EP2694672A1 - Method, loc and analysis device for the lysis of cells and pcr amplification - Google Patents
Method, loc and analysis device for the lysis of cells and pcr amplificationInfo
- Publication number
- EP2694672A1 EP2694672A1 EP12704033.5A EP12704033A EP2694672A1 EP 2694672 A1 EP2694672 A1 EP 2694672A1 EP 12704033 A EP12704033 A EP 12704033A EP 2694672 A1 EP2694672 A1 EP 2694672A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- filter
- pcr
- loc
- lysis
- 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.)
- Granted
Links
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- 238000012408 PCR amplification Methods 0.000 title claims abstract description 19
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0681—Filter
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0809—Geometry, shape and general structure rectangular shaped
- B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
- B01L2300/0877—Flow chambers
Definitions
- the invention relates to a method, a LOC (Lab-on-a-Chip) and an analysis device for lysing cells and PCR amplification of the DNA of the cells.
- LOC Lab-on-a-Chip
- MRSA Methicillin-resistant Staphylococcus aureus
- pneumonia Sepsis
- tuberculosis and fluoroquinolone resistance in Escherichia coli
- PCR polymerase chain reaction
- a temperature-stable DNA polymerase is added to the DNA to be amplified and a temperature cycle consisting of denaturation, i. Splitting DNA into single strands, at about 95 ° C;
- the DNA-containing material is purified before the PCR, following an assay procedure protocol.
- Gram-negative cells the PCR works even if cell material is used directly after a prolonged first high-temperature step without purification, as at temperatures near the boiling point the cells are destroyed and DNA is released.
- this does not work with Gram-positive cells, which is a particular
- a photolysis with a laser is described in EP 4342914 A1.
- a cell-containing solution is first placed on a metal oxide carrier, wherein the metal oxide binds cells.
- the metal oxide is irradiated with the laser, destroying the cell walls and releasing DNA. Subsequently, unbound cell components are washed out in a purification step.
- the LOC and the analysis device can be compared with the prior art little effort cells, in particular
- Lysing bacteria, fungi, viruses, blood cells and cell aggregates in a filter without enzymes is possibly also possible while avoiding purification after lysis.
- the invention is easily transferred to the current biochemical assay and provides a fully integrated LOC.
- the invention also makes it possible to amplify Gram-positive bacteria or other cells which are difficult to lyse, such as fungi or spores, which makes possible a platform with applications for urinary tract infections, MRSA, pneumonia, sepsis, mycoses, etc.
- a laser scanner for scanning a sample can be conveniently realized with a micromirror in a very small construction and allows a compact analyzer.
- the laser lysis can be realized compactly with standardized components for optics, scanners and lasers with a considerably lower outlay than lysis devices according to the prior art.
- the invention makes possible an LOC as ⁇ (Micro Total Analysis System), since the sequence of the processing and amplification steps before detection is simplified compared to the prior art and a transferable into a biochip,
- Fig. 1 shows a schematic representation of an LOC in an analysis apparatus for lysis of cells and PCR amplification, both according to one embodiment of the present invention.
- FIG. 2 shows a schematic representation of an LOC in an analysis device for lysis of cells and PCR amplification, both in each case according to another
- FIG. 3 shows a flowchart of the method for lysis of cells and PCR amplification according to an embodiment of the present invention without
- FIG. 4 shows a flow chart of the method for lysis of cells and PCR amplification according to another embodiment of the present invention with purification step.
- Fig. 1 shows an LOC 10 in a cell lysis and PCR amplification analyzer 30, both in accordance with an embodiment of the present invention for DNA evaluation with an array 18.
- the LOC 10 has a rigid, flat substrate 1 1 fluidic network 12 in the substrate 1 1 on.
- To the fluidic network 12 include microchannels 13 shown by way of example, valves 14 and chamber 15, and a multi-function chamber 19 with a filter 16, in which inter alia, a PCR takes place, and an array chamber 17 with the array 18.
- the filter 16 contains here as a filter medium, a silica Pad and is a first external temperature control 43 under the substrate 1 1 tempered.
- the filter 16 fills a flow-through cross section of the
- Multifunction chamber 19 fully off.
- the array chamber 17 is via a second
- Elements of the fluidic network 12 can also run in the interior of the LOC 10 and are liquid-tight at the top with a membrane or film, not shown.
- the actuators for the operation of the fluidic network 12 and a fluidic interface to the analyzer 30 are not shown.
- the analyzer 30 includes a photolysis light source 31 for irradiating the filter 16 and a controller 32 for performing lysis by irradiating the filter.
- the photolysis light source 31 in this embodiment has a laser 33, a beam expander 34, a micromirror 35 and a lens 36.
- a light beam 37 leaving the laser 33 passes over the beam expander 34 to the micromirror 35 and is deflected there and continues to pass through the lens 36 to the filter 16.
- the micromirror 35 is a controllable micromirror with which the laser light beam 37 can focus the filter 16 and scan in a raster shape according to the beam diameter.
- the analyzer 30 further includes a camera 39 and an array illumination lightwave 40, each directed to the array 18.
- the controller 32 is connected via electrical control lines 38 to the laser 33, the
- Micro-mirror 35 the camera 39, the array illumination lightwave 40, the micromirror 35 and the tempering elements 43 and 44 connected.
- the analysis device 30 further has a mechanical interface (not shown) with the LOC 10 with which the LOC 10 is positioned in the analysis device 30 in a defined manner. Due to the defined positioning of the filter 16 and the array 18 in the LOC, LOCs are interchangeable in the analyzer 30 without re-optical adjustment.
- the photolysis light source 31 is directed to the position of the filter 16 and the camera 39 is directed to the position of the array 18, here in the array chamber 17.
- the filter 16 is arranged above the first external tempering element 43, and the array chamber 17 is arranged above the second tempering 44.
- tempering Peltier elements micro-hotplates or convective heating / cooling elements, optionally electrical resistors, can also be used in combination.
- the mechanical interface allows a suitable heat transfer.
- the controller 32 controls the flow of the assay protocol on the LOC 10.
- the fluidic network 12 is controlled via the fluidic interface. Temperatures and temperature changes are controlled by the temperature control elements 43 and 44.
- the detection is carried out with an array / biochip and by means of the fluorescence exciting an array illumination light wave 40 and the fluorescent light
- the LOC 10 for lysis of cells and PCR amplification has a common, a
- Filter 16 having multi-function chamber 19 for cell accumulation, staining of the cells, cell lysis and PCR.
- FIG. 2 shows an LOC 46 in an analysis device 47 for lysing cells and PCR amplification, both in each case according to a further embodiment of the present invention for DNA evaluation.
- the elements known from Fig. 1 again have the same reference numerals.
- the detection takes place by means of real-time PCR in the same chamber as the lysis, in the multi-function chamber 19.
- the fluorescent dyes used in real-time PCR by means of a
- Fluorescence radiation possibly at different wavelengths observed.
- an array chamber is not required.
- FIG. 3 shows a flowchart 50 of the method for lysis of cells and PCR amplification of the DNA of the cells according to an embodiment of the present invention
- the method comprises the method steps:
- the DNA of the cells is present in sufficient quantity for DNA examinations.
- the method is suitable for Gram-negative and Gram-positive cells.
- g) Analysis of the amplified DNA This relates to the analysis by means of an array, corresponding to a device such as shown in Fig. 1.
- method steps f) and g) and method step e) in this case real-time PCR, are omitted directly in the multifunctional chamber 19 in FIG. 2 instead of.
- the individual process steps are followed by further explanations, optionally with reference to the analysis device 30 and the LOC 10 from FIG. 1 or 2.
- a cell-containing liquid usually body fluid such as urine, sputum, is placed on the filter for positioning the cells. Serum, plasma, smears, BAL (bronchoalveolar lavage), etc.
- a dye solution about 50 ⁇ , is added to the filter and incubated for about 30 s. It is then washed, e.g. first with 200 ⁇ ethanol and then with 200 ⁇ water.
- dyeing agents can be used:
- Periodic acid-Schiff reaction Fuchsinschwefelige acid for mushrooms, brilliant green, methyl green, ethyl green, brilliant blue, Coomassie purple, etc.
- These dyes have an ammonium group, typically in a quinoid system with other alkyl / arylamino groups.
- the dyes can also be stored as solids in depots on the LOC 10, for example lyophilized, and washed with water into the filter 16. The stained bacteria can be lysed with light of comparatively low intensities.
- the filter is filled with the PCR treatment solution, the so-called master mix.
- This PCR treatment solution consists of a mixture of DNTPs (deoxynucleotide triphosphates), primers, polymerase and buffer. Further, in the mixture, a substance for blocking the filter surface is contained, e.g. BSA (bovine serum albumin), PEG (polyethylene glycol), PPG
- the photolysis of the cells in process step d) is preferably carried out by means of laser lysis. During photolysis, the DNA of the cells is released.
- the stained cells are irradiated with a commercially available laser.
- the laser beam is preferably focused and guided with a micromirror on the filter, wherein as far as possible the entire filter surface is swept over completely with the laser beam.
- the filter can also be irradiated with high-energy LEDs, flash lamps or focused light sources with high energy density.
- the decisive factor is that the bacteria absorb as much light as possible, whereas the matrix and the surrounding liquid should absorb as little energy as possible. This is achieved by having the light used for photolysis substantially in a wavelength range which is strongly absorbed by the colored cells but weakly absorbed by water.
- the color of the stained cells is determined by the dye, but may be different from the color of the dye.
- the wavelength or wavelengths of the photolysis light are matched to the color of the stained cells, so that the absorption coefficient of the stained cell is a multiple of the absorption coefficient of a fluid or a stationary phase of a buffer medium present around the cells, usually water.
- a multiple may be, for example, at least the factor 2.5, for example 10.
- a high energy intake of the fluid or the stationary phase would result in a strong, poorly controlled increase in temperature, which leads to
- the photolysis light source advantageously has a Wavelength in the visible region which is complementary to the color of the stained cells is, for example, 532 nm in the case of red staining of the cells with methylene blue, so that the absorption by the stained bacteria is as high as possible.
- the content of the filter 16 is pumped into the array chamber 17, in which then the hybridization and detection takes place.
- the filter is subjected to the usual thermal cycles for the PCR. At these temperature cycles, the DNA is amplified as described above. Now the DNA of the cells is in sufficient quantity for DNA examinations.
- the method is suitable for Gram-negative and Gram-positive cells.
- the DNA can be eluted from the filter element. Thus, for example, from 10 ml of urine with 10 5 cells after 20 cycles, 10 11 DNA molecules are isolated in 50 ⁇ l.
- other amplifications, including isotherms, are also possible, for example NASBA (Nucleic Acid Sequence Based Amplification).
- the hybridization buffer can be
- Buffer system and a salt to increase a salt concentration include.
- the method according to the invention can be easily transferred both to the filter PCR and to a fully integrated LOC.
- Fig. 4 shows a flow chart 60 of the method for lysis of cells and PCR amplification of the DNA of the cells according to an embodiment of the present invention with a purification step.
- the method comprises the steps of: a) positioning the cells on a filter;
- the DNA of the cells is present in sufficient quantity for DNA examinations.
- the method is suitable for Gram-negative and Gram-positive cells.
- the analysis includes hybridization and detection.
- the method steps of the same name correspond to the method steps known from the description of FIG. 3, taking into account the following differences.
- the DNA of the cells is then purified in process step d1). This purification can be accomplished by one or more washes in which cell components other than DNA are removed. Because of the liquid exchange associated with the cleaning, the PCR treatment solution is added only after the purification - process step c) thus takes place after process step d1).
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dispersion Chemistry (AREA)
- Analytical Chemistry (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE102011007035A DE102011007035A1 (en) | 2011-04-08 | 2011-04-08 | Method, LOC and analyzer for cell lysis and PCR amplification |
PCT/EP2012/052072 WO2012136400A1 (en) | 2011-04-08 | 2012-02-08 | Method, loc and analysis device for the lysis of cells and pcr amplification |
Publications (2)
Publication Number | Publication Date |
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EP2694672A1 true EP2694672A1 (en) | 2014-02-12 |
EP2694672B1 EP2694672B1 (en) | 2018-09-12 |
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Application Number | Title | Priority Date | Filing Date |
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EP12704033.5A Active EP2694672B1 (en) | 2011-04-08 | 2012-02-08 | Method for the lysis of cells and pcr amplification |
Country Status (3)
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EP (1) | EP2694672B1 (en) |
DE (1) | DE102011007035A1 (en) |
WO (1) | WO2012136400A1 (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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DE102013203655B4 (en) * | 2013-03-04 | 2023-03-23 | Robert Bosch Gmbh | Method and device for the selective lysis of cellular particles |
DE102014207774B4 (en) * | 2014-04-25 | 2015-12-31 | Robert Bosch Gmbh | Method and device for purifying biological molecules |
EP3338889A1 (en) | 2016-12-23 | 2018-06-27 | IMEC vzw | Combined extraction and pcr systems |
DE102017123919A1 (en) | 2017-10-13 | 2019-04-18 | Gna Biosolutions Gmbh | Method and apparatus for lysing microorganisms |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US5028379A (en) | 1989-12-20 | 1991-07-02 | General Electric Company | Fuel handling system for nuclear reactor plants |
US6815209B2 (en) * | 2001-11-16 | 2004-11-09 | Cornell Research Foundation, Inc. | Laser-induced cell lysis system |
KR101157175B1 (en) * | 2005-12-14 | 2012-07-03 | 삼성전자주식회사 | Microfluidic device and method for concentration and lysis of cells or viruses |
KR100813271B1 (en) * | 2006-11-09 | 2008-03-13 | 삼성전자주식회사 | Method and apparatus for disrupting cell or virus and amplifying nucleic acids using gold nanorod |
-
2011
- 2011-04-08 DE DE102011007035A patent/DE102011007035A1/en not_active Withdrawn
-
2012
- 2012-02-08 WO PCT/EP2012/052072 patent/WO2012136400A1/en active Application Filing
- 2012-02-08 EP EP12704033.5A patent/EP2694672B1/en active Active
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Also Published As
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DE102011007035A1 (en) | 2012-10-11 |
WO2012136400A1 (en) | 2012-10-11 |
EP2694672B1 (en) | 2018-09-12 |
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