this apparatus is also desired.
本発明は前記の課題を解決することを目的としてなされたものであり、DNAを増幅させながら、所定のDNAの量を越えたことを検出して、その後、DNAを増幅させるための触媒を失活させ、DNAのSNPsを検出する過程を、簡単な操作で、自動で実施する装置である。以下、各請求項の説明をする。 The present invention has been made for the purpose of solving the above-mentioned problems. While amplifying the DNA, it is detected that the amount of the predetermined DNA has been exceeded, and then the catalyst for amplifying the DNA is lost. It is an apparatus that automatically performs a process of detecting and detecting SNPs of DNA by a simple operation. Hereinafter, each claim will be described.
本発明は、以下の通りである。
[1]
・測定試料を収容するための容器を格納するため容器格納部を少なくとも1つ有するサンプルホルダであって、前記サンプルホルダの容器格納部は、底部に容器格納部の深さ方向に1つの縦貫通孔と底部付近の側壁に容器格納部の深さ方向と直行する方向に容器格納部内で開口部が対向する2つの横貫通孔を有し、
・前記容器格納部内に格納した容器への光照射用光源、
・前記容器格納部内に格納した容器中の測定試料の濁度計測用素子、
・前記容器格納部内に格納した容器中の測定試料から発せられる蛍光計測用素子、
・前記容器格納部内に格納した容器の温度を調節するための温度調節装置、
を含む蛍光及び濁度測定装置であって、
-前記光源及び前記容器格納部は、前記2つの横貫通孔の一方を介して連通しており、
-前記容器格納部及び前記濁度計測用素子は、前記2つの横貫通孔の他方を介して連通しており、
-前記容器格納部及び前記蛍光計測用素子は、前記縦貫通孔を介して連通しており、
-前記容器格納部と前記蛍光計測用素子の間に、光源の光の少なくとも一部をカットし、容器から発せられる蛍光の少なくとも一部を透過する波長フィルタを含む、
前記装置。
[2]
-前記光源、前記容器格納部及び前記濁度計測用素子は、略水平関係に位置しており、
-前記蛍光計測素子は、前記容器格納部の略下方に位置する、
[1]に記載の装置。
[3]
-前記光源は、LEDであり、
-前記蛍光計測用素子は、フォトICであり、
-前記濁度計測用素子は、フォトダイオードである、
[1]または[2]に記載の装置。
[4]
前記LEDは、ピーク波長が465nmであり、半値幅が約20nmであり、
前記フォトICは、波長帯域が500~580nmであり、
前記フォトダイオードは、波長帯域が450~500nmであり、かつ
前記波長フィルタは、520nm以下の波長をカットするフィルタである、
[3]に記載の装置。
[5]
前記温度調節装置は、前記サンプルホルダの内部に設けられた加熱手段、前記サンプルホルダに向けて送風するための送風機、及び前記サンプルホルダの内部またはサンプルホルダに隣接して設けられた温度測定手段を含む、
[1]~[4]のいずれかに記載の装置。
[6]
前記サンプルホルダの容器格納部の開口に対向する位置に、前記サンプルホルダに接近及び乖離可能な、サンプルホルダの容器格納部に格納された容器の蓋を押さえるための容器蓋押さえ部材をさらに有し、前記容器蓋押さえ部材は、温度調節装置を含む、[1]~[5]のいずれかに記載の装置。
[7]
前記容器中の濁度及び蛍光を同時又は逐次に測定するために用いられる、[1]~[6]のいずれかに記載の装置。
[8]
前記容器中に含まれる核酸の増幅を濁度測定により検知するために用いる、[7]に記載の装置。
[9]
前記容器中において核酸増幅後に、一本鎖核酸のハイブリダイズ又は二本鎖核酸の解離を蛍光の発光又は消光を用いて検知するために用いられる、[7]又は[8]に記載の装置。
[10]
[1]~[6]のいずれかに記載の装置を用いる核酸分析方法であって、
(1)前記容器中に測定試料及び核酸増幅のための材料を含有する溶液を配置し、
(2)前記容器中の溶液の濁度を測定して、前記測定試料を鋳型とする核酸の増幅の度合を検知し、
(3)所定の増幅が確認された後に、前記容器中の溶液の蛍光を測定して、増幅した核酸の配列を確認する、ことを含み、
-前記容器中の溶液の蛍光測定は、溶液の温度を変化させることで、溶液中の一本鎖核酸同士をハイブリダイズさせて蛍光の発光又は消光を観察するか、又は溶液中の二本鎖核酸の解離を蛍光の発光又は消光を観察することで行う、前記方法。
[11]
前記容器中の溶液の蛍光測定は、溶液の温度を高温から低温に変化させることで、一本鎖核酸同士をハイブリダイズさせて蛍光の発光又は消光を観察することで行う、[10]に記載の方法。
[12]
前記容器中の溶液の蛍光測定は、溶液の温度を低温から高温に変化させることで、二本鎖核酸の解離を蛍光の発光又は消光を観察することで行う、[10]に記載の方法。
[13]
前記測定試料を鋳型とする核酸の増幅は等温核酸増幅法で行う、[10]~[12]のいずれかに記載の方法。
[14]
前記等温核酸増幅法がLAMP法である、[13]に記載の方法。
[15]
前記等温核酸増幅法による核酸増幅は、40~70℃の温度で行い、増幅完了後に溶液を90~100℃に加熱し、増幅した二本鎖核酸を解離させて一本鎖核酸とし、その後、溶液を冷却して一本鎖核酸のハイブリダイズによる蛍光の発光又は消光を観察する、[13]又は[14]に記載の方法。
[16]
前記測定試料に含まれる核酸の一塩基多型(SNPs)を測定するための、[10]~[15]のいずれかに記載の方法。
The present invention is as follows.
[1]
A sample holder having at least one container storage unit for storing a container for containing a measurement sample, wherein the container storage unit of the sample holder has one vertical penetration in the depth direction of the container storage unit at the bottom The side wall near the bottom and the bottom has two lateral through-holes whose openings are opposed to each other in the direction perpendicular to the depth direction of the container storage unit,
A light source for irradiating light to the container stored in the container storage unit,
An element for measuring turbidity of a measurement sample in a container stored in the container storage unit,
An element for fluorescence measurement emitted from a measurement sample in a container stored in the container storage unit,
A temperature adjusting device for adjusting the temperature of the container stored in the container storage unit,
A fluorescence and turbidity measuring device comprising:
The light source and the container storage are in communication via one of the two lateral through-holes;
The container storage and the turbidity measuring element communicate with each other through the other of the two lateral through holes;
The container storage unit and the fluorescence measuring element communicate with each other through the vertical through hole;
-A wavelength filter that cuts at least part of the light of the light source and transmits at least part of the fluorescence emitted from the container between the container housing and the fluorescence measuring element;
Said device.
[2]
The light source, the container storage and the turbidity measuring element are positioned in a substantially horizontal relationship;
The fluorescence measuring element is located substantially below the container storage;
The device according to [1].
[3]
The light source is an LED;
The fluorescence measuring element is a photo IC;
The turbidity measuring element is a photodiode;
The device according to [1] or [2].
[4]
The LED has a peak wavelength of 465 nm, a half width of about 20 nm,
The photo IC has a wavelength band of 500 to 580 nm,
The photodiode has a wavelength band of 450 to 500 nm, and the wavelength filter is a filter that cuts a wavelength of 520 nm or less.
The apparatus according to [3].
[5]
The temperature adjusting device includes a heating unit provided inside the sample holder, a blower for blowing air toward the sample holder, and a temperature measuring unit provided inside the sample holder or adjacent to the sample holder. Including,
The device according to any one of [1] to [4].
[6]
A container lid pressing member for pressing the lid of the container stored in the container storage part of the sample holder, which can approach and separate from the sample holder, at a position facing the opening of the container storage part of the sample holder. The device according to any one of [1] to [5], wherein the container lid pressing member includes a temperature adjusting device.
[7]
The apparatus according to any one of [1] to [6], which is used to measure turbidity and fluorescence in the container simultaneously or sequentially.
[8]
The apparatus according to [7], which is used for detecting amplification of a nucleic acid contained in the container by turbidity measurement.
[9]
The apparatus according to [7] or [8], which is used for detecting hybridization of single-stranded nucleic acid or dissociation of double-stranded nucleic acid using fluorescence emission or quenching after nucleic acid amplification in the container.
[10]
A nucleic acid analysis method using the apparatus according to any one of [1] to [6],
(1) A solution containing a measurement sample and a material for nucleic acid amplification is placed in the container,
(2) Measure the turbidity of the solution in the container to detect the degree of nucleic acid amplification using the measurement sample as a template,
(3) After the predetermined amplification is confirmed, the fluorescence of the solution in the container is measured to confirm the sequence of the amplified nucleic acid,
-Fluorescence measurement of the solution in the container can be performed by observing fluorescence emission or quenching by hybridizing single-stranded nucleic acids in the solution by changing the temperature of the solution, or double-stranded in the solution. The method, wherein the nucleic acid is dissociated by observing fluorescence emission or quenching.
[11]
Fluorescence measurement of the solution in the container is performed by changing the temperature of the solution from a high temperature to a low temperature to hybridize single-stranded nucleic acids and observe fluorescence emission or quenching, [10] the method of.
[12]
The method according to [10], wherein the fluorescence measurement of the solution in the container is performed by observing fluorescence emission or quenching by dissociating the double-stranded nucleic acid by changing the temperature of the solution from a low temperature to a high temperature.
[13]
The method according to any one of [10] to [12], wherein the nucleic acid amplification using the measurement sample as a template is performed by an isothermal nucleic acid amplification method.
[14]
The method according to [13], wherein the isothermal nucleic acid amplification method is a LAMP method.
[15]
Nucleic acid amplification by the isothermal nucleic acid amplification method is performed at a temperature of 40 to 70 ° C., and after completion of the amplification, the solution is heated to 90 to 100 ° C. to dissociate the amplified double-stranded nucleic acid into a single-stranded nucleic acid. The method according to [13] or [14], wherein the solution is cooled and fluorescence emission or quenching due to hybridization of the single-stranded nucleic acid is observed.
[16]
The method according to any one of [10] to [15], for measuring single nucleotide polymorphisms (SNPs) of nucleic acids contained in the measurement sample.
 本発明によれば、LAMP法によるDNAの増幅を検出及び確認できる機能とSNPsを検出する機能を併せ持った装置を提供することができる。本発明の装置を用いることで、LAMP法によるDNAの増幅を検出及び確認と、能とSNPsの検出を1つの装置で行う核酸分析方法を提供することができ、さらに、SNPsの検出を、DNAの増幅完了確認も含めて自動で行うこともできる。 に よ According to the present invention, it is possible to provide an apparatus having both a function of detecting and confirming DNA amplification by the LAMP method and a function of detecting SNPs. By using the apparatus of the present invention, it is possible to provide a nucleic acid analysis method for detecting and confirming amplification of DNA by the LAMP method and detecting ability and SNPs with one apparatus. It can also be performed automatically, including confirmation of completion of amplification.
図1は、本発明装置の測定部10の斜視図である。FIG. 1 is a perspective view of a measuring unit 10 of the apparatus of the present invention. 図2は、本発明装置の測定部10とファン50の斜視図である。FIG. 2 is a perspective view of the measuring unit 10 and the fan 50 of the apparatus of the present invention. 図3は、本発明装置の測定部10の斜視断面図である。FIG. 3 is a perspective sectional view of the measuring unit 10 of the apparatus of the present invention. 図4は、本発明装置の測定部10の略断面説明図である。FIG. 4 is a schematic cross-sectional explanatory view of the measuring unit 10 of the device of the present invention. 図5は、本発明装置のサンプルホルダ11の説明図である。FIG. 5 is an explanatory view of the sample holder 11 of the apparatus of the present invention. 図6は、本発明装置の容器(チューブ)蓋押え部14の説明図である。FIG. 6 is an explanatory view of the container (tube) lid pressing portion 14 of the apparatus of the present invention. 図7は、本発明の実施形態のDNAを入れた溶液を使用し、DNAの増幅過程とDNA増幅の検出過程、DNAを増やすための触媒の失活過程、DNAのSNPsの検出過程を示す図である。FIG. 7 is a diagram showing a DNA amplification process, a DNA amplification detection process, a catalyst deactivation process, and a DNA SNP detection process using the solution containing the DNA of the embodiment of the present invention. It is. 図8は、測定時の温度グラフである。FIG. 8 is a temperature graph at the time of measurement. 図9は、濁度測定時の光強度グラフである。FIG. 9 is a light intensity graph at the time of turbidity measurement. 図10は、濁度グラフである。FIG. 10 is a turbidity graph. 図11は、蛍光測定時の蛍光光強度グラフである。FIG. 11 is a fluorescence light intensity graph at the time of fluorescence measurement. 図12は、蛍光変化グラフである。FIG. 12 is a fluorescence change graph. 図13は、検出結果一覧表である。FIG. 13 is a list of detection results.
[蛍光及び濁度測定装置]
本発明の蛍光及び濁度測定装置(以下、本発明装置と呼ぶ)を図1~6を参照して説明する。
[Fluorescence and turbidity measuring device]
The fluorescence and turbidity measuring apparatus of the present invention (hereinafter referred to as the present apparatus) will be described with reference to FIGS.
本発明装置は、
・測定試料を収容するための容器1を格納するため容器格納部11aを少なくとも1つ有するサンプルホルダ11、
・前記容器格納部11a内に格納した容器1への光照射用光源20、
・前記容器格納部11a内に格納した容器1中の測定試料の濁度計測用素子30、
・前記容器格納部11a内に格納した容器1中の測定試料から発せられる蛍光計測用素子40、
・前記容器格納部11a内に格納した容器1を加熱及び冷却するための温度調節装置、
を含む。
The device of the present invention
A sample holder 11 having at least one container storage portion 11a for storing a container 1 for storing a measurement sample;
A light irradiating light source 20 for the container 1 stored in the container storage portion 11a,
The element 30 for measuring turbidity of the measurement sample in the container 1 stored in the container storage unit 11a,
The fluorescence measuring element 40 emitted from the measurement sample in the container 1 stored in the container storage unit 11a;
A temperature adjusting device for heating and cooling the container 1 stored in the container storage section 11a;
including.
<サンプルホルダ11>
図3のようにサンプルホルダ11は、測定試料を収容するための容器1を格納するため容器格納部11aを少なくとも1つ有するサンプルホルダ11である。測定試料を収容するための容器1は、容器1に収容した測定試料の濁度及び蛍光の測定を妨げないという観点から、少なくとも濁度及び蛍光の測定に関係する波長域では光透過性であることが適当であり、さらに、光源からの光についても、濁度及び蛍光の測定に関係する波長域の光に対しては、光透過性であることが適当である。そのような容器としては、可視光領域において透明な反応用チューブを挙げることができる。
<Sample holder 11>
As shown in FIG. 3, the sample holder 11 is a sample holder 11 having at least one container storage portion 11a for storing the container 1 for storing the measurement sample. The container 1 for storing the measurement sample is light transmissive at least in the wavelength range related to the measurement of turbidity and fluorescence from the viewpoint of not hindering the turbidity and fluorescence measurement of the measurement sample accommodated in the container 1. In addition, it is appropriate that the light from the light source is light-transmitting with respect to light in a wavelength range related to the measurement of turbidity and fluorescence. An example of such a container is a reaction tube that is transparent in the visible light region.
サンプルホルダ11の容器格納部11aは、底部に容器格納部の深さ方向に1つの縦貫通孔11cと底部付近の側壁に容器格納部の深さ方向と直行する方向に容器格納部内で開口部が対向する2つの横貫通孔11b、11dを有する。後述するように、一方の横貫通孔は、光源20と容器格納部11aとの間を連通させるためのものであり、光源20から容器格納部11aへの光照射が過不足なく確保できるような形状及び寸法を有するものであることができる。他方の横貫通孔は、容器格納部11aと濁度計測用素子30との間を連通させるためのものであり、容器格納部11aに格納された容器中の測定試料の濁度を濁度計測用素子30において、測定できるように、容器中の測定試料を透過した光が濁度計測用素子30に過不足なく到達できるような形状及び寸法を有するものであることができる。さらに、縦貫通孔は、容器格納部11aと蛍光計測用素子40との間を連通させるためのものであり、容器格納部11aに格納された容器中の測定試料から発せられる蛍光を蛍光計測用素子40において、測定できるように、容器中の測定試料から発せられた蛍光が蛍光計測用素子40に過不足なく到達できるような形状及び寸法を有するものであることができる。 The container holder 11a of the sample holder 11 has one vertical through hole 11c in the depth direction of the container storage portion at the bottom and an opening in the container storage portion in a direction perpendicular to the depth direction of the container storage portion in the side wall near the bottom. Has two lateral through holes 11b and 11d facing each other. As will be described later, one of the horizontal through-holes is for communicating between the light source 20 and the container storage portion 11a, so that light irradiation from the light source 20 to the container storage portion 11a can be ensured without excess or deficiency. It can have a shape and dimensions. The other lateral through-hole is for communicating between the container storage portion 11a and the turbidity measuring element 30, and the turbidity measurement of the measurement sample in the container stored in the container storage portion 11a is performed. The element 30 can be shaped and dimensioned so that the light transmitted through the measurement sample in the container can reach the turbidity measuring element 30 without excess or deficiency so that measurement can be performed. Further, the vertical through hole is for communicating between the container storage portion 11a and the fluorescence measurement element 40, and the fluorescence emitted from the measurement sample in the container stored in the container storage portion 11a is used for fluorescence measurement. The element 40 can have a shape and a dimension so that the fluorescence emitted from the measurement sample in the container can reach the fluorescence measurement element 40 without excess or deficiency so that measurement can be performed.
サンプルホルダ11に設けられる容器格納部の数は特に限定はなく、本発明装置において同時に測定したい測定試料の数に応じて適宜決定できる。サンプルホルダ11に設けられる容器格納部の数は、例えば、1~20の範囲であり、2~10の範囲であることができる。但し、これらの範囲に限定される意図ではなく、適宜決定できる事項である。また、各容器格納部は、それぞれ底部に容器格納部の深さ方向に1つの縦貫通孔11cと底部付近の側壁に容器格納部の深さ方向と直行する方向に容器格納部内で開口部が対向する2つの横貫通孔11b、11dを有する。容器格納部の形状や寸法は、容器格納部に格納する容器の形状及び寸法に応じて適宜決定できる。容器は、生体試料の実験用に汎用されているプラクチック製チューブであることができ、容器格納部の形状及び寸法は、プラクチック製チューブの形状及び寸法に応じて適宜決定できる。 The number of container storage portions provided in the sample holder 11 is not particularly limited, and can be appropriately determined according to the number of measurement samples to be simultaneously measured in the apparatus of the present invention. The number of container storage units provided in the sample holder 11 is, for example, in the range of 1 to 20, and can be in the range of 2 to 10. However, it is not intended to be limited to these ranges, but matters that can be determined as appropriate. In addition, each container storage portion has one vertical through hole 11c in the depth direction of the container storage portion at the bottom and an opening in the container storage portion in a direction perpendicular to the depth direction of the container storage portion in the side wall near the bottom. There are two lateral through- holes 11b and 11d facing each other. The shape and size of the container storage unit can be appropriately determined according to the shape and size of the container stored in the container storage unit. The container can be a plastic tube that is widely used for experiments on biological samples, and the shape and dimensions of the container storage portion can be appropriately determined according to the shape and dimensions of the plastic tube.
<光照射用光源20>
光照射用光源20は、容器格納部11a内に格納した容器1へ、濁度及び蛍光計測用の光を照射するためのものである。光照射用光源20は、波長と光量を有する光源であれば、特に限定されるものではないが、エネルギー変換効率が高く、所望の波長域の光照射が比較的簡単にできるという観点からLEDであることが好ましい。LEDは、例えば、ピーク波長が465nmであり、半値幅が約20nmであることが、濁度の測定及び蛍光の測定の両方の観点から好ましい。但し、このようなLEDに限定される意図ではなく、濁度の測定及び蛍光の測定が可能であれば、他のピーク波長と半値幅を有するLEDを適宜使用することもできる。
<Light source 20 for light irradiation>
The light irradiation light source 20 is for irradiating the container 1 stored in the container storage unit 11a with light for measuring turbidity and fluorescence. The light source 20 for light irradiation is not particularly limited as long as it is a light source having a wavelength and a light amount, but it is an LED from the viewpoint that energy conversion efficiency is high and light irradiation in a desired wavelength region can be relatively easily performed. Preferably there is. For example, the LED preferably has a peak wavelength of 465 nm and a full width at half maximum of about 20 nm from the viewpoint of both turbidity measurement and fluorescence measurement. However, the present invention is not intended to be limited to such an LED, and an LED having another peak wavelength and a full width at half maximum can be appropriately used as long as turbidity measurement and fluorescence measurement are possible.
光照射用光源20は、密閉された光源部遮光ユニット21内に収納され、光源部遮光ユニット21は、内部に光照射用光源20が格納され、光源部遮光ユニット21の開口は、容器格納部11aの横貫通孔11bの外部開口と対向して接続される。光照射用光源20からの照射光は、容器格納部11aの横貫通孔11bを介して、容器格納部11a内に格納された容器1に照射される。 The light irradiation light source 20 is housed in a hermetically sealed light source unit light shielding unit 21, and the light source light shielding unit 21 stores therein the light irradiation light source 20, and the opening of the light source unit light shielding unit 21 is a container storage unit. It is connected to the external opening of the lateral through hole 11b of 11a. Irradiation light from the light irradiation light source 20 is applied to the container 1 stored in the container storage portion 11a through the lateral through hole 11b of the container storage portion 11a.
<濁度計測用素子30>
濁度計測用素子30は、容器格納部11a内に格納した容器1中の測定試料の濁度を計測するための素子である。光照射用光源20から照射される光は、横貫通孔11bを介して、容器格納部11a内に格納された容器1に照射され、容器1内の測定試料を透過して、濁度計測用素子30により光強度として計測される。濁度計測用素子30は、密閉された濁度計測用遮光ユニット31内に収納され、濁度計測用遮光ユニット31の開口は、容器格納部11aの横貫通孔11dの外部開口と対向して接続される。容器格納部11aからの光は、容器格納部11aの横貫通孔11dを介して、濁度計測用遮光ユニット31内に収納された濁度計測用素子30に到達する。
<Turbidity measuring element 30>
The turbidity measuring element 30 is an element for measuring the turbidity of the measurement sample in the container 1 stored in the container storage portion 11a. The light irradiated from the light irradiation light source 20 is irradiated to the container 1 stored in the container storage portion 11a through the horizontal through hole 11b, passes through the measurement sample in the container 1, and is used for turbidity measurement. The light intensity is measured by the element 30. The turbidity measuring element 30 is housed in a sealed turbidity measuring light shielding unit 31, and the opening of the turbidity measuring light shielding unit 31 faces the external opening of the lateral through hole 11d of the container storage portion 11a. Connected. The light from the container storage portion 11a reaches the turbidity measuring element 30 accommodated in the turbidity measurement light-shielding unit 31 through the lateral through hole 11d of the container storage portion 11a.
以上のように、光源20、容器格納部11a及び濁度計測用素子30は、略直線上に位置する。それにより、光源30及び容器格納部11aは、一方の横貫通孔11bを介して連通する。さらに、容器格納部11a及び濁度計測用素子30は、他方の横貫通孔11dを介して連通する。この状態は図3及び4に示される。より具体的には、本発明装置内において、光源20、容器格納部11a及び濁度計測用素子30は、略水平の位置関係に配置することができる。 As described above, the light source 20, the container storage portion 11a, and the turbidity measuring element 30 are positioned on a substantially straight line. Thereby, the light source 30 and the container storage part 11a communicate with each other through one lateral through hole 11b. Further, the container storage portion 11a and the turbidity measuring element 30 communicate with each other through the other lateral through hole 11d. This state is shown in FIGS. More specifically, in the device of the present invention, the light source 20, the container storage portion 11a, and the turbidity measuring element 30 can be arranged in a substantially horizontal positional relationship.
本発明装置においては、濁度計測用素子30は、フォトダイオードであることができる。フォトダイオードでは、入射した光を電荷に変換するため、光強度を電圧として計測することが可能である。フォトダイオードは、感度が高く高精度の光強度測定が可能である。容器1中の測定試料が透明な場合、光強度が大きく、容器1中の測定試料が濁った場合、光強度が小さくなる。光強度の変化の度合いを濁度として計測する。フォトダイオードは、光源LEDのピーク波長が465nmである場合、波長帯域が例えば、450~500nmであることができる。但し、これに限定される意図ではない。 In the device of the present invention, the turbidity measuring element 30 can be a photodiode. In the photodiode, incident light is converted into electric charge, so that the light intensity can be measured as a voltage. The photodiode has high sensitivity and can measure the light intensity with high accuracy. When the measurement sample in the container 1 is transparent, the light intensity is high, and when the measurement sample in the container 1 is cloudy, the light intensity is low. The degree of change in light intensity is measured as turbidity. When the peak wavelength of the light source LED is 465 nm, the wavelength band of the photodiode can be, for example, 450 to 500 nm. However, it is not the intention limited to this.
<蛍光計測用素子40>
蛍光計測用素子40は、容器格納部11a内に格納した容器1中の測定試料から発せられる蛍光を計測するための素子である。光照射用光源20から照射される光は、横貫通孔11bを介して、容器格納部11a内に格納された容器1に照射され、容器1内の測定試料がこの照射光により蛍光を発する場合、蛍光計測用素子40により計測される。蛍光計測用素子40は、密閉された蛍光計測用遮光ユニット41内に収納され、蛍光計測用遮光ユニット41の開口は、容器格納部11aの縦貫通孔11cの外部開口と対向して接続される。容器格納部11aからの蛍光は、容器格納部11aの縦貫通孔11cを介して、蛍光計測用遮光ユニット41内に収納された蛍光計測用素子40に到達する。
<Fluorescence measurement element 40>
The fluorescence measurement element 40 is an element for measuring fluorescence emitted from the measurement sample in the container 1 stored in the container storage portion 11a. The light irradiated from the light irradiation light source 20 is irradiated to the container 1 stored in the container storage portion 11a through the horizontal through hole 11b, and the measurement sample in the container 1 emits fluorescence by this irradiation light. Measured by the fluorescence measuring element 40. The fluorescence measurement element 40 is housed in a sealed fluorescence measurement light-shielding unit 41, and the opening of the fluorescence measurement light-shielding unit 41 is connected to face the external opening of the vertical through hole 11c of the container storage portion 11a. . The fluorescence from the container storage portion 11a reaches the fluorescence measurement element 40 housed in the fluorescence measurement light-shielding unit 41 via the vertical through hole 11c of the container storage portion 11a.
本発明装置においては、容器格納部11a及び蛍光計測用素子40は、縦貫通孔11cを介して連通する。光源20、容器格納部11a及び蛍光計測用素子40は、光源20と容器格納部11aを結ぶ直線と容器格納部11aと蛍光を計測するための素子40を結ぶ直線とが、略垂直関係になるように位置する。
より具体的には、本発明装置内において、蛍光計測用素子40は、容器格納部11aの略下方に位置するように配置することができる。
In the device of the present invention, the container storage portion 11a and the fluorescence measurement element 40 communicate with each other through the vertical through hole 11c. In the light source 20, the container storage unit 11a, and the fluorescence measurement element 40, a straight line connecting the light source 20 and the container storage unit 11a and a straight line connecting the container storage unit 11a and the element 40 for measuring fluorescence are in a substantially vertical relationship. Is located.
More specifically, in the device of the present invention, the fluorescence measurement element 40 can be arranged so as to be positioned substantially below the container storage portion 11a.
本発明装置においては、蛍光計測用素子40は、フォトICであることができる。フォトICでは、入射した光を電荷に変換するため、光強度を電圧として計測することが可能である。フォトICは、自身で電荷を増幅するため、微弱な蛍光であっても計測することが可能であり、より高精度、高感度の蛍光測定が可能である。容器1中の測定試料に蛍光体が含まれている場合、光源LEDの光を蛍光体に照射することで励起された光が全方向に発光する。光源LEDのピーク波長が465nmである場合、例えば、蛍光体の励起波長が465nm近傍であり、蛍光体の発光波長が530nm近傍である場合、フォトICは、波長帯域が例えば、500~580nmであることができる。但し、これに限定される意図ではない。 In the device of the present invention, the fluorescence measurement element 40 can be a photo IC. In the photo IC, incident light is converted into electric charge, so that the light intensity can be measured as a voltage. Since the photo IC amplifies charges by itself, it is possible to measure even weak fluorescence, and fluorescence measurement with higher accuracy and higher sensitivity is possible. When the fluorescent substance is contained in the measurement sample in the container 1, the light excited by irradiating the fluorescent substance with the light from the light source LED emits light in all directions. When the peak wavelength of the light source LED is 465 nm, for example, when the excitation wavelength of the phosphor is around 465 nm and the emission wavelength of the phosphor is around 530 nm, the photo IC has a wavelength band of, for example, 500 to 580 nm. be able to. However, it is not the intention limited to this.
<波長フィルタ44>
本発明装置においては、容器格納部11aと蛍光計測用素子40の間に、光源の光の少なくとも一部をカットし、容器1中の測定試料から発せられる蛍光の少なくとも一部を透過する波長フィルタ44を含む。これにより、容器1中の測定試料から発せられる蛍光のみを測定することが可能になり、測定精度を向上させることができる。
<Wavelength filter 44>
In the device of the present invention, a wavelength filter that cuts at least part of the light of the light source and transmits at least part of the fluorescence emitted from the measurement sample in the container 1 between the container storage portion 11a and the fluorescence measurement element 40. 44. Thereby, it becomes possible to measure only the fluorescence emitted from the measurement sample in the container 1, and the measurement accuracy can be improved.
本発明装置においては、波長フィルタ44は、光源LEDのピーク波長が465nmである場合、例えば、520nm以下の波長をカットするフィルタであることができる。波長フィルタ44は、光源LEDのピーク波長や容器1中の測定試料から発せられる蛍光のピーク波長などを考慮して、適宜選択することができる。 In the device of the present invention, the wavelength filter 44 can be a filter that cuts a wavelength of 520 nm or less, for example, when the peak wavelength of the light source LED is 465 nm. The wavelength filter 44 can be appropriately selected in consideration of the peak wavelength of the light source LED and the peak wavelength of fluorescence emitted from the measurement sample in the container 1.
<温度調節装置>
本発明装置は、容器格納部11a内に格納した容器1の温度を調節するための温度調節装置を含む。より具体的には、温度調節用の加熱のための装置として、サンプルホルダ11は、例えば、内部にヒーターを内蔵することができ、サンプルホルダ11が複数の容器格納部11aを有する場合には、図1のようにサンプルホルダ11の長手方向に棒状ヒーター12を内蔵することができる。棒状ヒーター12は単一のヒーターであっても、複数のヒーターであってもよい。サンプルホルダ11の内部にヒーターを内蔵させることで、容器格納部11a内に格納した容器1をより所望の温度に的確に加熱することができる。但し、サンプルホルダ11の外側に追加でまたは内部ヒーターに代えてヒーターを設けることもできる。その際、棒状以外の形状のヒーターであってもよい。
<Temperature control device>
The device of the present invention includes a temperature adjusting device for adjusting the temperature of the container 1 stored in the container storage portion 11a. More specifically, as an apparatus for heating for temperature control, the sample holder 11 can incorporate a heater, for example, and when the sample holder 11 has a plurality of container storage portions 11a, As shown in FIG. 1, a rod heater 12 can be built in the longitudinal direction of the sample holder 11. The rod heater 12 may be a single heater or a plurality of heaters. By incorporating a heater in the sample holder 11, the container 1 stored in the container storage portion 11a can be heated more accurately to a desired temperature. However, a heater may be provided outside the sample holder 11 or in place of the internal heater. In that case, a heater having a shape other than a rod shape may be used.
さらに温度調節用の冷却のための装置として、図2に示すように、測定部10に向けて送風するための送風用のファン50を設けることができる。送風用のファン50は、測定部10の例えば、下部に設けることができ、このファン50は、サンプルホルダ11等に装置の外部の空気を送り、サンプルホルダ11を効率よく、かつ均一に冷却することができる。即ち、ファン50を稼働すると装置外の空気を装置内に吸気して、測定部10の方向に送風して、サンプルホルダ11を冷却する。 Furthermore, as a device for cooling for temperature adjustment, as shown in FIG. 2, a fan 50 for blowing air for blowing air toward the measuring unit 10 can be provided. The fan 50 for blowing air can be provided, for example, in the lower part of the measuring unit 10, and the fan 50 sends air outside the apparatus to the sample holder 11 and the like to cool the sample holder 11 efficiently and uniformly. be able to. That is, when the fan 50 is operated, air outside the apparatus is sucked into the apparatus and blown in the direction of the measurement unit 10 to cool the sample holder 11.
さらに、前記温度調節装置は、サンプルホルダ11の内部またはサンプルホルダ11に隣接して設けられた温度測定手段、例えば、温度センサを含むことができる。 Further, the temperature adjusting device may include temperature measuring means, for example, a temperature sensor, provided inside the sample holder 11 or adjacent to the sample holder 11.
<容器蓋押さえ部14>
本発明装置は、サンプルホルダ11の容器格納部11aの、容器1を容器格納部11aに挿入して格納するため記載開口に対向する位置に、サンプルホルダの容器格納部に格納された容器の蓋を押さえるための容器蓋押さえ部14をさらに有することができる。容器蓋押さえ部14は、サンプルホルダに接近及び乖離可能であり、容器格納部11aに容器1を挿入するときは、サンプルホルダから乖離した位置を取り、容器格納部11aに容器1を挿入して格納した後は、サンプルホルダに接近し、さらに容器格納部11aに格納された容器1の蓋を押さえることができる。容器蓋押さえ部14は、温度調節装置を含むことができる。ここでの温度調節装置はヒーターであり、より具体的には容器蓋押さえ部14の内部に設けたヒーターであることができる。容器蓋押さえ部14は、サンプルホルダ11の平面形状と対応する形状であることが、サンプルホルダ11に設けられた容器格納部11aに格納される全ての容器1(複数の場合)の蓋押さえることができるという観点で好ましい。サンプルホルダ11が複数の容器格納部11aを有し、容器蓋押さえ部14がそれに対応する形状を有する場合、蓋押さえ部14の内部に設けるヒーターは、棒状ヒーター15であることができる。蓋押さえ部14がヒーターを内装することで、容器1の蓋を加熱することができ、それにより容器内の溶液の蒸発を抑え、容器の上部や蓋内に水滴が付着することを抑え、容器内での反応条件を一定に維持することができる。但し、蓋押さえ部14の外側に追加でまたは内部ヒーターに代えてヒーターを設けることもできる。その際、棒状以外の形状のヒーターであってもよい。
<Container lid holding part 14>
The apparatus according to the present invention is configured so that the container storage portion 11a of the sample holder 11 has a lid for the container stored in the container storage portion of the sample holder at a position facing the described opening for inserting and storing the container 1 into the container storage portion 11a. The container lid pressing part 14 for pressing can be further provided. The container lid holding part 14 can approach and separate from the sample holder. When the container 1 is inserted into the container storage part 11a, the container cover holding part 14 takes a position away from the sample holder and inserts the container 1 into the container storage part 11a. After the storage, the sample holder can be approached and the lid of the container 1 stored in the container storage unit 11a can be pressed. The container lid pressing part 14 can include a temperature adjusting device. The temperature adjusting device here is a heater, and more specifically, a heater provided inside the container lid pressing portion 14. The container lid holding portion 14 has a shape corresponding to the planar shape of the sample holder 11 to hold the lid of all the containers 1 (in a plurality of cases) stored in the container storage portion 11 a provided in the sample holder 11. It is preferable from the viewpoint that When the sample holder 11 has a plurality of container storage portions 11 a and the container lid pressing portion 14 has a corresponding shape, the heater provided inside the lid pressing portion 14 can be a rod-shaped heater 15. The lid holding part 14 includes a heater, so that the lid of the container 1 can be heated, thereby preventing evaporation of the solution in the container and preventing water droplets from adhering to the upper part of the container or the lid. The reaction conditions inside can be kept constant. However, a heater may be provided outside the lid pressing portion 14 or in place of the internal heater. In that case, a heater having a shape other than a rod shape may be used.
本発明装置について図1~3に加えて、図4~6を用いて、本発明装置をより具体的に説明する。図4~6においては、容器1として反応用の透明チューブを示す。 The apparatus of the present invention will be described more specifically with reference to FIGS. 4 to 6 in addition to FIGS. 4 to 6, a transparent tube for reaction is shown as the container 1.
図5は、サンプルホルダ11の上面を示す図であり、チューブ1を8個挿入できる形状の例を示している。13は温度センサであり、チューブ周辺のサンプルホルダの温度を測定する。温度センサ13は、サンプルホルダ11の側面のチューブ1を挿入する穴の間に配置することにより、温度センサ13を4個で、チューブ1の8個の温度を測定している。棒状ヒーター12は電圧を印加すると発熱し、電圧を変えると発熱量が変わる。周期的に温度センサ13を測定し、測定した温度に合わせて棒状ヒーター13への印加電圧を制御することにより、サンプルホルダ11の温度を制御する。 FIG. 5 is a view showing the upper surface of the sample holder 11 and shows an example of a shape into which eight tubes 1 can be inserted. A temperature sensor 13 measures the temperature of the sample holder around the tube. The temperature sensors 13 are arranged between the holes for inserting the tubes 1 on the side surface of the sample holder 11, thereby measuring four temperatures of the temperature sensors 13 and eight temperatures of the tubes 1. The bar heater 12 generates heat when a voltage is applied, and the amount of heat generated changes when the voltage is changed. The temperature of the sample holder 11 is controlled by periodically measuring the temperature sensor 13 and controlling the voltage applied to the rod heater 13 according to the measured temperature.
 図6は、容器(チューブ)蓋押さえ部14の上面を示す図である。容器(チューブ)蓋押さえ部14は、チューブ1の蓋を上から押さえる部材である。チューブ蓋押さえ部14は、棒状ヒーター15を内装しており、チューブ1の蓋を加熱することができ、チューブ内の溶液の蒸発を抑え、チューブ内に水滴が付着することを抑える。図6は、チューブ蓋押さえ部14の上面を示す図である。16は温度センサであり、チューブ蓋押さえ部14の温度を測定する。棒状ヒーター15は電圧を印加すると発熱し、電圧を変えると発熱量が変わる。周期的に温度センサ16を測定し、測定した温度に合わせて棒状ヒーター15への印加電圧を制御することにより、チューブ蓋押さえ部14の温度を制御する。 FIG. 6 is a view showing the upper surface of the container (tube) lid pressing portion 14. The container (tube) lid pressing portion 14 is a member that presses the lid of the tube 1 from above. The tube lid holding portion 14 includes a rod-shaped heater 15 that can heat the lid of the tube 1, suppresses evaporation of the solution in the tube, and prevents water droplets from adhering to the tube. FIG. 6 is a view showing the upper surface of the tube lid pressing portion 14. Reference numeral 16 denotes a temperature sensor, which measures the temperature of the tube lid pressing portion 14. The bar heater 15 generates heat when a voltage is applied, and the amount of generated heat changes when the voltage is changed. The temperature of the tube lid holding part 14 is controlled by measuring the temperature sensor 16 periodically and controlling the voltage applied to the rod heater 15 according to the measured temperature.
 図4は測定部の断面図である。サンプルホルダ11の容器格納部11aの先端部付近には、水平方向の対面に2個と下方向に1個、穴(貫通孔)が空いている。光源部遮光ユニット21にLED基板23が取り付けてあり、LED22から照射する光は光源部遮光ユニット21の内部を進み、チューブ1及びDNAが入った溶液2に照射する。この場合、LED22から照射する光は、光源部遮光ユニット21で遮光され、光の進行方向から外れた方向へは照射されない構造である。 FIG. 4 is a cross-sectional view of the measurement unit. In the vicinity of the tip of the container storage portion 11a of the sample holder 11, there are two holes (through holes) in the horizontal direction and one in the downward direction. The LED substrate 23 is attached to the light source unit light shielding unit 21, and the light emitted from the LED 22 travels inside the light source unit light shielding unit 21 to irradiate the solution 1 containing the tube 1 and DNA. In this case, the light emitted from the LED 22 is shielded by the light source unit light shielding unit 21 and is not irradiated in a direction deviating from the traveling direction of the light.
濁度測定部遮光ユニット31に濁度測定用光センサ基板33が取り付けてあり、DNAが入った溶液2を透過若しくは拡散した光は、チューブ1を透過し、濁度測定部遮光ユニット31の内部を進み、濁度測定用光センサ32で受光する。この場合、外部の光は濁度測定部遮光ユニット31で遮光される構造である。
蛍光測定部遮光ユニット41に蛍光測定用光センサ基板43と波長フィルタ44が取り付けてあり、DNAが入った溶液2で蛍光発光した光は、チューブ1を透過し、波長フィルタ44を透過し、蛍光測定部遮光ユニット41の内部を進み、蛍光測定用光センサ42で受光する。この場合、外部の光は蛍光測定部遮光ユニット41で遮光される構造である。また、波長フィルタ44の透過波長の光のみ蛍光測定用光センサ42に入射し、他の波長の光は遮光される構造である。
The turbidity measurement light-shielding unit 31 is attached to the turbidity measurement part light-shielding unit 31, and the light transmitted or diffused through the solution 2 containing the DNA is transmitted through the tube 1 and the interior of the turbidity measurement part light-shielding unit 31. The turbidity measuring optical sensor 32 receives light. In this case, the external light is shielded by the turbidity measuring unit light shielding unit 31.
The fluorescence measurement light-shielding unit 41 and the wavelength filter 44 are attached to the fluorescence measurement unit light-shielding unit 41. The light emitted by the solution 2 containing DNA passes through the tube 1, passes through the wavelength filter 44, and is fluorescent. The light travels inside the measurement unit light shielding unit 41 and is received by the fluorescence measurement optical sensor 42. In this case, external light is shielded by the fluorescence measurement unit light shielding unit 41. Further, only light having a transmission wavelength of the wavelength filter 44 is incident on the fluorescence measurement optical sensor 42, and light of other wavelengths is shielded.
水平対面方向の穴には一方に、21の光源部遮光ユニット、22のLED、23のLED基板、もう一方に、31の濁度測定部遮光ユニット、32の濁度測定用光センサ、33の濁度測定用光センサ基板が取り付けてあり、垂直下方向の穴には、41の蛍光測定部遮光ユニット、44の波長フィルタ、42の蛍光測定用光センサ、43の蛍光測定用光センサ基板が取り付けてある。 In the hole in the horizontal facing direction, 21 light source unit light shielding units, 22 LEDs, 23 LED boards, 31 turbidity measuring unit light shielding units, 32 turbidity measuring light sensors, 33 A turbidity measuring photosensor substrate is attached, and in the vertically downward hole, there are 41 fluorescence measuring section light shielding units, 44 wavelength filters, 42 fluorescence measuring photosensors, and 43 fluorescence measuring photosensor substrates. It is attached.
LED22から発光した光は、光源部遮光ユニット21の内部を進み、サンプルホルダ11の先端部分の水平方向の1つの穴を通り、チューブ1を透過し、DNAが入った溶液2に含まれる物質に当たり、光が透過若しくは拡散して、透過光及び拡散光の一部が水平方向の対面側へ進み、さらにチューブ1を透過して、サンプルホルダ11の容器格納部11aの先端部分の水平方向の対面にあるもう1つの穴を通り、濁度測定部遮光ユニット31の内部を進み、濁度測定用光センサ32で受光する。 The light emitted from the LED 22 travels inside the light source light blocking unit 21, passes through one horizontal hole at the tip of the sample holder 11, passes through the tube 1, and hits a substance contained in the solution 2 containing DNA. , The light is transmitted or diffused, part of the transmitted light and diffused light travels to the opposite side in the horizontal direction, further passes through the tube 1, and faces the front end of the container storage portion 11 a of the sample holder 11 in the horizontal direction. The light passes through another hole in the turbidity measurement unit, passes through the inside of the turbidity measurement unit light-shielding unit 31, and is received by the turbidity measurement optical sensor 32.
LED22から発光した光は、光源部遮光ユニット21の内部を進み、サンプルホルダ11の先端部分の水平方向の1つの穴を通り、チューブ1を透過して、DNAが入った溶液2に含まれる蛍光物質に当たり蛍光発光して、さらに、蛍光発光した光の一部がサンプルホルダ11の下方向に進み、チューブ1を透過して、サンプルホルダ11の先端部分の下方向の穴を通り、蛍光測定部遮光ユニット41の内部を進み、蛍光測定用光センサ42で受光する。 The light emitted from the LED 22 travels inside the light source block unit 21, passes through one horizontal hole at the tip of the sample holder 11, passes through the tube 1, and is contained in the solution 2 containing DNA. Fluorescent light is emitted upon hitting the substance, and further, a part of the fluorescent light travels downward in the sample holder 11, passes through the tube 1, passes through the downward hole in the distal end portion of the sample holder 11, and the fluorescence measuring unit The light passes through the light blocking unit 41 and is received by the fluorescence measuring optical sensor 42.
 濁度測定用光センサ32で受光した光強度と、蛍光測定用光センサ42で受光した光強度は、同時に取得することもできる。これにより、濁度測定と蛍光測定が同時に実施可能である。 The light intensity received by the turbidity measurement optical sensor 32 and the light intensity received by the fluorescence measurement optical sensor 42 can be acquired simultaneously. Thereby, turbidity measurement and fluorescence measurement can be performed simultaneously.
本発明装置は、容器1中の濁度及び蛍光を同時又は逐次に測定するために用いられる。さらに本発明装置は、容器1中のDNAが入った溶液2に含まれる核酸の増幅を濁度測定により検知するために用いることができる。より具体的には、容器1中のDNAが入った溶液2において核酸増幅後に、一本鎖核酸のハイブリダイズ又は二本鎖核酸の解離を蛍光の発光又は消光を用いて検知するために用いることができる。この点については、後述する。 The apparatus of the present invention is used to measure turbidity and fluorescence in the container 1 simultaneously or sequentially. Furthermore, the device of the present invention can be used to detect amplification of nucleic acids contained in the solution 2 containing DNA in the container 1 by turbidity measurement. More specifically, it is used to detect hybridization of single-stranded nucleic acid or dissociation of double-stranded nucleic acid using fluorescence emission or quenching after nucleic acid amplification in solution 2 containing DNA in container 1. Can do. This point will be described later.
[核酸分析方法]
本発明の核酸分析方法は、前記本発明装置を用いる核酸分析方法であって、以下の(1)~(3)の工程を含む。
(1)前記容器中に測定試料及び核酸増幅のための材料を含有する溶液を配置する。
(2)前記容器中の溶液の濁度を測定して、前記測定試料を鋳型とする核酸の増幅の度合を検知する。
(3)所定の増幅が確認された後に、前記容器中の溶液の蛍光を測定して、増幅した核酸の配列を確認する。
[Nucleic acid analysis method]
The nucleic acid analysis method of the present invention is a nucleic acid analysis method using the apparatus of the present invention, and includes the following steps (1) to (3).
(1) A solution containing a measurement sample and a material for nucleic acid amplification is placed in the container.
(2) The turbidity of the solution in the container is measured to detect the degree of nucleic acid amplification using the measurement sample as a template.
(3) After the predetermined amplification is confirmed, the fluorescence of the solution in the container is measured to confirm the sequence of the amplified nucleic acid.
 さらに本発明の核酸分析方法では、前記容器中の溶液の蛍光測定は、溶液の温度を変化させることで、溶液中の一本鎖核酸をハイブリダイズさせて蛍光の発光又は消光を観察するか、又は溶液中の二本鎖核酸の解離を蛍光の発光又は消光を観察することで行う。 Furthermore, in the nucleic acid analysis method of the present invention, the fluorescence measurement of the solution in the container is performed by changing the temperature of the solution to hybridize single-stranded nucleic acid in the solution and observing fluorescence emission or quenching, Alternatively, the double-stranded nucleic acid in the solution is dissociated by observing fluorescence emission or quenching.
工程(1)
容器1中に測定試料及び核酸増幅のための材料を含有する溶液を配置する。
容器1は、前述の本発明装置において説明した通りである。
測定試料は、例えば、生体(例えば、動物や植物)から抽出したDNAであることができる。但し、LAMP法で増幅できる者であれば、DNA以外の核酸であってもよい。核酸増幅のための材料は、増幅対象である核酸の種類や増幅方法に応じて適宜選択できる。
核酸分析のための反応系に用いる、測定試料及び核酸増幅のための材料を含有する溶液は、既知のものをそのまま利用できる。核酸増幅のための材料としては、例えば、WO2014/167377に記載のLAMP-FLP法として説明した反応系(例えば、段落0019~0036参照)を利用することができる。また、測定試料としては、WO2014/167377の例えば、段落0060~0064の記載を参照できる。WO2014/167377の全記載は、ここに特に開示として援用される。
Process (1)
A solution containing a measurement sample and a material for nucleic acid amplification is placed in the container 1.
The container 1 is as described in the above-described device of the present invention.
The measurement sample can be, for example, DNA extracted from a living body (for example, an animal or a plant). However, a nucleic acid other than DNA may be used as long as it can be amplified by the LAMP method. The material for nucleic acid amplification can be appropriately selected according to the type of nucleic acid to be amplified and the amplification method.
As a solution containing a measurement sample and a material for nucleic acid amplification used in a reaction system for nucleic acid analysis, a known solution can be used as it is. As a material for nucleic acid amplification, for example, the reaction system described as the LAMP-FLP method described in WO2014 / 167377 (for example, see paragraphs 0019 to 0036) can be used. For the measurement sample, reference can be made to, for example, the descriptions in paragraphs 0060 to 0064 of WO2014 / 167377. The entire description of WO2014 / 167377 is hereby specifically incorporated by reference.
前記溶液を含有する容器1は本発明装置のサンプルホルダにセットする。
本発明装置は、複数のサンプルホルダを有することができ、各サンプルホルダに異なる溶液(特に異なる測定試料を含む溶液)を含む容器をセットすることで、これらの測定試料についての核酸分析を同時に実施することができる。
The container 1 containing the solution is set in the sample holder of the device of the present invention.
The apparatus of the present invention can have a plurality of sample holders, and by setting a container containing different solutions (particularly solutions containing different measurement samples) in each sample holder, nucleic acid analysis of these measurement samples is simultaneously performed. can do.
工程(2)
前記容器中の溶液の濁度を測定して、前記測定試料を鋳型とする核酸の増幅の度合を検知する。濁度測定は連続的にまたは断続的に行うことができる。測定された濁度は、電気信号として記録することができ、さらに必要に応じて、ディスプレイや紙媒体などに表示することができる。さらに、測定された濁度に、既知のデータに基づいて閾値を設定し、この閾値を超えたら、核酸の増幅が所定(所望)の度合まで進行した、と認定することもできる。さらに、前記閾値を超えたことをディスプレイや紙媒体に表示して、測定者に知らせることもできる。
Step (2)
The turbidity of the solution in the container is measured to detect the degree of nucleic acid amplification using the measurement sample as a template. Turbidity measurements can be made continuously or intermittently. The measured turbidity can be recorded as an electrical signal, and can be displayed on a display or a paper medium, if necessary. Furthermore, a threshold value is set for the measured turbidity based on known data, and when this threshold value is exceeded, it can be recognized that the nucleic acid amplification has progressed to a predetermined (desired) degree. Furthermore, it is possible to notify the measurer that the threshold value has been exceeded by displaying it on a display or a paper medium.
工程(3)
所定の増幅が確認された後に、前記容器中の溶液の蛍光を測定して、増幅した核酸の配列を確認する。蛍光測定は連続的にまたは断続的に行うことができる。測定された蛍光は、信号として記録することができ、さらに必要に応じて、ディスプレイや紙媒体などに表示することができる。
Process (3)
After the predetermined amplification is confirmed, the fluorescence of the solution in the container is measured to confirm the sequence of the amplified nucleic acid. Fluorescence measurements can be made continuously or intermittently. The measured fluorescence can be recorded as a signal, and can be displayed on a display or a paper medium, if necessary.
蛍光測定は、例えば、溶液の温度を高温から低温に変化させることで、一本鎖核酸同士をハイブリダイズさせて蛍光の発光又は消光を観察することで行うことができる。あるいは、蛍光測定は、溶液の温度を低温から高温に変化させることで、二本鎖核酸の解離を蛍光の発光又は消光を観察することで行うことができる。尚、溶液の温度の「高温」及び「低温」は、ハイブリダイズさせる一本鎖核酸の配列、解離させる二本鎖核酸の配列に応じて、適宜設定できる。 The fluorescence measurement can be performed, for example, by changing the temperature of the solution from a high temperature to a low temperature to hybridize single-stranded nucleic acids and observe fluorescence emission or quenching. Alternatively, fluorescence measurement can be performed by observing fluorescence emission or quenching by dissociating double-stranded nucleic acids by changing the temperature of the solution from a low temperature to a high temperature. The “high temperature” and “low temperature” of the solution temperature can be appropriately set according to the sequence of the single-stranded nucleic acid to be hybridized and the sequence of the double-stranded nucleic acid to be dissociated.
測定試料を鋳型とする核酸の増幅は等温核酸増幅法で行うことができる。等温核酸増幅法は、例えば、LAMP法であることができる。LAMP法による核酸増幅方法は、例えば、前述のWO2014/167377を参照できる。但し、等温核酸増幅法及びLAMP法に限定される意図ではなく、他の方法も適宜使用することはできる。 Amplification of a nucleic acid using a measurement sample as a template can be performed by an isothermal nucleic acid amplification method. The isothermal nucleic acid amplification method can be, for example, the LAMP method. For the nucleic acid amplification method by the LAMP method, for example, the above-mentioned WO2014 / 167377 can be referred to. However, it is not intended to be limited to the isothermal nucleic acid amplification method and the LAMP method, and other methods can be used as appropriate.
等温核酸増幅法による核酸増幅は、例えば、40~70℃の温度で行い、増幅完了後に溶液を90~100℃に加熱し、増幅した二本鎖核酸を解離させて一本鎖核酸とし、その後、溶液を冷却して一本鎖核酸のハイブリダイズによる蛍光の発光又は消光を観察することができる。但し、この条件に限定される意図ではない。 Nucleic acid amplification by the isothermal nucleic acid amplification method is performed, for example, at a temperature of 40 to 70 ° C. After the amplification is completed, the solution is heated to 90 to 100 ° C. to dissociate the amplified double-stranded nucleic acid into a single-stranded nucleic acid, and then The solution can be cooled and fluorescence emission or quenching due to hybridization of the single-stranded nucleic acid can be observed. However, it is not intended to be limited to this condition.
本発明の核酸分析方法は、前記測定試料に含まれる核酸の一塩基多型(SNPs)を測定するために用いることができる。この点を以下により具体的に説明する。 The nucleic acid analysis method of the present invention can be used for measuring single nucleotide polymorphisms (SNPs) of nucleic acids contained in the measurement sample. This point will be described more specifically below.
 図7は、本発明の実施形態のDNAを入れた溶液を使用し、DNAの増幅過程とDNA増幅の検出過程、DNAを増やすための触媒の失活過程、DNAのSNPsの検出過程を示す図である。1は溶液を入れるためのチューブである。2は生体から抽出したDNA、DNA増幅に必要な触媒、DNAと結合する蛍光物質、DNAと結合した際に濁る物質を混合した溶液である。DNAの入った混合溶液2を透明チューブ1に入れ、以下の処理を実施する。 FIG. 7 is a diagram showing a DNA amplification process, a DNA amplification detection process, a catalyst deactivation process, and a DNA SNP detection process using the solution containing the DNA of the embodiment of the present invention. It is. Reference numeral 1 denotes a tube for containing the solution. Reference numeral 2 denotes a solution in which DNA extracted from a living body, a catalyst necessary for DNA amplification, a fluorescent substance that binds to DNA, and a substance that becomes turbid when bound to DNA are mixed. The mixed solution 2 containing DNA is put into the transparent tube 1 and the following treatment is performed.
DNAの増幅過程とDNA増幅の検出過程では、所定の温度(DNAごとに異なる50℃から70℃弱のいずれかの温度)で所定の時間(DNAごとに異なる数分から数時間のいずれかの時間)加熱することにより、DNAが増幅する。DNAが増幅すると、蛍光物質が混合している場合、蛍光の光強度が大きくなるため、蛍光の明るさや光強度の変化が大きくなることを目視や検知することにより確認でき、濁る物質が混合している場合、溶液が濁ってくるため、物質の色の変化や、溶液を透過してくる光の光強度が小さくなることを目視や機器で検出することにより確認できる。 In the DNA amplification process and the DNA amplification detection process, a predetermined time (any temperature ranging from 50 ° C. to less than 70 ° C., which varies depending on the DNA) and a predetermined time (a time ranging from several minutes to several hours, which varies depending on the DNA) ) DNA is amplified by heating. When DNA is amplified, the fluorescent light intensity increases when fluorescent substances are mixed. Therefore, it can be confirmed by visual observation or detection that the change in the brightness or light intensity of the fluorescent light increases. In this case, since the solution becomes cloudy, it can be confirmed visually or by detecting with a device that the light intensity of the light transmitted through the solution becomes small.
DNAを増やすための触媒の失活過程では、所定の温度(DNAごとに異なる80℃から100℃のいずれかの温度)で所定の時間(DNAごとに異なる2分から10分のいずれかの時間)加熱して、DNAを増幅するために用いた触媒を失活させ、溶液中のDNAを安定させる。 In the deactivation process of the catalyst for increasing the DNA, a predetermined time at a predetermined temperature (any temperature between 80 ° C. and 100 ° C., which differs for each DNA) (any time between 2 minutes and 10 minutes, which differs for each DNA) Heating deactivates the catalyst used to amplify the DNA and stabilizes the DNA in the solution.
 DNAのSNPsの検出過程では、所定の温度範囲(100℃から40℃)で温度を変化させることにより、ある温度でDNAの二本鎖が切り離され2つの一本鎖に分離し、別の温度で二本鎖に結合する現象を利用する。DNAの二本鎖が切り離され2つの一本鎖に分離した際に、その一本鎖の一部と結合する物質を用い、その物質に蛍光体を結合させておき、DNAの二本鎖が切り離されたり、結合したりした時の蛍光光の光強度を検出することにより、SNPsを検出する。 In the detection process of DNA SNPs, by changing the temperature within a predetermined temperature range (100 ° C. to 40 ° C.), the DNA double strand is separated at one temperature and separated into two single strands, and another temperature. Uses the phenomenon of binding to double strands. When a double strand of DNA is cut and separated into two single strands, a substance that binds to a part of the single strand is used, and a phosphor is bound to the substance so that the double strand of DNA SNPs are detected by detecting the light intensity of the fluorescent light when it is separated or combined.
以下、本発明を実施例に基づいて更に詳細に説明する。但し、実施例は本発明の例示であって、本発明は実施例に限定される意図ではない。 Hereinafter, the present invention will be described in more detail based on examples. However, the examples are illustrative of the present invention, and the present invention is not intended to be limited to the examples.
 測定例を挙げ、DNAの増幅の検出方法と、DNAのSNPsの検出方法について説明する。サンプルとして人工DNAを使い、DNA増幅に必要な触媒、DNAと結合する蛍光物質、DNAと結合した際に濁る物質などを混合したものを使用した。今回、No.1からNo.8の8個のサンプルを測定した。 A measurement example will be given to explain a method for detecting DNA amplification and a method for detecting DNA SNPs. Artificial DNA was used as a sample, and a mixture of a catalyst necessary for DNA amplification, a fluorescent substance that binds to DNA, and a substance that becomes turbid when bound to DNA was used. This time, No. 1 to No. Eight samples of eight were measured.
 図8は測定時の温度グラフである。縦軸が温度(℃)、横軸が時間(秒)である。室温(約25℃)から63℃へ上昇し、63℃で30分(1800秒)温度を維持し、98℃へ上昇し、98℃で5分(300秒)温度を維持し、35℃へ下降し、35℃で測定を終了した。
 63℃で30分温度を維持している期間は、濁度測定時であり、サンプルの濁度光強度を測定する。
 98℃から35℃へ温度下降している期間は、蛍光測定時である、サンプルの蛍光光強度を測定する。尚、ここでは、温度下降時に蛍光測定する事例を説明しているが、温度上昇時に蛍光測定することも可能である。
FIG. 8 is a temperature graph at the time of measurement. The vertical axis is temperature (° C.), and the horizontal axis is time (seconds). Rise from room temperature (about 25 ° C) to 63 ° C, maintain temperature at 63 ° C for 30 minutes (1800 seconds), rise to 98 ° C, maintain temperature at 98 ° C for 5 minutes (300 seconds), to 35 ° C Then, the measurement was completed at 35 ° C.
The period during which the temperature is maintained at 63 ° C. for 30 minutes is during turbidity measurement, and the turbidity light intensity of the sample is measured.
During the period when the temperature is lowered from 98 ° C. to 35 ° C., the fluorescence intensity of the sample, which is the time of fluorescence measurement, is measured. Note that, here, an example in which fluorescence is measured when the temperature falls is described, but fluorescence measurement can also be performed when the temperature rises.
 図9は濁度測定時の光強度グラフである。縦軸が32濁度測定用光センサの電圧値(mV)、横軸が時間(秒)である。LAMP法では、濁度Tとして光強度の自然対数(LN)を使う。濁度測定開始からn番目の濁度Tは、濁度測定開始直後の濁度測定用光センサの電圧値Vを分母、濁度測定開始からn番目の濁度測定用光センサの電圧値Vを分子とした値をLN計算した値(式1)である。 FIG. 9 is a light intensity graph at the time of turbidity measurement. The vertical axis represents the voltage value (mV) of the 32 turbidity measuring photosensor, and the horizontal axis represents time (seconds). In the LAMP method, the natural logarithm (LN) of the light intensity is used as the turbidity T. The nth turbidity T n from the start of turbidity measurement is the denominator of the voltage value V 0 of the turbidity measurement photosensor immediately after the start of turbidity measurement, and the voltage of the nth turbidity measurement photosensor from the start of turbidity measurement. This is a value obtained by LN calculation using the value V n as a numerator (formula 1).
   T=LN(V/V) ・・・式1
     T:濁度測定開始からn番目の濁度
     V:濁度測定開始直後の濁度測定用光センサの電圧値(mV)
     V:濁度測定開始からn番目の濁度測定用光センサの電圧値(mV)
T n = LN (V 0 / V n ) Equation 1
T n : nth turbidity from the start of turbidity measurement V 0 : voltage value (mV) of the turbidity measuring photosensor immediately after the start of turbidity measurement
V n: n-th turbidity voltage value of the measuring light sensor from the turbidity measurement start (mV)
図9に示す濁度グラフ(縦軸が濁度T、横軸が時間(秒))において、濁度が閾値を越えた場合、DNAが増幅したと判定する。濁度の閾値は使用する条件、例えば、LAMP法ではプライマー配列により異なるため、DNAを用いた測定結果を基に、任意の値を設定する。本例では、閾値を1000とした。さらに、濁度測定開始から閾値を越えた時までの経過時間Tt、濁度のピーク値Dfを計算する。 In the turbidity graph shown in FIG. 9 (the ordinate is turbidity T and the abscissa is time (seconds)), when the turbidity exceeds a threshold value, it is determined that DNA is amplified. The turbidity threshold varies depending on the conditions used, for example, the primer sequence in the LAMP method, and therefore an arbitrary value is set based on the measurement result using DNA. In this example, the threshold is 1000. Further, the elapsed time Tt from the start of turbidity measurement until the threshold value is exceeded, and the turbidity peak value Df are calculated.
尚、閾値の設定は、増幅が陽性の場合には必ず一度は濁度がこの値を越え、陰性の場合には必ずこの値を下回り、一度もこの値を上回ることが無いように行う。さらに、閾値以上の濁度を示した場合、増幅陽性として増幅反応終了時に、装置本体に表示され、閾値未満の濁度を示した場合である増幅陰性の場合も同様に、装置本体に表示される。具体的には、増幅反応終了時に、当該ウェルのDET1(検出1)のLEDランプが判定結果の表示に用い、増幅陽性の場合は例えば、緑色、増幅陰性の場合は例えば、赤色がそれぞれ点灯するようにすることができる。これにより、増幅の有無 (閾値を越えたか越えていないか) を即時確認できる。 Note that the threshold is set so that the turbidity always exceeds this value once when the amplification is positive, and is always below this value when the amplification is negative, and never exceeds this value. Furthermore, when turbidity above the threshold is shown, amplification positive is displayed at the end of the amplification reaction, and in the case of amplification negative where turbidity below the threshold is shown, it is also displayed on the apparatus main body. The Specifically, at the end of the amplification reaction, the LED lamp of the DET1 (detection 1) of the well is used to display the determination result. For example, green is lit when amplification is positive, and red is lit when amplification is negative. Can be. As a result, the presence / absence of amplification (whether the threshold is exceeded or not exceeded) can be immediately confirmed.
 図10は蛍光測定時の光強度グラフである。縦軸が42蛍光測定用光センサの電圧値(mV)、横軸が温度(℃)である。図11は蛍光測定時の蛍光変化グラフである。縦軸が42蛍光測定用光センサの電圧差(mV)、横軸が温度(℃)である。蛍光変化量は、42蛍光測定用光センサの、測定した電圧値と1回前に測定した電圧値の差である。電圧差をそのまま利用するとノイズの影響が大きいため、平均化処理を行い、蛍光変化グラフを滑らかにする(図12)。蛍光変化グラフの複数のピークを検出し、その温度を検出する。ピークの温度が所定の温度範囲内にあれば、該当するSNPと判定する。ピークの温度範囲は使用するプローブにより異なるため、まず、ピークの自動検出設定のために、当該SNPをメジャータイプとした人工DNAと当該SNPをマイナータイプとした人工DNAを用いて、各タイプのピーク温度を確認する。そして、実際に検出されたピークがメジャータイプと同程度であればメジャータイプ、マイナータイプと同様であればマイナータイプと判断するように設定する。同程度とは、例えば±3℃である。 FIG. 10 is a light intensity graph at the time of fluorescence measurement. The vertical axis represents the voltage value (mV) of the 42 fluorescence measurement photosensor, and the horizontal axis represents the temperature (° C.). FIG. 11 is a fluorescence change graph at the time of fluorescence measurement. The vertical axis represents the voltage difference (mV) of the 42 fluorescence measurement optical sensor, and the horizontal axis represents the temperature (° C.). The amount of change in fluorescence is the difference between the measured voltage value of the 42 fluorescence measurement optical sensor and the voltage value measured once before. Since the influence of noise is large if the voltage difference is used as it is, an averaging process is performed to smooth the fluorescence change graph (FIG. 12). A plurality of peaks in the fluorescence change graph are detected, and their temperatures are detected. If the peak temperature is within a predetermined temperature range, it is determined as the corresponding SNP. Since the temperature range of the peak differs depending on the probe used, first, for the automatic detection setting of the peak, each type of peak is determined using artificial DNA having the SNP as a major type and artificial DNA having the SNP as a minor type. Check the temperature. If the actually detected peak is similar to the major type, the major type is determined. If the detected peak is similar to the minor type, the minor type is determined. The same level is, for example, ± 3 ° C.
メジャータイプ及びマイナータイプの判定のためには、ピークの形成される温度すなわちプローブの蛍光が消光する温度の範囲を予め設定する。メジャーアリルとマイナーアリルでピーク形成温度に差が生じるように設計されたプローブを用いることにより、SNPsタイピングにおいては最大二つのピークを形成することができる。このピーク位置は前述の通り人工DNAを用いた検証により決定され、温度範囲の誤差は±3℃の範囲である。装置本体に、この二つのピークを検出するための温度範囲設定の項目を予め備えておく。例えば、高温側のピークを55から64℃、低温側のピークを45-54℃と設定しておくことにより、それぞれの範囲にピークがあるかどうかを判定させることができる。但し、ノイズをピークとして検出しないようにするため、ここでも閾値を設定しておくことも可能である。 For the determination of the major type and the minor type, a temperature range at which a peak is formed, that is, a temperature range where the fluorescence of the probe is quenched is set in advance. By using a probe designed to produce a difference in peak formation temperature between major allyl and minor allyl, a maximum of two peaks can be formed in SNPs typing. This peak position is determined by verification using artificial DNA as described above, and the temperature range error is within ± 3 ° C. The apparatus main body is previously provided with a temperature range setting item for detecting these two peaks. For example, by setting the peak on the high temperature side to 55 to 64 ° C. and the peak on the low temperature side to 45 to 54 ° C., it can be determined whether or not there is a peak in each range. However, in order not to detect noise as a peak, it is also possible to set a threshold here.
本例では、2種類の温度範囲(1つ目として40.5℃から46.5℃の範囲(メジャータイプ)、2つ目として50.0℃から56.0℃の範囲(マイナータイプ))を設定し、メジャータイプのSNP、マイナータイプのSNP、メジャータイプとマイナータイプが共存するタイプのSNPの、3種類のSNPsタイプを検出可能とした。 In this example, two temperature ranges (first range from 40.5 ° C to 46.5 ° C (major type), second range from 50.0 ° C to 56.0 ° C (minor type)) The three types of SNPs can be detected: a major type SNP, a minor type SNP, and a SNP in which the major type and minor type coexist.
より具体的には、前述した二つの範囲のいずれかあるいは両方にピークが検出され、その組合せは3通りである。3通りの結果の表示は例えば、以下のように行うことができる。高温側のみピークを検出した場合は、当該ウェルのDET1のLEDが緑色に点灯する。一方で、低温側のみピークを検出した場合は、当該ウェルのDET2のLEDが青色に点灯する。さらに、両温度でピークを検出した場合、すなわちヘテロのアリルの場合には、当該ウェルのLEDはDET1の緑色、DET2の青色ともに点灯する。なお、ピークが検出されなかった場合は当該ウェルのNGのLEDが赤色に点灯するように設定できる。このように、SNPs判定結果を反応終了後に装置本体に表示することで、容易に視認することができる。 More specifically, a peak is detected in one or both of the two ranges described above, and there are three combinations. The display of the three results can be performed as follows, for example. When a peak is detected only on the high temperature side, the LED of DET1 of the well is lit in green. On the other hand, when a peak is detected only on the low temperature side, the DET2 LED of the well is lit in blue. Further, when a peak is detected at both temperatures, that is, in the case of hetero allyl, the LED of the well is lit both in green for DET1 and blue for DET2. If no peak is detected, the NG LED of the well can be set to light red. Thus, the SNPs determination result can be easily visually recognized by displaying it on the apparatus main body after the reaction is completed.
 図13は検出結果一覧表である。No.1とNo.2はDNA増幅していないサンプルである。No.3とNo.6はメジャータイプのSNP(温度範囲40.5℃から46.5℃でのみ蛍光測定時のピーク検出)、No.4とNo.7はマイナータイプのSNP(温度範囲50.0℃から56.0℃でのみ蛍光測定時のピーク検出)、No.5とNo.8はメジャータイプとマイナータイプが共存するSNP(温度範囲40.5℃から46.5℃と、温度範囲50.0℃から56.0℃の両方で蛍光測定時のピーク検出)のサンプルである。目的のSNPsが検出可能であることが確認できた。 FIG. 13 is a list of detection results. No. 1 and No. 2 is a sample which has not been amplified. No. 3 and no. No. 6 is a major type SNP (peak detection at the time of fluorescence measurement only in a temperature range of 40.5 ° C. to 46.5 ° C.). 4 and no. No. 7 is a minor type SNP (peak detection at the time of fluorescence measurement only in a temperature range of 50.0 ° C. to 56.0 ° C.), No. 7 5 and No. 8 is a sample of SNP (peak detection at the time of fluorescence measurement in both temperature range 40.5 ° C to 46.5 ° C and temperature range 50.0 ° C to 56.0 ° C) in which major type and minor type coexist. . It was confirmed that the target SNPs can be detected.
本発明は、核酸分析用装置及び分析方法の分野において有用である。 The present invention is useful in the field of nucleic acid analysis devices and analysis methods.
1   容器(チューブ)
2   DNA含有溶液
10  測定部
11  サンプルホルダ
11a 容器格納部
11b、11d 横貫通孔
11c 縦貫通孔
12  棒状ヒーター(サンプルホルダ用)
13  温度センサ(サンプルホルダ用)
14  容器(チューブ)蓋押さえ部
15  棒状ヒーター(チューブ蓋押さえ部用)
16  温度センサ(チューブ蓋押さえ部用)
20  光源部
21  光源部遮光ユニット
22  LED
23  LED基板
30  濁度測定部
31  濁度測定部遮光ユニット
32  濁度測定用光センサ
33  濁度測定用光センサ基板
40  蛍光測定部
41  蛍光測定部遮光ユニット
42  蛍光測定用光センサ
43  蛍光測定用光センサ基板
44  波長フィルタ
50  ファン
 
 
1 Container (tube)
2 DNA-containing solution 10 Measuring unit 11 Sample holder 11a Container storage unit 11b, 11d Horizontal through hole 11c Vertical through hole 12 Bar heater (for sample holder)
13 Temperature sensor (for sample holder)
14 Container (tube) lid holding part 15 Bar heater (for tube lid holding part)
16 Temperature sensor (for tube lid retainer)
20 Light source part 21 Light source part light shielding unit 22 LED
23 LED substrate 30 Turbidity measurement unit 31 Turbidity measurement unit light-shielding unit 32 Turbidity measurement optical sensor 33 Turbidity measurement optical sensor substrate 40 Fluorescence measurement unit 41 Fluorescence measurement unit light-shielding unit 42 Fluorescence measurement optical sensor 43 Fluorescence measurement Optical sensor substrate 44 Wavelength filter 50 Fan

Claims (16)

  1. ・測定試料を収容するための容器を格納するため容器格納部を少なくとも1つ有するサンプルホルダであって、前記サンプルホルダの容器格納部は、底部に容器格納部の深さ方向に1つの縦貫通孔と底部付近の側壁に容器格納部の深さ方向と直行する方向に容器格納部内で開口部が対向する2つの横貫通孔を有し、
    ・前記容器格納部内に格納した容器への光照射用光源、
    ・前記容器格納部内に格納した容器中の測定試料の濁度計測用素子、
    ・前記容器格納部内に格納した容器中の測定試料から発せられる蛍光計測用素子、
    ・前記容器格納部内に格納した容器の温度を調節するための温度調節装置、
    を含む蛍光及び濁度測定装置であって、
    -前記光源及び前記容器格納部は、前記2つの横貫通孔の一方を介して連通しており、
    -前記容器格納部及び前記濁度計測用素子は、前記2つの横貫通孔の他方を介して連通しており、
    -前記容器格納部及び前記蛍光計測用素子は、前記縦貫通孔を介して連通しており、
    -前記容器格納部と前記蛍光計測用素子の間に、光源の光の少なくとも一部をカットし、容器から発せられる蛍光の少なくとも一部を透過する波長フィルタを含む、
    前記装置。
    A sample holder having at least one container storage unit for storing a container for containing a measurement sample, wherein the container storage unit of the sample holder has one vertical penetration in the depth direction of the container storage unit at the bottom The side wall near the bottom and the bottom has two lateral through-holes whose openings are opposed to each other in the direction perpendicular to the depth direction of the container storage unit,
    A light source for irradiating light to the container stored in the container storage unit,
    An element for measuring turbidity of a measurement sample in a container stored in the container storage unit,
    An element for fluorescence measurement emitted from a measurement sample in a container stored in the container storage unit,
    A temperature adjusting device for adjusting the temperature of the container stored in the container storage unit,
    A fluorescence and turbidity measuring device comprising:
    The light source and the container storage are in communication via one of the two lateral through-holes;
    The container storage and the turbidity measuring element communicate with each other through the other of the two lateral through holes;
    The container storage unit and the fluorescence measuring element communicate with each other through the vertical through hole;
    -A wavelength filter that cuts at least part of the light of the light source and transmits at least part of the fluorescence emitted from the container between the container housing and the fluorescence measuring element;
    Said device.
  2. -前記光源、前記容器格納部及び前記濁度計測用素子は、略水平関係に位置しており、
    -前記蛍光計測素子は、前記容器格納部の略下方に位置する、
    請求項1に記載の装置。
    The light source, the container storage and the turbidity measuring element are positioned in a substantially horizontal relationship;
    The fluorescence measuring element is located substantially below the container storage;
    The apparatus of claim 1.
  3. -前記光源は、LEDであり、
    -前記蛍光計測用素子は、フォトICであり、
    -前記濁度計測用素子は、フォトダイオードである、
    請求項1または2に記載の装置。
    The light source is an LED;
    The fluorescence measuring element is a photo IC;
    The turbidity measuring element is a photodiode;
    The apparatus according to claim 1 or 2.
  4. 前記LEDは、ピーク波長が465nmであり、半値幅が約20nmであり、
    前記フォトICは、波長帯域が500~580nmであり、
    前記フォトダイオードは、波長帯域が450~500nmであり、かつ
    前記波長フィルタは、520nm以下の波長をカットするフィルタである、
    請求項3に記載の装置。
    The LED has a peak wavelength of 465 nm, a half width of about 20 nm,
    The photo IC has a wavelength band of 500 to 580 nm,
    The photodiode has a wavelength band of 450 to 500 nm, and the wavelength filter is a filter that cuts a wavelength of 520 nm or less.
    The apparatus of claim 3.
  5. 前記温度調節装置は、前記サンプルホルダの内部に設けられた加熱手段、前記サンプルホルダに向けて送風するための送風機、及び前記サンプルホルダの内部またはサンプルホルダに隣接して設けられた温度測定手段を含む、
    請求項1~4のいずれかに記載の装置。
    The temperature adjusting device includes a heating unit provided inside the sample holder, a blower for blowing air toward the sample holder, and a temperature measuring unit provided inside the sample holder or adjacent to the sample holder. Including,
    The apparatus according to any one of claims 1 to 4.
  6. 前記サンプルホルダの容器格納部の開口に対向する位置に、前記サンプルホルダに接近及び乖離可能な、サンプルホルダの容器格納部に格納された容器の蓋を押さえるための容器蓋押さえ部材をさらに有し、前記容器蓋押さえ部材は、温度調節装置を含む、請求項1~5のいずれかに記載の装置。 A container lid pressing member for pressing the lid of the container stored in the container storage part of the sample holder, which can approach and separate from the sample holder, at a position facing the opening of the container storage part of the sample holder. The device according to any one of claims 1 to 5, wherein the container lid pressing member includes a temperature adjusting device.
  7. 前記容器中の濁度及び蛍光を同時又は逐次に測定するために用いられる、請求項1~6のいずれかに記載の装置。 The apparatus according to any one of claims 1 to 6, which is used for measuring turbidity and fluorescence in the container simultaneously or sequentially.
  8. 前記容器中に含まれる核酸の増幅を濁度測定により検知するために用いる、請求項7に記載の装置。 The apparatus according to claim 7, which is used for detecting amplification of nucleic acid contained in the container by turbidity measurement.
  9. 前記容器中において核酸増幅後に、一本鎖核酸のハイブリダイズ又は二本鎖核酸の解離を蛍光の発光又は消光を用いて検知するために用いられる、請求項7又は8に記載の装置。
     
    The apparatus according to claim 7 or 8, which is used for detecting hybridization of single-stranded nucleic acid or dissociation of double-stranded nucleic acid using fluorescence emission or quenching after nucleic acid amplification in the container.
  10. 請求項1~6のいずれかに記載の装置を用いる核酸分析方法であって、
    (1)前記容器中に測定試料及び核酸増幅のための材料を含有する溶液を配置し、
    (2)前記容器中の溶液の濁度を測定して、前記測定試料を鋳型とする核酸の増幅の度合を検知し、
    (3)所定の増幅が確認された後に、前記容器中の溶液の蛍光を測定して、増幅した核酸の配列を確認する、ことを含み、
    -前記容器中の溶液の蛍光測定は、溶液の温度を変化させることで、溶液中の一本鎖核酸同士をハイブリダイズさせて蛍光の発光又は消光を観察するか、又は溶液中の二本鎖核酸の解離を蛍光の発光又は消光を観察することで行う、前記方法。
    A nucleic acid analysis method using the apparatus according to any one of claims 1 to 6,
    (1) A solution containing a measurement sample and a material for nucleic acid amplification is placed in the container,
    (2) Measure the turbidity of the solution in the container to detect the degree of nucleic acid amplification using the measurement sample as a template,
    (3) After the predetermined amplification is confirmed, the fluorescence of the solution in the container is measured to confirm the sequence of the amplified nucleic acid,
    -Fluorescence measurement of the solution in the container can be performed by observing fluorescence emission or quenching by hybridizing single-stranded nucleic acids in the solution by changing the temperature of the solution, or double-stranded in the solution. The method, wherein the nucleic acid is dissociated by observing fluorescence emission or quenching.
  11. 前記容器中の溶液の蛍光測定は、溶液の温度を高温から低温に変化させることで、一本鎖核酸同士をハイブリダイズさせて蛍光の発光又は消光を観察することで行う、請求項10に記載の方法。 The fluorescence measurement of the solution in the container is performed by changing the temperature of the solution from a high temperature to a low temperature to hybridize single-stranded nucleic acids and observe fluorescence emission or quenching. the method of.
  12. 前記容器中の溶液の蛍光測定は、溶液の温度を低温から高温に変化させることで、二本鎖核酸の解離を蛍光の発光又は消光を観察することで行う、請求項10に記載の方法。 The method according to claim 10, wherein the fluorescence measurement of the solution in the container is performed by observing fluorescence emission or quenching by dissociating the double-stranded nucleic acid by changing the temperature of the solution from a low temperature to a high temperature.
  13. 前記測定試料を鋳型とする核酸の増幅は等温核酸増幅法で行う、請求項10~12のいずれかに記載の方法。 The method according to any one of claims 10 to 12, wherein the nucleic acid amplification using the measurement sample as a template is performed by an isothermal nucleic acid amplification method.
  14. 前記等温核酸増幅法がLAMP法である、請求項13に記載の方法。 The method according to claim 13, wherein the isothermal nucleic acid amplification method is a LAMP method.
  15. 前記等温核酸増幅法による核酸増幅は、40~70℃の温度で行い、増幅完了後に溶液を90~100℃に加熱し、増幅した二本鎖核酸を解離させて一本鎖核酸とし、その後、溶液を冷却して一本鎖核酸のハイブリダイズによる蛍光の発光又は消光を観察する、請求項13又は14に記載の方法。 Nucleic acid amplification by the isothermal nucleic acid amplification method is performed at a temperature of 40 to 70 ° C., and after completion of the amplification, the solution is heated to 90 to 100 ° C. to dissociate the amplified double-stranded nucleic acid into a single-stranded nucleic acid. The method according to claim 13 or 14, wherein the solution is cooled and fluorescence emission or quenching due to hybridization of single-stranded nucleic acid is observed.
  16. 前記測定試料に含まれる核酸の一塩基多型(SNPs)を測定するための、請求項10~15のいずれかに記載の方法。
     
    The method according to any one of claims 10 to 15, for measuring single nucleotide polymorphisms (SNPs) of nucleic acids contained in the measurement sample.
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