[go: up one dir, main page]

WO2016104601A1 - Procédé pour analyser un acide nucléique et dispositif de mesure de la fluorescence/turbidité qui y est utilisé - Google Patents

Procédé pour analyser un acide nucléique et dispositif de mesure de la fluorescence/turbidité qui y est utilisé Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
container
fluorescence
nucleic acid
measurement
dna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2015/086024
Other languages
English (en)
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
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nippon Gene KK
Tateyama Machine Co Ltd
Original Assignee
Nippon Gene KK
Tateyama Machine Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nippon Gene KK, Tateyama Machine Co Ltd filed Critical Nippon Gene KK
Priority to JP2016566440A priority Critical patent/JP6751352B2/ja
Publication of WO2016104601A1 publication Critical patent/WO2016104601A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems 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.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plasma & Fusion (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)

Abstract

La présente invention concerne un dispositif qui permet, dans un seul dispositif, de mesurer la turbidité en vue de la détection et de la confirmation de l'amplification d'ADN et de mesurer la fluorescence pour détection de SNP. L'utilisation de ce dispositif permet également de détecter automatiquement les SNP de l'ADN. Ce dispositif d'analyse d'acide nucléique peut être utilisé en combinaison avec le procédé LAMP pour amplifier l'ADN et le procédé de FLP pour détecter les SNP de l'ADN. La présente invention concerne un dispositif présentant à la fois une fonction pour détecter les SNP et une fonction pouvant détecter et confirmer l'amplification d'ADN par le procédé LAMP et concerne un procédé pour analyser un acide nucléique à l'aide du dispositif.
PCT/JP2015/086024 2014-12-26 2015-12-24 Procédé pour analyser un acide nucléique et dispositif de mesure de la fluorescence/turbidité qui y est utilisé Ceased WO2016104601A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2016566440A JP6751352B2 (ja) 2014-12-26 2015-12-24 核酸分析方法及びそれに用いる蛍光・濁度測定装置

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2014-265940 2014-12-26
JP2014265940 2014-12-26

Publications (1)

Publication Number Publication Date
WO2016104601A1 true WO2016104601A1 (fr) 2016-06-30

Family

ID=56150614

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/086024 Ceased WO2016104601A1 (fr) 2014-12-26 2015-12-24 Procédé pour analyser un acide nucléique et dispositif de mesure de la fluorescence/turbidité qui y est utilisé

Country Status (2)

Country Link
JP (1) JP6751352B2 (fr)
WO (1) WO2016104601A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025031327A1 (fr) * 2023-08-04 2025-02-13 广州国家实验室 Module d'amplification et de test pour acides nucléiques et dispositif tout-en-un d'amplification et de test pour acides nucléiques

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS58174833A (ja) * 1982-04-07 1983-10-13 Hitachi Ltd 蛍光光度計
JPS60263838A (ja) * 1984-06-12 1985-12-27 Hitachi Ltd 光度計
JPH07191033A (ja) * 1993-12-27 1995-07-28 Hitachi Electron Eng Co Ltd リポソームを用いた免疫測定法
JP2000146975A (ja) * 1998-11-04 2000-05-26 Mitsubishi Chemicals Corp 抗原・抗体の測定方法
JP2004283161A (ja) * 2003-03-04 2004-10-14 Eiken Chem Co Ltd 核酸増幅の有無を検出する方法および装置
JP2012034617A (ja) * 2010-08-06 2012-02-23 Sony Corp 核酸増幅反応装置
JP2014082987A (ja) * 2012-10-23 2014-05-12 Sony Corp 塩基配列解析方法、塩基配列解析装置及び塩基配列解析プログラム

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS58174833A (ja) * 1982-04-07 1983-10-13 Hitachi Ltd 蛍光光度計
JPS60263838A (ja) * 1984-06-12 1985-12-27 Hitachi Ltd 光度計
JPH07191033A (ja) * 1993-12-27 1995-07-28 Hitachi Electron Eng Co Ltd リポソームを用いた免疫測定法
JP2000146975A (ja) * 1998-11-04 2000-05-26 Mitsubishi Chemicals Corp 抗原・抗体の測定方法
JP2004283161A (ja) * 2003-03-04 2004-10-14 Eiken Chem Co Ltd 核酸増幅の有無を検出する方法および装置
JP2012034617A (ja) * 2010-08-06 2012-02-23 Sony Corp 核酸増幅反応装置
JP2014082987A (ja) * 2012-10-23 2014-05-12 Sony Corp 塩基配列解析方法、塩基配列解析装置及び塩基配列解析プログラム

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025031327A1 (fr) * 2023-08-04 2025-02-13 广州国家实验室 Module d'amplification et de test pour acides nucléiques et dispositif tout-en-un d'amplification et de test pour acides nucléiques

Also Published As

Publication number Publication date
JPWO2016104601A1 (ja) 2017-10-12
JP6751352B2 (ja) 2020-09-02

Similar Documents

Publication Publication Date Title
CN101241075B (zh) 带有一个可移动的检测模件的荧光检测系统和方法
EP2791655B1 (fr) Dispositif pour mesurer optiquement la fluorescence d'acides nucléiques dans des échantillons de test et utilisation du dispositif
ES2384170T3 (es) Sistema mejorado de PCR multicolor a tiempo real
KR102400907B1 (ko) 휴대용 멀티플렉스 pcr 장치
US11173491B2 (en) Apparatus for high-speed nucleic acid amplification and method for temperature control of nucleic acid amplification reaction
EP1059523A2 (fr) Procédé automatisé d'analyse en temps réel d'amplification d'acide nucléique
US9322784B2 (en) Microplate-reader with a controlled gas atmosphere, corresponding method and use of same
US12018319B2 (en) Method and device for digital PCR measurement
KR101866195B1 (ko) 휴대용 핵산 증폭장치 및 이를 이용한 핵산 증폭방법
WO2005100538A1 (fr) Systeme pour l'amplification rapide et la detection d'acides nucleiques
JP6754420B2 (ja) 対流ｐｃｒ装置
US20220364164A1 (en) Digital pcr measurement method and measurement device
JP6174591B2 (ja) マイクロ環境での温度測定を検証する方法およびマイクロ環境での温度測定を検証するシステム
CN113518631A (zh) 具有颜色传感器的灭菌指示器读取装置
CN114836317A (zh) 一种便携式实时荧光定量核酸诊断仪及其检测方法
CN108139328A (zh) 光学结构及光学光检测系统
JP6751352B2 (ja) 核酸分析方法及びそれに用いる蛍光・濁度測定装置
US9464329B2 (en) Portable systems and methods for amplifying nucleotides and detecting nucleotide sequences
JP6961700B2 (ja) 生体サンプル中の遺伝物質を検出する方法及びその実施のための装置
JP6374967B2 (ja) 多孔質基材における核酸増幅の検出
US11366116B1 (en) Real time autonomous surveillance of pathogens
US20170023555A1 (en) Devices and kits for measuring biological results
US20240344120A1 (en) Dna detection method and dna detection system
JP2018507694A5 (fr)
CN113969238A (zh) 一种用于基因扩增荧光检测的便携式可视化成像系统

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15873169

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2016566440

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15873169

Country of ref document: EP

Kind code of ref document: A1