WO2016136464A1 - Dispositif d'analyse et procédé d'analyse utilisant ce dernier - Google Patents

Dispositif d'analyse et procédé d'analyse utilisant ce dernier Download PDF

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Publication number
WO2016136464A1
WO2016136464A1 PCT/JP2016/053880 JP2016053880W WO2016136464A1 WO 2016136464 A1 WO2016136464 A1 WO 2016136464A1 JP 2016053880 W JP2016053880 W JP 2016053880W WO 2016136464 A1 WO2016136464 A1 WO 2016136464A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
acid analyzer
sample
light
detector
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PCT/JP2016/053880
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English (en)
Japanese (ja)
Inventor
優 日下
勇夫 古矢
大輔 森島
麻奈美 南木
康則 庄司
忠雄 藪原
浩子 藤田
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株式会社 日立ハイテクノロジーズ
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Application filed by 株式会社 日立ハイテクノロジーズ filed Critical 株式会社 日立ハイテクノロジーズ
Priority to JP2017502048A priority Critical patent/JP6476275B2/ja
Publication of WO2016136464A1 publication Critical patent/WO2016136464A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/47—Scattering, i.e. diffuse reflection
    • G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
    • G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
    • 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
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor

Definitions

  • the present invention relates to an analysis apparatus and an analysis method thereof, for example, an analysis apparatus and an analysis method for analyzing a biological sample by amplifying a nucleic acid contained in the biological sample.
  • PCR Polymerase Chain Reaction
  • a fluorescent label whose fluorescence intensity varies depending on the amount of PCR product is mixed with the reaction solution, irradiated with excitation light, and emitted from the fluorescent label.
  • a method for measuring the fluorescence intensity is used.
  • Patent Document 1 discloses a carousel rotatable around a rotation axis, a plurality of reaction containers held along a circumferential edge of the carousel, a light source for irradiating the reaction container with excitation light, There is a description of an analyzer having at least one detector having a detection element for detecting fluorescence from a reaction solution.
  • Patent Document 2 describes a method for reducing the influence of bubbles and dust in scattered light measurement on an automatic analyzer.
  • JP 2013-148590 A Japanese Unexamined Patent Publication No. 2014-202523
  • Patent Document 1 describes a method of amplifying a reaction solution using a PCR method, but does not describe noise.
  • Patent Document 2 describes the influence of bubbles and dust in the measurement of scattered light on an automatic analyzer, it cannot be applied as it is to a nucleic acid analyzer that detects by a method different from the automatic analyzer.
  • the present invention has been made in view of the above, and one of its purposes is to provide a nucleic acid analyzer capable of rapidly detecting contamination in a reaction solution and a nucleic acid analysis method thereof. There is.
  • a holding member that can hold a reaction container containing a sample, a light source that emits light to a predetermined position of the holding member, and fluorescence emitted from a predetermined position of the holding member in response to light irradiation from the light source
  • a nucleic acid analyzer comprising: a first detector; and a second detector that detects scattered light emitted from a predetermined position of the holding member in response to light irradiation from the light source.
  • FIG. 1 and FIG. 2 are schematic block diagrams of main structural examples of the nucleic acid analyzer of FIG. 1 and FIG.
  • FIG. 5 is a flowchart showing an example of processing contents of the analysis processing unit in FIG.
  • the figure which shows an example of each detection signal obtained by a fluorescence detector and a scattered light detector The figure which shows an example at the time of providing an output value based on each detection signal obtained by a fluorescence detector and a scattered light detector
  • the figure showing an example of the time transition of each detection signal when there is no increase in blind fluorescent substance during measurement A figure showing an example of the time transition of each detection signal when there is an increase in blind fluorescent substance during measurement
  • the constituent elements are not necessarily indispensable unless otherwise specified and apparently essential in principle. Needless to say.
  • the shapes, positional relationships, etc. of the components, etc. when referring to the shapes, positional relationships, etc. of the components, etc., the shapes are substantially the same unless otherwise specified, or otherwise apparent in principle. And the like are included. The same applies to the above numerical values and ranges.
  • Embodiment 1 of the present invention will be described with reference to FIG.
  • FIG. 2 is a cross-sectional view illustrating a configuration example between A-A ′ in FIG. 1.
  • a plurality of temperature control blocks 1 (12 in this example) are arranged around the center axis of the carousel 2 along the outer periphery, and rotate around the rotation axis 3.
  • Peltier elements 4 are arranged between the plurality of temperature control blocks 1 and the carousel 2, respectively.
  • the temperature of the temperature control block 1 is adjusted by controlling the Peltier element 4 while monitoring the temperature with a temperature sensor 5 mounted in the temperature control block 1.
  • a pair of Peltier elements 4 and temperature sensors 5 corresponding to each of the plurality of temperature control blocks 1 the temperatures of the plurality of temperature control blocks 1 are independently adjusted.
  • a photometer 6 is disposed on the outer periphery of the carousel 2.
  • two photometers 6 using light of different wavelengths are shown, but one or three or more photometers 6 may be arranged on the outer periphery of the carousel 2. Since all the temperature control blocks 1 move on the same circumference by rotational drive, the relative positions of the photometer 6 and the temperature control block 1 when passing in front of the photometer 6 are the same in all the temperature control blocks 1. become.
  • the plurality of temperature control blocks 1 are covered with a shielding plate 7 including the carousel 2 in order to reduce optical disturbance when analyzed by the photometer 6.
  • a tube (reaction vessel) 10 containing a reaction solution (sample) in which a reagent or the like is mixed with nucleic acid is held by a temperature control block (holding member) 1.
  • All temperature control blocks 1 are provided with an excitation light irradiation window 8 for receiving excitation light from the photometer 6 and a fluorescence detection window 9 for the photometer 6 to capture fluorescence.
  • the excitation light irradiation window 8 is arranged on the lower surface side of the temperature control block 1 and the fluorescence detection window 9 is arranged on the side surface side of the temperature control block 1, the arrangement of the windows can be freely set according to the structure of the photometer. It is possible to set.
  • FIG. 3 is a schematic diagram illustrating a detailed configuration example of a photometer in the nucleic acid analyzer of FIGS. 1 and 2.
  • excitation light emitted from a light source LED (Light Emitting Diode) 11 passes through a lens 12 to become parallel light, and needs to pass through a first wavelength selection filter 13. Only the correct wavelength component is extracted. The light that has passed through the first wavelength selection filter 13 is collected by the lens 14 and enters the excitation light irradiation window 8 of the temperature control block (holding member) 1.
  • a light source LED Light Emitting Diode
  • a tube (reaction vessel) 10 containing a reaction solution (sample) in which a reagent or the like is mixed with nucleic acid is held.
  • the excitation light collected by the lens 14 is irradiated to the temperature control block 1 in a state where the tube 10 is held, the fluorescence is emitted from the fluorescent component of the reaction solution in the tube 10 in response to the excitation light.
  • the reaction liquid in the tube contains a scattered light component
  • the scattered light is emitted from the scattered component in response to the excitation light.
  • the fluorescence and scattered light emitted from the fluorescence detection window 9 of the temperature control block 1 are converted into parallel light again by the lens 15.
  • the scattered light detector 19 is composed of, for example, a photoelectric conversion diode (PD).
  • the remaining light that has passed through the optical splitter 16 passes through the third wavelength selection filter 20 and only the necessary wavelength components are extracted.
  • the light that has passed through the third wavelength selection filter 20 is collected by the lens 21 and enters the fluorescence detector (first detector) 22.
  • the fluorescence detector 22 is composed of, for example, a photoelectric conversion diode (PD).
  • the LED 11 that is a light source always emits excitation light
  • the scattered light detector 19 and the fluorescence detector 22 always perform detection.
  • the light detected by the scattered light detector 19 and the fluorescence detector 22 generates a detection signal (current or voltage) corresponding to the intensity of the light, and the detection signal is A / D converted through a signal amplification circuit, It is transmitted to the signal processing unit 27.
  • the load on the nucleic acid analyzer 31 is heavy, so the nucleic acid analyzer 31 actually triggers just before the temperature control block 1 passes in front of the photometer 6.
  • the control for stopping the acquisition of the detection signal is performed immediately after passing.
  • a detection signal as shown in FIG. 4 is typically obtained.
  • FIG. 4 is a diagram showing an example of temporal transition of each detection signal obtained when analyzing the nucleic acid in the scattered light detector 19 and the fluorescence detector 22 of FIG.
  • the detection signals from the scattered light detector 19 and the fluorescence detector 22 become a signal having a mountain waveform over time, and the center line of the temperature control block 1 to be measured passes through the optical axis of the LED 11 of the photometer 6. Peak at the moment you do.
  • the detection signal from the scattered light detector 19 is hidden by electrical noise and shows a substantially constant value as shown in FIG.
  • nucleic acid analyzers in order to reduce the influence of electrical noise contained in the detection signal, an approximation curve is obtained by curve fitting the detection signal waveform according to a certain rule, and the peak value of the approximation curve is obtained.
  • the nucleic acid is analyzed by observing the change.
  • FIG. 5 is a schematic block diagram showing an example of a main configuration in terms of the functions of the nucleic acid analyzer of FIGS. 1 and 2.
  • a nucleic acid analyzer 31 shown in FIG. 5 includes an analysis processing unit 36 that performs control and the like in addition to the plurality of temperature control blocks 1, the carousel 2, and the photometer 6 described above.
  • the analysis processing unit 36 is mainly configured by a computer system or the like, and is mainly a temperature processing unit 38 that adjusts the temperature of each temperature control block 1 based on a predetermined processing sequence, and a rotation that performs rotation control of the carousel 2.
  • a processing unit 39 and a signal processing unit 27 for controlling the photometer 6 and the like are included.
  • the signal processing unit 27 processes a signal obtained by each detector such as the fluorescence detector 22 and the scattered light detector 19 in the photometer 6. Further, the result of the analysis processing unit 36 is displayed on the display unit 40.
  • FIG. 6 is a flowchart showing an example of processing contents of the analysis processing unit 36 in FIG.
  • the analysis processing unit 36 is activated, for example, immediately after the nucleic acid analyzer 31 is turned on, and executes the process of FIG. 6 immediately after the start of measurement.
  • the analysis processing unit 36 includes an LED (light source) 11 in the photometer 6 and a temperature control block (holding member) 1 in a state where the tube (reaction vessel) 10 shown in FIG. Irradiate excitation light toward
  • the light source is unstable immediately after lighting, and may be irradiated in advance.
  • what is necessary is just to irradiate LED (light source) 11 previously also when letting the temperature control block 1 pass.
  • the fluorescence detector 22 is made to detect the intensity of the fluorescence emitted from the tube (reaction vessel) 10. Further, the scattered light detector 19 detects the intensity of the scattered light generated from the tube (reaction vessel) 10.
  • the scattered light may be generated not only in the blind fluorescent material such as bubbles and dust in the tube (reaction vessel) 10 but also in various other places.
  • the signal processing unit 37 of the analysis processing unit 36 detects the presence / absence of a blind fluorescent substance based on the detected fluorescence intensity and scattered light intensity.
  • FIG. 7 is a diagram illustrating an example of each detection signal obtained by the fluorescence detector and the scattered light detector.
  • the detection signal from the fluorescence detector 19 includes a fluorescent material and shows a value greater than or equal to a certain value as compared to the background when the blind fluorescent material is not mixed.
  • the scattered light detector 22 is detected when a blind fluorescent material is mixed.
  • the detection value of fluorescence may be reduced due to the mixing of the blind fluorescent substance as compared with the case where it is not mixed.
  • the fluorescence intensity and the scattered light intensity are constant for the sake of explanation, but the fluorescence intensity and the scattered light intensity change depending on the content of the fluorescent substance or the blind fluorescent substance or the progress of the PCR cycle. There is.
  • the diagnosis is performed with the light emission power of the LED 11 set to the same level as in FIG. 4, but depending on the case, the light emission power of the LED 11 is increased to reduce the scattered light. You may detect the presence or absence of a blind fluorescent substance in the increased state.
  • FIG. 8 is a diagram showing an example of a case where a threshold value is provided for the detection signal obtained by the scattered light detector.
  • a threshold value is set in advance in the level of the scattered light detection signal in FIG. 7, and when this threshold value is exceeded, it is determined that the blind fluorescent material is mixed, and an alarm is output to the display unit 40. Then, it can be easily determined that the blind fluorescent substance is contained in the reaction container in the tube.
  • FIG. 9 is a diagram showing an example of the temporal transition of each detection signal when there is no blind fluorescent substance during measurement, or when the amount of blind fluorescent substance is very small and the blind fluorescent substance does not increase by PCR. In such a case, the scattered light intensity does not increase, and an output value indicating the presence or absence of foreign matter is not detected.
  • FIG. 10 is a diagram showing an example of temporal transition of each detection signal when there is a blind fluorescent substance during measurement and the blind fluorescent substance increases by PCR.
  • the blind fluorescent material contained in the reaction vessel increases, the scattered light intensity increases.
  • an output signal that exceeds this threshold value and is determined to contain a blind fluorescent substance can be obtained.
  • FIG. 11 is a top view showing a schematic configuration example of the nucleic acid analyzer according to the present embodiment.
  • the nucleic acid analyzer 32 in FIG. 11 is a nucleic acid extraction unit 33 that extracts nucleic acid from a specimen, a reagent mixing unit 34 that dispenses and mixes reagents into the extracted nucleic acid, and the temperature of the mixed reaction solution to control fluorescence. And a nucleic acid analysis unit 35 for detecting.
  • the nucleic acid extraction unit 33 is composed of a sample erection unit 41, a centrifuge unit 42, a retraction chamber 43, a tube erection unit 44, an extraction reagent storage 45, a consumables storage 46, and the like. It is responsible for removing components and extracting only the nucleic acids required for analysis.
  • the reagent mixing unit 34 includes an analysis reagent storage 47, a consumable storage 48, a mixing unit 49, and the like. Although not described in detail, the reagent for analysis is mixed with the nucleic acid extracted by the nucleic acid extraction unit 33. Take on the function.
  • the configuration of the nucleic acid analysis unit 35 is the same as that of the nucleic acid analyzer 31 shown in FIG. The transfer of the tubes between the units is performed by the robot arm 50.
  • the person performing the analysis starts up the nucleic acid analyzer 32, sets consumables such as specimens, reagents, and tubes, and starts analysis.
  • the analysis processing unit indicates that the result is that the blind fluorescent substance is mixed
  • the user can confirm that there is no contamination with a blind fluorescent substance before the pretreatment for analysis (specifically, before mixing the reagents, more preferably before extracting the nucleic acid), The sample is not wasted.
  • the nucleic acid extraction unit 33, the reagent mixing unit 34, and the nucleic acid analysis unit 35 are configured by different devices, there may occur a situation in which the reagents are already mixed.
  • the present invention made by the present inventor has been specifically described based on the embodiment.
  • the present invention is not limited to the embodiment, and various modifications can be made without departing from the scope of the invention.
  • the above-described embodiment has been described in detail for easy understanding of the present invention, and is not necessarily limited to one having all the configurations described.
  • a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment. .
  • the nucleic acid analyzer which is a particularly useful application example of the apparatus diagnosis method according to the present embodiment has been described.
  • the present invention is not necessarily limited to the nucleic acid analyzer.
  • the same effect can be applied to any apparatus that sets a reaction vessel on a holding member and analyzes a sample in the reaction vessel using a photometer. May be obtained.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
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  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)
  • Investigating Or Analysing Materials By Optical Means (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un dispositif d'analyse d'acide nucléique avec lequel une rapide détection de contaminants ou analogues dans un récipient à réaction est possible. Ce dispositif d'analyse d'acide nucléique est caractérisé par le fait qu'il comprend un élément de retenue qui peut retenir un récipient à réaction dans lequel un échantillon a été placé, une source de lumière destinée à éclairer un emplacement prescrit de l'élément de retenue avec de la lumière, un premier détecteur destiné à détecter la fluorescence émise depuis l'emplacement prescrit de l'élément de retenue à la suite de l'éclairage avec de la lumière provenant de la source de lumière, et un second détecteur destiné à détecter la lumière diffusée émise depuis l'emplacement prescrit de l'élément de retenue à la suite d'un éclairage avec de la lumière provenant de la source de lumière, l'échantillon dans le récipient à réaction étant évalué sur la base de l'intensité de la lumière diffusée.
PCT/JP2016/053880 2015-02-27 2016-02-10 Dispositif d'analyse et procédé d'analyse utilisant ce dernier WO2016136464A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018205087A (ja) * 2017-06-02 2018-12-27 浜松ホトニクス株式会社 光計測装置
CN111747019A (zh) * 2020-07-03 2020-10-09 江西奥普照明有限公司 一种led灯管的自动检测流水线

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5723844A (en) * 1980-07-19 1982-02-08 Chugai Pharmaceut Co Ltd Method and apparatus for measuring light scattering removed of influence of unnecessary scattered light in solution
JPH08240529A (ja) * 1995-03-02 1996-09-17 Orc Mfg Co Ltd 液体収納容器内の微小異物の検出装置およびその方法
JP2011149822A (ja) * 2010-01-21 2011-08-04 Sony Corp 光学的測定装置及び光学的測定方法
JP2013148590A (ja) * 2013-04-05 2013-08-01 Hitachi High-Technologies Corp 核酸分析装置及び核酸分析方法
JP2014021008A (ja) * 2012-07-20 2014-02-03 Hitachi High-Technologies Corp 自動分析装置及び自動分析方法

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP4170947B2 (ja) * 2004-04-09 2008-10-22 株式会社日立ハイテクノロジーズ 生体試料成分検出法及びその装置
JP2014143927A (ja) * 2013-01-28 2014-08-14 Hitachi High-Technologies Corp 核酸増幅装置および温度調節機能の異常検出方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5723844A (en) * 1980-07-19 1982-02-08 Chugai Pharmaceut Co Ltd Method and apparatus for measuring light scattering removed of influence of unnecessary scattered light in solution
JPH08240529A (ja) * 1995-03-02 1996-09-17 Orc Mfg Co Ltd 液体収納容器内の微小異物の検出装置およびその方法
JP2011149822A (ja) * 2010-01-21 2011-08-04 Sony Corp 光学的測定装置及び光学的測定方法
JP2014021008A (ja) * 2012-07-20 2014-02-03 Hitachi High-Technologies Corp 自動分析装置及び自動分析方法
JP2013148590A (ja) * 2013-04-05 2013-08-01 Hitachi High-Technologies Corp 核酸分析装置及び核酸分析方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2018205087A (ja) * 2017-06-02 2018-12-27 浜松ホトニクス株式会社 光計測装置
CN111747019A (zh) * 2020-07-03 2020-10-09 江西奥普照明有限公司 一种led灯管的自动检测流水线

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