EP1924842A1 - Dispositif pour effectuer des reactions pcr en temps reel - Google Patents
Dispositif pour effectuer des reactions pcr en temps reelInfo
- Publication number
- EP1924842A1 EP1924842A1 EP06791787A EP06791787A EP1924842A1 EP 1924842 A1 EP1924842 A1 EP 1924842A1 EP 06791787 A EP06791787 A EP 06791787A EP 06791787 A EP06791787 A EP 06791787A EP 1924842 A1 EP1924842 A1 EP 1924842A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- light
- emitting diode
- light emitting
- beam path
- measured
- 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.)
- Withdrawn
Links
- 238000003753 real-time PCR Methods 0.000 title claims abstract description 10
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 230000003287 optical effect Effects 0.000 claims abstract description 12
- 238000005286 illumination Methods 0.000 claims abstract description 10
- 238000005259 measurement Methods 0.000 claims abstract description 9
- 230000005284 excitation Effects 0.000 claims abstract description 6
- 238000011156 evaluation Methods 0.000 claims abstract 2
- 239000013307 optical fiber Substances 0.000 claims description 7
- 239000011521 glass Substances 0.000 claims description 5
- 230000007935 neutral effect Effects 0.000 claims description 4
- 230000001419 dependent effect Effects 0.000 abstract description 3
- 238000013459 approach Methods 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009529 body temperature measurement Methods 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000000295 emission spectrum Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 239000003269 fluorescent indicator Substances 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 230000000191 radiation effect Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
Classifications
-
- 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
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/27—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands using photo-electric detection ; circuits for computing concentration
- G01N21/274—Calibration, base line adjustment, drift correction
-
- 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
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/251—Colorimeters; Construction thereof
- G01N21/253—Colorimeters; Construction thereof for batch operation, i.e. multisample apparatus
-
- 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
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
- B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
Definitions
- the invention relates to a device according to the preamble of claim 1.
- PCR reactions nucleic acid amplification methods
- PCR products amplification products
- This special form of PCR is called real-time PCR reaction.
- test runs are usually measured which contain fluorescence indicators which, upon excitation, emit fluorescence signals whose intensity is dependent on the amount of PCR product formed.
- fluorescence indicators which, upon excitation, emit fluorescence signals whose intensity is dependent on the amount of PCR product formed.
- the increase in PCR products can be monitored by an increase in the intensity of the measured fluorescence signals.
- a well-known example of a suitable fluorescence indicator is, for example, the dye Sybrgreen, which inserts nonspecifically into double-stranded DNA and emits a fluorescence signal in the embedded state.
- suitable fluorescent indicators which are known to the person skilled in the art and which will not be discussed in detail here. It is supplemented by the publication of "Neusser; Transkript Laborwelt No. 2/2000;” PCR procedure for the quantification of PCR products "", which describes in detail the different possibilities for real-time PCR reactions.
- thermocycler with a reaction region in which several temperature-controllable receptacles for reaction vessels are provided.
- an illumination device associated with the reaction area is provided which has a plurality of light-emitting diodes, usually one diode per receptacle.
- a detector is provided which generates measured values as a function of a measured light intensity.
- the detector may e.g. be a CCD chip or a Fotomultiplier or contain.
- the device furthermore has suitable optical devices which define a beam path leading from the illumination device to the reaction space and from there to the detector.
- the optics include e.g. a dichroic mirror arranged between the illuminating device and the receptacle, which allows the excitation light emanating from the illuminating device to pass to the receptacles and a longer-wavelength fluorescence signal emitted from the reaction region onto the e.g. reflected detector laterally.
- a number of other e.g. the detector upstream filters and lenses, etc. provided.
- a problem with known real-time PCR devices is that temperature or current fluctuations can affect the measurement. It is conceivable, for example, that the excitation light generated by the light-emitting diodes becomes weaker as the operating time increases, or that the optical devices experience a drift during operation of the device at different temperatures, to name just a few examples. From WO 01/35079, it is known, for example, that a reference device is provided for the standardization of the light-emitting diodes, which has a separate detector in the form of a photodiode with which the light-emitting diodes are measured and the measured reference value is offset with the sample measured value.
- a disadvantage of the known device is that a check of the detector is omitted.
- the object of the invention is, starting from the prior art, to provide a device with which a possible measured value drift can be detected and compensated in a simple manner.
- a reference device is therefore provided in the device, which has a separate from the illumination device reference light emitting diode whose light is coupled behind the reaction area in the beam path.
- the invention uses a separate light-emitting diode to quantify and compensate for any drifting due to temperature fluctuations or fluctuations in the power supply.
- a diode whose emission spectrum is wider than that of the light-emitting diodes (14) of the illumination device is used as the reference light-emitting diode.
- the lighting device diodes are usually used, which are e.g. produce narrow-band blue light having a wavelength which is below the detection wavelengths are irradiated with the provided in the detector means multiplier.
- a diode is provided which produces broadband white light. With such a reference light-emitting diode, all multipliers can be directly irradiated in all filter positions of the detector device.
- a reference light-emitting diode which with respect to its properties with the light-emitting diodes of the illumination device matches. If, for example, a reference light-emitting diode is used which conforms to the LEDs with regard to its specification and the operating conditions, then it can be assumed that influences which cause a measured-value drift in the light-emitting diodes also do so in the same way with the reference light-emitting diode, so that direct compensation of the Measurement results is possible.
- an optical filter device connected downstream of the reference light-emitting diode, in particular a neutral-glass filter, is provided.
- the intensity of the light emitted by the reference light-emitting diode can be adjusted to a desired intensity before being coupled into the beam path.
- a neutral glass filter is selected, which adjusts the intensity so that at medium sensitivity of the detector an optimized reference signal reaches the multipliers, which is strong enough for a favorable signal-to-noise ratio and at the same time is not in the saturation range.
- the coupling of the light of the reference light-emitting diode into the beam path takes place behind the reaction space.
- the light emanating from the reference light-emitting diode can be detected by means of a mirror or other suitable optical means, e.g. to couple one of the reference light emitting diode associated optical fiber in the beam path.
- the last-mentioned use of a light guide is particularly suitable for those devices whose optical devices have light guides with which the fluorescent light emitted from the reaction space is recorded.
- the light entry surfaces of the light guides are e.g. each associated with a receptacle, while the light exit surfaces are arranged side by side bundled.
- a further light guide can be provided with little effort, the light inlet opening of the reference light emitting diode is assigned and whose light exit opening is located in particular in the middle of the other light guide.
- the recordings are each individually excited and measured one after the other in known devices.
- a series of measurement runs takes place in which the images or the images in the images are measured.
- the reference light-emitting diode according to the invention can be switched on in the same frequency and with the same duration as the light-emitting diodes, so that a comparable load and wear occurs. In this control, each measurement run can be compensated for a possible drift.
- the reference light-emitting diode and the light-emitting diodes of the illumination device are therefore connected to the same power source such that all diodes experience a matching power supply. Due to the fact that the reference light-emitting diode is influenced equally in this embodiment, current fluctuations are calculated.
- the device 10 has a schematically illustrated conventional thermal cycler 11 with receptacles 12.
- 12 reaction vessels are used in the recordings, in each of which a PCR approach with the above-mentioned fluorescence indicator, or the indicators is included.
- a cover housing 13 is mounted with a lighting device with multiple light emitting diodes 14. Each a light emitting diode
- the LEDs 14 is associated with a receptacle 12.
- the LEDs 14 are arrayed. In the measurement, the LEDs 14 are preferably switched so that only one associated recording 12 is always irradiated.
- the light 15 is emitted by the light-emitting diode 14, and then initially passes a short-pass filter 16, with the long-wave components to be filtered out. After that, the light enters
- the light emitted by the light-emitting diode 14 15 is intended to excite a fluorescence indicator located in the receptacle 12 in a PCR approach, whereupon it emits a fluorescence signal 15 '.
- the beam splitter 17 is designed so that the fluorescence signal 15 'is reflected to the side.
- a dichroic mirror is used as the beam splitter 17, which transmits the excitation light, however, reflects the emitted longer-wavelength fluorescence signal.
- the reflected fluorescence signal 15 ' is then detected by a detector 27.
- the detector 27 is preceded by optical devices with which the fluorescence signal 15 'can be imaged on the detector 27.
- the detected signal is detected by means of one, usually several, e.g. amplified wave length specific not shown Multipliern.
- the optical devices comprises a series of optical fibers 20 with light entry surfaces 21, each of which is associated with a receptacle 12 or the fluorescence signals 15 'emitted from the receptacles 12 and reflected at the beam splitter 17.
- the entrance surfaces 21 are in turn arrayed like the light emitting diodes 14.
- a further diode is now provided in the cover housing in spatial proximity to the light-emitting diodes 14 as a reference light-emitting diode 140.
- the light generated by the reference light-emitting diode 140 is laterally deflected by a mirror 220, then passes through a neutral glass filter 230 and arrives at a light entry surface 210 of a light serving as a reference optical fiber 200.
- the mirror 220 may be, for example, a ceramic mirror.
- the neutral glass filter is used to set the intensity of the reference signal to a good detectable value.
- optical fibers 20 and the Referenzanderleitmaschine 200 are combined at its outlet end into a bundle 23, wherein advantageously to minimize lateral radiation effects, the exit end of the reference optical fiber 200 is located centrally in the bundle 23.
- the fluorescent signal 15 'and the light of the reference light emitting diode 140 is then transmitted from the optical fiber bundle 23 via further optical devices, e.g. a lens 24, a long-pass filter 25 and another lens 26 imaged on the detector 27.
- optical devices e.g. a lens 24, a long-pass filter 25 and another lens 26 imaged on the detector 27.
- a reference light-emitting diode can be provided with constructively relatively little effort and coupled into the beam path.
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Theoretical Computer Science (AREA)
- Mathematical Physics (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP10010978A EP2282194A1 (fr) | 2005-09-13 | 2006-09-01 | Dispositif pour éffectuer des réactions PCR en temps réel |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102005043834A DE102005043834A1 (de) | 2005-09-13 | 2005-09-13 | Vorrichtung zur Durchführung von Real-Time PCR-Reaktionen |
PCT/EP2006/008559 WO2007031203A1 (fr) | 2005-09-13 | 2006-09-01 | Dispositif pour effectuer des reactions pcr en temps reel |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1924842A1 true EP1924842A1 (fr) | 2008-05-28 |
Family
ID=37428617
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP06791787A Withdrawn EP1924842A1 (fr) | 2005-09-13 | 2006-09-01 | Dispositif pour effectuer des reactions pcr en temps reel |
EP10010978A Withdrawn EP2282194A1 (fr) | 2005-09-13 | 2006-09-01 | Dispositif pour éffectuer des réactions PCR en temps réel |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP10010978A Withdrawn EP2282194A1 (fr) | 2005-09-13 | 2006-09-01 | Dispositif pour éffectuer des réactions PCR en temps réel |
Country Status (8)
Country | Link |
---|---|
US (1) | US20090218518A1 (fr) |
EP (2) | EP1924842A1 (fr) |
JP (1) | JP2009507501A (fr) |
CN (1) | CN101273262A (fr) |
AU (1) | AU2006291698A1 (fr) |
CA (1) | CA2622139A1 (fr) |
DE (1) | DE102005043834A1 (fr) |
WO (1) | WO2007031203A1 (fr) |
Families Citing this family (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5339838B2 (ja) * | 2008-10-01 | 2013-11-13 | キヤノン株式会社 | 遺伝子検査装置 |
JP5370286B2 (ja) * | 2010-06-29 | 2013-12-18 | 株式会社島津製作所 | 蛍光検出装置 |
EP2615462B1 (fr) * | 2010-11-15 | 2016-12-14 | F. Hoffmann-La Roche AG | Instrument et procédé pour le traitement thermique automatique d'échantillons liquides |
US20140127701A1 (en) * | 2011-04-08 | 2014-05-08 | Stokes Bio Limited | End-Point Optical System and Method of Use |
EP2525211B1 (fr) | 2011-05-16 | 2018-01-03 | F. Hoffmann-La Roche AG | Instrument et procédé de détection d'analytes |
IN2014DN03441A (fr) | 2011-09-30 | 2015-06-05 | Life Technologies Corp | |
EP2581728B1 (fr) * | 2011-10-10 | 2013-09-18 | CYCLERtest B.V. | Dispositif d'étalonnage pour cycleur thermique |
CN102565016B (zh) * | 2011-12-30 | 2014-05-07 | 北京农业智能装备技术研究中心 | 基于荧光淬灭传感器的检测的温度效应补偿装置及方法 |
AU2013202808B2 (en) * | 2012-07-31 | 2014-11-13 | Gen-Probe Incorporated | System and method for performing multiplex thermal melt analysis |
SG11201506481QA (en) | 2013-02-22 | 2015-09-29 | Life Technologies Corp | Optical systems and methods for biological analysis |
DE102014108143A1 (de) * | 2014-06-10 | 2015-12-10 | Kist Europe-Korea Institute of Science and Technologie Europe Forschungsgesellschaft mbh | An optical system for detecting fluorescent or luminescent signals of at least two samples |
DE102016200271A1 (de) | 2016-01-13 | 2017-07-13 | Institut Dr. Foerster Gmbh & Co. Kg | Vorrichtung zur Erzeugung und Messung einer Emission |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3575048A (en) * | 1968-07-10 | 1971-04-13 | Union Carbide Corp | Calorimeter for high power lasers |
DE3407754A1 (de) * | 1984-03-02 | 1985-09-12 | Boehringer Mannheim Gmbh, 6800 Mannheim | Geraet zur bestimmung des diffusen reflexionsvermoegens einer probenflaeche kleiner abmessungen |
US4750837A (en) * | 1986-04-11 | 1988-06-14 | Sclavo Inc. | Fluorometer with reference light source |
CA2129787A1 (fr) * | 1993-08-27 | 1995-02-28 | Russell G. Higuchi | Surveillance de plusieurs reactions d'amplification simultanement et analyse de ces reactions simultanement |
US5670375A (en) * | 1996-02-21 | 1997-09-23 | Biomerieux Vitek, Inc. | Sample card transport method for biological sample testing machine |
CN1664562A (zh) * | 1998-05-16 | 2005-09-07 | 阿普尔拉公司 | 用于监测dna聚合酶链反应的仪器 |
DE60016170D1 (de) * | 1999-01-08 | 2004-12-30 | Ibsen Photonics As Farum | Spektrometer |
US6852986B1 (en) * | 1999-11-12 | 2005-02-08 | E. I. Du Pont De Nemours And Company | Fluorometer with low heat-generating light source |
JP4846152B2 (ja) * | 1999-11-12 | 2011-12-28 | イー・アイ・デュポン・ドウ・ヌムール・アンド・カンパニー | 低発熱光源を有する蛍光測定器 |
DE10131687A1 (de) * | 2001-06-29 | 2003-01-16 | Eppendorf Ag | Vorrichtung zur Durchführung von Nukleinsäure-Amplifikationsreaktionen bei gleichzeitiger Verfolgung der Bildung von Amplifikationsprodukten |
DE20122266U1 (de) * | 2001-06-29 | 2004-11-04 | Eppendorf Ag | Vorrichtung zur Durchführung von Nukleinsäure-Amplifikationsreaktionen bei gleichzeitiger Verfolgung der Bildung von Amplifikationsprodukten |
US6480392B1 (en) * | 2001-07-17 | 2002-11-12 | Lite-On Enclosure Inc. | Retaining and fixing structure of interface card |
NL1023680C2 (nl) * | 2003-06-17 | 2004-12-20 | Tno | Sensor met polymeren componenten. |
US7788039B2 (en) * | 2003-09-25 | 2010-08-31 | Roche Molecular Systems, Inc. | Quantitation of nucleic acids using growth curves |
-
2005
- 2005-09-13 DE DE102005043834A patent/DE102005043834A1/de not_active Withdrawn
-
2006
- 2006-09-01 JP JP2008530372A patent/JP2009507501A/ja active Pending
- 2006-09-01 CA CA002622139A patent/CA2622139A1/fr not_active Abandoned
- 2006-09-01 CN CNA2006800333821A patent/CN101273262A/zh active Pending
- 2006-09-01 EP EP06791787A patent/EP1924842A1/fr not_active Withdrawn
- 2006-09-01 WO PCT/EP2006/008559 patent/WO2007031203A1/fr active Application Filing
- 2006-09-01 EP EP10010978A patent/EP2282194A1/fr not_active Withdrawn
- 2006-09-01 US US12/066,425 patent/US20090218518A1/en not_active Abandoned
- 2006-09-01 AU AU2006291698A patent/AU2006291698A1/en not_active Abandoned
Non-Patent Citations (1)
Title |
---|
See references of WO2007031203A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO2007031203A1 (fr) | 2007-03-22 |
US20090218518A1 (en) | 2009-09-03 |
AU2006291698A1 (en) | 2007-03-22 |
JP2009507501A (ja) | 2009-02-26 |
DE102005043834A1 (de) | 2007-03-22 |
EP2282194A1 (fr) | 2011-02-09 |
CN101273262A (zh) | 2008-09-24 |
CA2622139A1 (fr) | 2007-03-22 |
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